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https://openalex.org/W3196710090	https://europepmc.org/articles/pmc8472215?pdf=render	English	null	A Fetus with Congenital Microcephaly, Microphthalmia and Cataract Was Detected with Biallelic Variants in the OCLN Gene: A Case Report	Diagnostics	2,021	cc-by	7,259	Citation: Ng, V.K.S.; Lau, T.K.; Kan, A.S.Y.; Chung, B.H.Y.; Luk, H.M.; Ng, W.F.; Shi, M.; Choy, K.W.; Cao, Y.; Leung, W.C. A Fetus with Congenital Microcephaly, Microphthalmia and Cataract Was Detected with Biallelic Variants in the OCLN Gene: A Case Report. Diagnostics 2021, 11, 1576. https://doi.org/10.3390/diagnostics 11091576 Abstract: Microcephaly and microphthalmia are both rare congenital abnormalities, while concur- rently, these two are even rarer. The underlying etiology would be complex interplaying between heterogeneous genetic background and the environmental pathogens, particularly during critical periods of early tissue development. Here, we reported a prenatal case with microcephaly, microph- thalmia, and bilateral cataracts detected by ultrasonography and conﬁrmed by autopsy. Various routine infection-related tests and invasive genetic testing were negative. Whole genome sequencing of fetus and parents revealed OCLN gene defects may be associated with these multiple congenital abnormalities. Keywords: OCLN gene; whole genome sequencing; microphthalmia; microcephaly; cataract; prena- tal diagnosis Vivian Kwun Sin Ng 1, Tze Kin Lau 2, Anita Sik Yau Kan 3 , Brian Hon Yin Chung 4, Ho Ming Luk 5, Wai Fu Ng 6, Mengmeng Shi 2, Kwong Wai Choy 2, Ye Cao 2,7,* and Wing Cheong Leung 1,* 1 Department of Obstetrics and Gynaecology, Kwong Wah Hospital, Hong Kong, China; vivian_nks@hotmail.com 1 Department of Obstetrics and Gynaecology, Kwong Wah Hospital, Hong Kong, China; 2 Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China; tklau2019@gmail.com (T.K.L.); shimengmeng@link.cuhk.edu.hk (M.S.); richardchoy@cuhk.edu.hk (K.W.C.) 2 Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China; tklau2019@gmail.com (T.K.L.); shimengmeng@link.cuhk.edu.hk (M.S.); richardchoy@cuhk.edu.hk (K.W.C.) 3 3 Department of Obstetrics and Gynaecology, Queen Mary Hospital, Hong Kong, China; kansya@hku.hk 3 Department of Obstetrics and Gynaecology, Queen Mary Hospital, Hong Kong, China; kansya@hku.hk 4 Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China; bhychung@hku.hk 4 Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China bhychung@hku.hk 5 Clinical Genetic Service, Department of Health, Hong Kong, China; luksite@gmail.com 6 Department of Anatomical and Cellular Pathology, Hong Kong Children Hospital, Hong Kong, China; ngwaifu@ha.org.hk g g 7 Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, China g g 7 Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, China * Correspondence: yecao@cuhk.edu.hk (Y.C.); leungwc@ha.org.hk (W.C.L.); Tel.: +852-3505-2859 (Y.C.) diagnostics diagnostics diagnostics Case Report A Fetus with Congenital Microcephaly, Microphthalmia and Cataract Was Detected with Biallelic Variants in the OCLN Gene: A Case Report Vivian Kwun Sin Ng 1, Tze Kin Lau 2, Anita Sik Yau Kan 3 , Brian Hon Yin Chung 4, Ho Ming Luk 5, Wai Fu Ng 6, Mengmeng Shi 2, Kwong Wai Choy 2, Ye Cao 2,7,* and Wing Cheong Leung 1,* diagnostics diagnostics 1. Introduction Congenital microcephaly is deﬁned as occipitofrontal head circumference (OFC) more than two standard deviations (SD) below the mean adjusted for age, sex, and race, which includes approximately 2% of the population [1]. In severe (true) case, it is deﬁned by OFC ≤−3 SD from the mean, which includes approximately 0.1% of the population [1]. The overall prevalence of microcephaly in the absence of Zika virus infection was 3 to 7.4 per 10,000 live births [2,3]. The etiology is diverse but can be divided into environmental (e.g., congenital infection, teratogen exposure, trauma resulting in ischaemia) and genetic causes (e.g., chromosomal abnormalities leading to cortical malformation, single gene mutation leading to neuronal migration disorders and syndromes) [4]. 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: © 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/). g g y Microphthalmia is deﬁned by the reduced total axial length of the globe within the orbit. It may be unilateral or bilateral. Together with anophthalmia (absence of ocular tis-sue within orbit), the prevalence of congenital microphthalmia is approximately one to three per 10,000 live births, according to several countries’ or regions’ population-based assessments [5–7]. It accounts up to 11% of blind children [8]. It may occur in isolation or as part of a syndrome in more than 50% (published range 33–95%) of cases that present extraocular ﬁndings of variable severity, most commonly involving craniofacial region https://www.mdpi.com/journal/diagnostics Diagnostics 2021, 11, 1576. https://doi.org/10.3390/diagnostics11091576 Diagnostics 2021, 11, 1576 2 of 8 with anomalies, musculoskeletal system [9]. The etiology of microphthalmia is complex, as it may be caused by genetic abnormalities (e.g., gene mutation, chromosomal aberration), infection, or exposure of teratogens. Several risk factors, including advanced maternal age, elevated maternal pre-pregnancy body mass index (BMI), maternal smoking during pregnancy, multiple births, preterm birth, and low birth weight have been suggested [7]. Prenatal detection of microcephaly and microphthalmia is challenging but possible with the use of high-resolution ultrasound, magnetic resonance imaging [10,11]. 1. Introduction The pres- ence of orbit shown in imaging does not guarantee normal visual function, as it depends not only on orbit but also on retinal development and other intra- or extra-ocular character- istics. Moreover, the neurological outcome of prenatal detected microcephaly cannot be ascertained since fetal brain is still under development. Microcephaly or microphthalmia could arise independently or together as presentations of certain rare syndromes. Therefore, establishing molecular diagnosis toward an informative prenatal counselling regarding multiple congenital abnormalities is important but difﬁcult considering their rarity and heterogeneous etiologies. The formation of brain and orbit involves multiple processes of induction and differ- entiation during embryogenesis, which involved hundreds of genes [12,13]. Its genetic heterogeneity and rarity make diagnosis challenging and usually requires a comprehensive genome-wide genetic testing which stand a better chance to pinpoint the diagnosis. Here, we present an undiagnosed case of fetal bilateral microphthalmia, in which microcephaly carries biallelic variants in the OCLN gene identiﬁed by whole genome sequencing. 2. Case Report Both orbital and lens diameters were much smaller than 10th centile. Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Goldstein et al. Both orbital and lens diameters were much smaller than 10th centile. Goldstein et al. Both orbital and l ns d Go dstei et a . ot o bita a d e s dia ete s we e uc s a e t a 0t ce ti e. Figure 3. Postmortem examination of abortus confirmed ultrasound diagnosis of microcephaly and microphthalmia. (a) Gross feature of the abortus brain shows no evidence of other anomalies, dis- ruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpe- bral fissure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens Figure 3. Postmortem examination of abortus confirmed ultrasound diagnosis of microcephaly and microphthalmia. (a) Gross feature of the abortus brain shows no evidence of other anomalies, dis- ruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpe- bral fissure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens of our abortus with comparison to controls at 22 weeks (right) and 24 weeks (left). Figure 3. Postmortem examination of abortus confirmed ultrasound diagnosis of microcephaly and microphthalmia. 2. Case Report Our patient was a 37 year-old G2P1 Chinese female. Her ﬁrst pregnancy was a pre- term delivery at 35 weeks of gestation of an otherwise healthy girl. During this pregnancy, she suffered from roseola and was exposed to Erythromycin at very early gestational age. At ﬁrst trimester, non-invasive prenatal testing (NIPT) for common aneuploidies and mi-cro-deletion/duplications showed low-risk results. The fetal morphology scan at 21 weeks of gestation showed a unilateral choroid plexus cyst in the transventricular plane (Figure 1a), which was resolved later. The diameters of both orbits and lens (Figure 2a,b) were smaller than 10th centile using the nomogram by Goldstein et al. [14]. Invasive genetic testing was suggested, and the patient agreed to amniocentesis. Quantitative ﬂuorescence polymerase chain reaction (QF-PCR), karyotyping, and chromosomal microarray analysis (CMA) all indicated a chromosomally normal male fetus. Screening for congenital infection (toxoplasmosis, rubella, cytomegalovirus (CMV), herpes simplex virus (HSV), human immunodeﬁciency virus) from maternal blood and urine as well as amniotic ﬂuid was negative at the time of diagnosis. In the later scan at 23 weeks of gestation, the fetal head circumference (Figure 1b) was found three weeks smaller than expected. The patient decided to terminate the pregnancy at 23 weeks of gestation after counselling. The fetal postmortem examination (Figure 3a–e) conﬁrmed microcephaly in the absence of brain anatomy disruption, calciﬁcation, and fetal akinesia. Bilateral microphthalmia with poorly formed anterior chamber and lens closely opposed to the corneal surface. The corneas and lens were opaciﬁed suggestive of bilateral cataract. There was no evidence of infection in any organs. Diagnostics 2021, 11, 1576 Diagnostics 2021, 11, x FOR 3 of 8 3 of 8 Figure 1. Ultrasound scan at 21 and 23 weeks of gestation. (a) Transventricular plane at 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 k (b) Bi i t l di t d h d i f t 23 k Figure 1. Ultrasound scan at 21 and 23 weeks of gestation. (a) Transventricular plane at 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 weeks; (b) Biparietal diameter and head circumference at 23 weeks. Figure 1. Ultrasound scan at 21 and 23 weeks of gestation. (a) Transventricular plane at 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 weeks; (b) Biparietal diameter and head circumference at 23 weeks. Figure 1. 2. Case Report (a) Gross feature of the abortus brain shows no evidence of other anomalies, dis- ruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpe- bral fissure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens of our abortus with comparison to controls at 22 weeks (right) and 24 weeks (left). Figure 3. Postmortem examination of abortus confirmed ultrasound diagnosis of microcephaly and microphthalmia. (a) Gross feature of the abortus brain shows no evidence of other anomalies, dis- ruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpe- bral fissure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens of our abortus with comparison to controls at 22 weeks (right) and 24 weeks (left). Figure 3. Postmortem examination of abortus conﬁrmed ultrasound diagnosis of microcephaly and microphthalmia. (a) Gross feature of the abortus brain shows no evidence of other anomalies, disruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpebral ﬁssure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens of our abortus with comparison to controls at 22 weeks (right) and 24 weeks (left). of our abortus with comparison to controls at 22 weeks (right) and 24 weeks (left). 2. Case Report Ultrasound scan at 21 and 23 weeks of gestation. (a) Transventricular plane at 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 weeks; (b) Biparietal diameter and head circumference at 23 weeks. 23 k f i ( ) T i Figure 1. Ultrasound scan at 21 and 23 weeks of gestation. (a) Transventricular plane at 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 Figure 1. Ultrasound scan at 21 and 23 weeks of gestation. (a) Transventricular plane at 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 weeks; (b) Biparietal diameter and head circumference at 23 weeks. Figure 1. Ultrasound scan at 21 and 23 weeks of gestation. (a) Transventricular plane at 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 weeks; (b) Biparietal diameter and head circumference at 23 weeks. igu e . U asou sca a a 3 ee s o ges a io (a) a s e icu a p a e a 21 weeks showed unilateral choroid plexus cyst which was resolved later in scan at 23 weeks; (b) Biparietal diameter and head circumference at 23 weeks. p Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Goldstein et al. Both orbital and lens diameters were much smaller than 10th centile. Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Goldstein et al. Both orbital and lens diameters were much smaller than 10th centile. Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Goldstein et al. Both orbital and lens diameters were much smaller than 10th centile. Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Goldstein et al. Both orbital and lens diameters were much smaller than 10th centile. Figure 2. Orbital (a) and lens (b) diameters measured at 21 weeks with reference to growth chart by Goldstein et al. 2. Case Report (a) Gross feature of the abortus brain shows no evidence of other anomalies, dis- ruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpe- bral fissure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens of our abortus with comparison to controls at 22 weeks (right) and 24 weeks (left). Figure 3. Postmortem examination of abortus conﬁrmed ultrasound diagnosis of microcephaly and microphthalmia. (a) Gross feature of the abortus brain shows no evidence of other anomalies, disruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpebral ﬁssure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens of our abortus with comparison to controls at 22 weeks (right) and 24 weeks (left). Figure 3. Postmortem examination of abortus confirmed ultrasound diagnosis of microcephaly and microphthalmia. (a) Gross feature of the abortus brain shows no evidence of other anomalies, dis- ruption or evidence of infection to support syndromic conditions or congenital infection; (b) Palpe- bral fissure of abortus is shorter compared to control 1 (left) and 2 (right) at similar gestation; (c) Lateral view of the eyeball of abortus with comparison to controls at 22 weeks (right) and 24 weeks (left); (d) Pupil of abortus comparing with controls at 22 weeks (right) and 24 weeks (left). The pupils were not well seen in abortus; (e) Dissected eyeball demonstrating the small and opacity lens Figure 3. Postmortem examination of abortus confirmed ultrasound diagnosis of microcephaly and microphthalmia. 2. Case Report This trio WGS data analysis did not detect any clinically significant chromosomal balanced rearrangement or loss of heterozygosity (>5 Mb) but revealed rare compound heterozygous variants in the OCLN gene (NM_002538) that might be related to the fetal abnormalities: maternally in- herited c.458T>C (p.L153S) and paternal inherited c.-68-37T>C (Figure 4). Defects in OCLN cause Pseudo-TORCH syndrome 1 [MIM:251290], an autosomal recessive neurologic dis- order with characteristic features including congenital microcephaly, intracranial calcifi- cations, and severe developmental delay, which mimic intrauterine TORCH infection in the absence of evidence for infection [18]. The OCLN gene c.458T>C (p.L153S) variant was extremely rare with minor allele frequency 5.5 × 10−4 in the East Asian ethnic population according to the gnomAD. Multiple in silico algorithm predicted this missense having deleterious impact on protein (Polyphen: probably_damaging; SIFT: deleterious, REVEL: 0.51; CADD: 25.5). This variant is highly conservative throughout different species with phyloP100wayAll score up to 7.887. The c.-68-37T>C variant was located in the intron 1 with a CADD score of 8.1. This variant has never been reported before. Both variants were classified as variants of uncertain significance (VUS) according to the ACMG guidelines (criteria PM2 was applied for both variants) [16]. Figure 4. The schematic of mutations in OCLN gene, Occludin detected in this fetus. Number 1-9 indicate the Exon 1-9 of the OLCN gene (NM_002538). TM: transmembrane domain. Figure 4. The schematic of mutations in OCLN gene, Occludin detected in this fetus. Number 1-9 indicate the Exon 1-9 of the OLCN gene (NM_002538). TM: transmembrane domain. Figure 4. The schematic of mutations in OCLN gene, Occludin detected in this fetus. Number 1-9 indicate the Exon 1-9 of the OLCN gene (NM_002538). TM: transmembrane domain. Figure 4. The schematic of mutations in OCLN gene, Occludin detected in this fetus. Number 1-9 indicate the Exon 1-9 of the OLCN gene (NM_002538). TM: transmembrane domain. On the other hand, this patient was pregnant soon after the termination and was re- ferred for genetic counselling by clinical geneticist. The couple was informed of the WGS findings that variants of uncertain significance in the OCLN gene were identified. This result suggested that these variants could not explain clearly the fetal abnormalities. It would be due to the gaps in our current knowledge that impede an accurate interpretation of their pathogenicity. 2. Case Report In the absence of chromosomal abnormalities identiﬁed by NIPT, QF-PCR, and kary- otyping and the lack of pathogenic copy number variant detected by CMA, trio whole exome sequencing was performed but did not yield relevant alternations. In order to further explore the possibility of an underlying genetic cause, the couple was counselled 4 of 8 d kar whole Diagnostics 2021, 11, 1576 and agreed to whole genome sequencing (WGS, 30×) in a research setting. The analysis of single nucleotide variants (SNVs), small indels, copy number variants (CNVs), and struc- tural variants (SVs) were conducted by our published in-house pipelines [15]. The clinical signiﬁcance of the detected variants was interpreted in accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG) [16,17]. This trio WGS data analysis did not detect any clinically signiﬁcant chromosomal balanced rearrangement or loss of heterozygosity (>5 Mb) but revealed rare compound heterozygous variants in the OCLN gene (NM_002538) that might be related to the fetal abnormalities: maternally inherited c.458T>C (p.L153S) and paternal inherited c.-68-37T>C (Figure 4). Defects in OCLN cause Pseudo-TORCH syndrome 1 [MIM:251290], an autosomal recessive neurologic disorder with characteristic features including congenital microcephaly, intracranial calciﬁ- cations, and severe developmental delay, which mimic intrauterine TORCH infection in the absence of evidence for infection [18]. The OCLN gene c.458T>C (p.L153S) variant was extremely rare with minor allele frequency 5.5 × 10−4 in the East Asian ethnic population according to the gnomAD. Multiple in silico algorithm predicted this missense having deleterious impact on protein (Polyphen: probably_damaging; SIFT: deleterious, REVEL: 0.51; CADD: 25.5). This variant is highly conservative throughout different species with phyloP100wayAll score up to 7.887. The c.-68-37T>C variant was located in the intron 1 with a CADD score of 8.1. This variant has never been reported before. Both variants were classiﬁed as variants of uncertain signiﬁcance (VUS) according to the ACMG guidelines (criteria PM2 was applied for both variants) [16]. agreed to whole genome sequencing (WGS, 30×) in a research setting. The analysis of sin- gle nucleotide variants (SNVs), small indels, copy number variants (CNVs), and structural variants (SVs) were conducted by our published in-house pipelines [15]. The clinical sig- nificance of the detected variants was interpreted in accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG) [16,17]. 2. Case Report Prenatal invasive diagnosis for the compound heterozygous vari- ants was not indicated if sonographic evidence did not show microcephaly, microphthal- mia, or other abnormalities. The couple understood the genetic results and the limitation of current testing. They preferred a conservative approach with ultrasound monitoring after genetic counselling. Her pregnancy went on uneventfully with serial ultrasounds showing normal growth of both fetal orbits and lens. She had delivery at term by lower segment caesarean section in the private sector There was no abnormality found in the On the other hand, this patient was pregnant soon after the termination and was referred for genetic counselling by clinical geneticist. The couple was informed of the WGS ﬁndings that variants of uncertain signiﬁcance in the OCLN gene were identiﬁed. This result suggested that these variants could not explain clearly the fetal abnormalities. It would be due to the gaps in our current knowledge that impede an accurate interpretation of their pathogenicity. Prenatal invasive diagnosis for the compound heterozygous variants was not indicated if sonographic evidence did not show microcephaly, microphthalmia, or other abnormalities. The couple understood the genetic results and the limitation of current testing. They preferred a conservative approach with ultrasound monitoring after genetic counselling. Her pregnancy went on uneventfully with serial ultrasounds showing normal growth of both fetal orbits and lens. She had delivery at term by lower segment caesarean section in the private sector. There was no abnormality found in the newborn. g newborn. 3. Discussion Here, we presented a prenatal case with microcephaly, microphthalmia, and bilateral cataracts detected with rare biallelic changes in the OCLN gene through whole genome sequencing analysis. The OCLN gene identiﬁed encodes an integral membrane protein at tight junctions (TJs), Occludin, which is functional in endothelium in early fetal de- 5 of 8 Diagnostics 2021, 11, 1576 velopment and maintenance of blood–brain barrier in postnatal life [18,19]. Recently, Bendriem et al. revealed that OCLN regulated the centrosome organization and dynamics which is required by the early corticogenesis [19]. Multiple genes related with centro- some associated functions are known to cause microcephaly disorders [18]. The complex role of Occludin has been demonstrated in mice models with occludin deﬁciency that heterogeneous phenotypic effects impact gastric epithelium, brain, testes, salivary gland, as well as compact bone [19]. Occludin deﬁciency due to OCLN gene mutation leads to abnormal cerebral vasculature and blood–brain barrier, which results in immune cell mediated insults and ischaemia and thus cortical malformation. The variant c.458T>C (p.L153S) was predicted as disease-causing by multiple in silico algorithms. It was located in the highly conserved MARVEL domain of Occludin which was commonly detected with mutations in patients [20]. Currently limited cases with biallelic changes in the OCLN genes were reported [21–23]. The patients mainly present with microcephaly, simpliﬁed gyration and polymicrogyria (PMG), intracranial calciﬁcations, early onset seizures, and severe developmental delay which mimic congenital TORCH infection [21]. Some patients present a characteristic prominent band of cortical gray matter calciﬁcation in the brain MRI [21]. Besides the core phenotypes, other uncommon phenotypes include congenital cataract and/or microphthalmia, central diabetes insipidus, and renal dysfunction [20–23]. In general, the prognosis of patients was poor, and early death was frequently reported [22]. In comparison, our case presented partial core phenotypes, including microcephaly with general normal anatomy and microphthalmia in the 21 weeks of gestational age, with no signs regarding intracranial calciﬁcation. However, there is a paucity of data regarding pre- natal presentations of OCLN-related syndrome. Although in silico analysis is supportive, biological studies of these variants are needed to elucidate how these mutants impact the protein function and related to patient’s phenotype. These two variants in the OCLN gene were classiﬁed as variants of uncertain signiﬁcance, which possibly expands the phenotype spectrum to fetal microcephaly, microphthalmia, and bilateral cataracts. p p y p Microcephaly has been stratiﬁed based on the etiology and the timing of onset. g newborn. 3. Discussion Besides infections, genetic factors would be the common causes for congenital microcephaly. Di- verse genomic abnormalities cause abnormal neuronal development and migration, which commonly lead to microcephaly [1,4]. In addition, Zika virus infection could also lead to congenital microcephaly. Studies found that Zika virus affects the junctional integrity of human brain microvascular endothelial cells to enter the brain, which suggested the important physiological role of the tight junction proteins in human epithelial cells [24,25]. It also correlates the potential relationship between the abnormal tight junctions and mi- crocephaly. However, the certain TJ protein-encoding genes such as JAM3 produce brain hemorrhage rather than microcephaly, which the OCLN gene may have developmental functions unanticipated for a TJ protein [19]. p J p Microphthalmia is a rare congenital abnormality, and prenatal diagnosis of this con- dition is complex and tends to be identiﬁed late as the fetal orbit and lens can only be visualized sonographically in the second trimester. A normal ultrasound ﬁnding does not guarantee that the optic nerve and visual function preserves. Eye development mainly takes place between week 3 and week 10 of embryo and involves multiple progenitors, including ectoderm, neural crest cells, and mesenchyme, which differentiate into various components of the eye and orbit [26]. Microphthalmia may not always lead to poor vision, but is associated with other ocular defects such as congenital cataracts, which are due to improper growth of the lens ﬁbers. Increasing numbers of genetic changes associated with both syndromic and isolated microphthalmia have been reported [27]. Recently, studies suggest that the disruption of signaling in neural crest cells can lead to alterations in neural epithelial derived optic cup formation, resulting in microphthalmia [28,29]. TJ proteins, especially those with MARVEL (MAL and related proteins for vesicle trafﬁc and membrane link) domain are usually regulators or components of many important signaling pathway. MARVEL domain proteins may modulate junctional permeability properties of cells and send signals through tight junctions into the cell interior [30]. MarvelD3, which belong to Diagnostics 2021, 11, 1576 6 of 8 the closely associated MARVEL protein family, the same as Occludin, has recently been identiﬁed to be required to attenuate JNK signaling pathway where direct JNK stimulation disrupts neural crest development and links tight junctions and modulation of the JNK pathway to eye morphogenesis [30,31]. g newborn. 3. Discussion Occludin also contributes to the compartmentaliza- tion of the ocular micro-environments by controlling the ﬂux through the retinal–blood barrier in Xenopus [31]. These imply the potential pathophysiological roles of Occludin in human eye development. y p The genome sequencing technology is migrating rapidly from research studies in the clinical application. However, accurate assessment of genetic variations to prioritize the disease-causing mutations remains challenging. Whole genome sequencing could in-crease the diagnostic yield to 40–50% of cases; this improvement is substantial compared with ~25% of exome sequencing [32,33]. Still, more than half of cases would receive uncertain or negative results, which means no explanatory genetic variant detected. A negative or uncertain result may be due to current interpretive limitations that prioritize the disease- causing variants from overwhelmingly large targets [34]. Currently, a phenotype-driven strategy is widely adapted for process of genome scale sequencing data to help select relevant disease genes on the basis of known phenotype–gene associations [35]. However, prenatal presentations of most genetic diseases were very limited, as in this case, which may hamper data interpretations of prenatal genetic ﬁndings. The functional impacts of those identiﬁed variants through experimental characterization or in silico approaches would be an important step in genome interpretation. Nevertheless, a future reanalysis could be considered to reassess the genetic causes with multilayered and more updated information [36]. [ ] In conclusion, here, we reported a prenatal case with microcephaly, microphthalmia, and bilateral cataracts, with detection of rare biallelic OCLN variants by whole genome sequencing. Given WGS provided a more comprehensive genome-wide investigation compared to CMA and whole exome sequencing in prenatal diagnosis, and less DNA was required for the experiment, our case report supports that WGS would be considered the second-tier genetic testing assay in prenatal diagnosis and the potential migration to early gestational week while proper pre- and post-test genetic counseling is warranted. Author Contributions: Conceptualization, V.K.S.N., K.W.C., Y.C. and W.C.L.; methodology, V.K.S.N., T.K.L., A.S.Y.K., B.H.Y.C., H.M.L., W.F.N., M.S., Y.C. and W.C.L.; validation, M.S.; formal analysis: V.K.S.N., T.K.L., A.S.Y.K., B.H.Y.C., H.M.L., W.F.N., M.S., Y.C. and W.C.L.; investigation, V.K.S.N. and Y.C.; resources, W.C.L.; data curation, V.K.S.N., M.S. and Y.C.; writing—original draft preparation, V.K.S.N. and Y.C.; writing—review and editing, V.K.S.N., M.S., Y.C. and W.C.L.; visualization, V.K.S.N. and W.F.N.; supervision, K.W.C., Y.C. and W.C.L.; project administration, Y.C. and W.C.L. All authors have read and agreed to the published version of the manuscript. g newborn. 3. Discussion Funding: This research was partially funded by the Food and Health Bureau—Health and Medical Research Fund grant (project No. 04152666). Institutional Review Board Statement: This study protocol was approved by the Ethics Committee of the Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee (CREC Ref. No. 2016.713). Institutional Review Board Statement: This study protocol was approved by the Ethics Committee of the Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee (CREC Ref. No. 2016.713). Informed Consent Statement: Informed consent was obtained from patient involved in the study. Written informed consent has been obtained from the patient to publish this paper. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy and ethical restrictions. Conﬂicts of Interest: The authors declare no conﬂict of interest. g p y 2. 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https://openalex.org/W2113689842	https://europepmc.org/articles/pmc2712465?pdf=render	English	null	Recent advances in biomedical applications of accelerator mass spectrometry	Journal of biomedical science	2,009	cc-by	12,213	Introduction l analysis. However, equivalent discussions refer equally well with changes in chemistry [4], to several other long- lived isotopes that can be quantitated by AMS: 3H, 10Be, 16Al, 36Cl, 41Ca, 56Ni, 99Tc, 129I, and 239Pu. Introduction Accelerator mass spectrometry (AMS; see Figure 1 for AMS schematic diagram) is an extremely sensitive nuclear physics technique for detection of very low-abundant, sta- ble and long-lived isotopes, initially developed in the mid-70's as a method of determining isotope ratios for geochronology and archaeological research [1,2]. The technique utilizes a tandem van de Graaff accelerator in order to generate the potential energy, allowing for sepa- ration of elemental isotopes at the single atom level. Therefore, AMS can be applied to quantitating the concen- trations of long-lived radioisotopes, such as 14C, for which decay counting is an inefficient method of quantitation because of its relatively long half-life of 5760 years [3]. Much of this discussion concerns 14C because it is the pre- dominant isotope for biomedical/bioanalytical probe The cost of publication in Journal of Biom is bourne by the National Science Coun Accelerator mass spectrometry (AMS; see Figure 1 for AMS schematic diagram) is an extremely sensitive nuclear physics technique for detection of very low-abundant, sta- ble and long-lived isotopes, initially developed in the mid-70's as a method of determining isotope ratios for geochronology and archaeological research [1,2]. The technique utilizes a tandem van de Graaff accelerator in order to generate the potential energy, allowing for sepa- ration of elemental isotopes at the single atom level. Therefore, AMS can be applied to quantitating the concen- trations of long-lived radioisotopes, such as 14C, for which decay counting is an inefficient method of quantitation because of its relatively long half-life of 5760 years [3]. Much of this discussion concerns 14C because it is the pre- dominant isotope for biomedical/bioanalytical probe The sensitivity of AMS for radiocarbon can be emphasized by its use in geochronology or carbon dating of historical artefacts [1,2]. As widely known, small amounts of 14C are constantly being formed from 14N by bombardment with cosmic radiation in the upper atmosphere, and this for- mation gives rise to 14CO2, and maintains the atmosphere at a nearly constant radiocarbon concentration of about 1.2 × 10-10% 14C (primarily as CO2) [5-7]. Plants fix atmospheric 14CO2, animals eat the plants and thus all higher living organisms contain 14C in equilibrium. Journal of Biomedical Science Open Access BioMed Central BioMed Central Published: 17 June 2009 This article is available from: http://www.jbiomedsci.com/content/16/1/54 © 2009 Hah; 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. © 2009 Hah; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit © 2009 Hah; 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 The use of radioisotopes has a long history in biomedical science, and the technique of accelerator mass spectrometry (AMS), an extremely sensitive nuclear physics technique for detection of very low-abundant, stable and long-lived isotopes, has now revolutionized high-sensitivity isotope detection in biomedical research, because it allows the direct determination of the amount of isotope in a sample rather than measuring its decay, and thus the quantitative analysis of the fate of the radiolabeled probes under the given conditions. Since AMS was first used in the early 90's for the analysis of biological samples containing enriched 14C for toxicology and cancer research, the biomedical applications of AMS to date range from in vitro to in vivo studies, including the studies of 1) toxicant and drug metabolism, 2) neuroscience, 3) pharmacokinetics, and 4) nutrition and metabolism of endogenous molecules such as vitamins. In addition, a new drug development concept that relies on the ultrasensitivity of AMS, known as human microdosing, is being used to obtain early human metabolism information of candidate drugs. These various aspects of AMS are reviewed and a perspective on future applications of AMS to biomedical research is provided. Open A Review Recent advances in biomedical applications of accelerator mass spectrometry Sang Soo Hah Address: Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University 1 Hoegi-dong 701, Korea Email: Sang Soo Hah - sshah@khu.ac.kr Email: Sang Soo Hah - sshah@khu.ac.kr Received: 6 December 2008 Accepted: 17 June 2009 The cost of publication in Journal of Biomedical Science is bourne by the National Science Council, Taiwan. Introduction l When an organism dies, there is no longer any carbon exchange, Schematic Figure 1 g p y ( ) g Schematic diagram of an accelerator mass spectrometry (AMS). Cesium (Cs) sputter ion source (A) contains the wheel with the graphite samples under high vacuum. Atomic Cs vapor is produced from a heated Cs reservoir and sprayed on to a heated ionizer surface, producing Cs+ ions that are accelerated towards the target held at -8 kV. The Cs+ ions sputter car- bon atoms and ions from the target that are ionized to C- ions as they pass through a condensed Cs layer on the cathode. Neg- ative ions at m/z 13 (13C-) and 14 (14C-) are pulsed through an injection magnet or low energy mass spectrometer (B) into a tandem electrostatic accelerator (C). Negative C- ions are accelerated towards the high-voltage terminal (+518 kV) in the center of the accelerator where they are converted to positive ions, C+ being the most abundant. The high-energy ion beam is focused to collide with argon gas electron stripper or a thin carbon foil, 0.02–0.05 μm thick (D) in a collision cell. Molecular charged ions such as 13CH- and 12CH2- do not survive the electron stripping process and are converted to atomic species, and 14N- ions decay on a femtosecond time-scale. The positive ions are repelled toward the high-energy exit of the accelerator held at 0 V. 13C+ and 14C+ ions are separated by momentum using a high-energy analyzing magnet or mass spectrometer (E). The beam currents of relatively abundant 12C and 13C are measured with Faraday cups (F). The 14C beam is focused by a quadruple and electrostatic cylinder analyzer and the atoms are counted in a gas ionization detector (G). The advantage of a gas ionization detector is that it measures energy loss in terms of ΔE/E which facilitates isotope separation. It is possible to optimize the detector to the energy-loss separation of the isotope. and 14C decays over time. Thus, the 12C:14C ratio can be correlated with the amount of time elapsed after an organ- ism's death, which is the basis of carbon dating. Carbon dating now extends beyond 50,000 years back in time [3]. as well as other LCS-utilized biomedical research because of the technique's sensitivity and precision. The workings of an AMS instrument are outlined elsewhere [8-10]. Page 2 of 14 (page number not for citation purposes) Page 1 of 14 (page number not for citation purposes) Page 1 of 14 (page number not for citation purposes) The cost of publication in Journal of Biomedical Science is bourne by the National Science Council, Taiwan. Journal of Biomedical Science 2009, 16:54 http://www.jbiomedsci.com/content/16/1/54 Schematic diagram of an accelerator mass spectrometry (AMS) Figure 1 Schematic diagram of an accelerator mass spectrometry (AMS). Cesium (Cs) sputter ion source (A) contains the wheel with the graphite samples under high vacuum. Atomic Cs vapor is produced from a heated Cs reservoir and sprayed on to a heated ionizer surface, producing Cs+ ions that are accelerated towards the target held at -8 kV. The Cs+ ions sputter car- bon atoms and ions from the target that are ionized to C- ions as they pass through a condensed Cs layer on the cathode. Neg- ative ions at m/z 13 (13C-) and 14 (14C-) are pulsed through an injection magnet or low energy mass spectrometer (B) into a tandem electrostatic accelerator (C). Negative C- ions are accelerated towards the high-voltage terminal (+518 kV) in the center of the accelerator where they are converted to positive ions, C+ being the most abundant. The high-energy ion beam is focused to collide with argon gas electron stripper or a thin carbon foil, 0.02–0.05 μm thick (D) in a collision cell. Molecular charged ions such as 13CH- and 12CH2- do not survive the electron stripping process and are converted to atomic species, and 14N- ions decay on a femtosecond time-scale. The positive ions are repelled toward the high-energy exit of the accelerator held at 0 V. 13C+ and 14C+ ions are separated by momentum using a high-energy analyzing magnet or mass spectrometer (E). The beam currents of relatively abundant 12C and 13C are measured with Faraday cups (F). The 14C beam is focused by a quadruple and electrostatic cylinder analyzer and the atoms are counted in a gas ionization detector (G). The advantage of a gas ionization detector is that it measures energy loss in terms of ΔE/E which facilitates isotope separation. It is possible to optimize the detector to the energy-loss separation of the isotope. http://www.jbiomedsci.com/content/16/1/54 http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 LSC becomes experimentally limiting, while the tech- nique of AMS has now changed the experimental para- digm because its extremely sensitive detection limit virtually removed the previous experimental barriers. samples derived from radiotracer studies with humans soon became apparent, since AMS produces very specific quantitation with simple analysis [19]. Any isotope con- centration greater than the known stable natural 14C back- ground must arise from an introduced isotope label ("introduced" includes contamination, which must be carefully controlled and avoided). In the simplest experi- mental design, there is only one external radioactive source, perhaps a radiolabeled compound introduced into the biological system at a specific time. The isotope ratio of the isolated sample is then easily converted to the concentration of the labeled compound and its metabo- lites per g or ml of the analyte. The high sensitivity of AMS indeed affects experimental designs in several ways. First, the radioisotopic dose can be reduced to inconsequential levels of radiolysis, hazard- ous waste streams, and human subject exposure. Sec- ondly, the chemical dose to a biological system, including humans of all ages and health status, is minimized to sub- physiological and sub-toxic doses. This allows a realistic analysis of the effects arising from low chemical doses. For example, children and women of child-bearing ages, who are important targets of increased health-related research, are suitable subjects at the low doses afforded by AMS [10,11], since the administration of such low levels of 14C are considered non-radioactive from a regulatory point of view. Finally, even if the sampled material needs fraction- ation to specific biomolecules prior to quantitation, the sample sizes are reduced to amounts that can be obtained from well-defined, and often non-invasive procedures. Not surprisingly, AMS has soon become a tool of choice for pharmacokinetic analyses [10,11,16]. All the metabo- lites of the compound that contain the labeled moiety can be directly quantified in chromatographic separations without resorting either to secondary standards or to prior knowledge of metabolic pathways. Although some fluo- rescent methods quantitate into the amol levels [20,21], they require derivatization procedures that are not suita- ble for in vivo tracing, create tracers that are not chemically equivalent, and are less general in applicability across many biological systems. Conversely, AMS is specific only to the labeled compound in any chemical or biological medium. http://www.jbiomedsci.com/content/16/1/54 Such specificity requires neither prior speciation nor the introduction of either molecular modifications or internal standards. With AMS, it is possible to conduct radiotracer studies in human with the administration of such low levels of 14C [10,11]. For a practical AMS measurement, biological samples con- taining 0.2–5 mg of carbon must be converted to solid carbon (graphite or fullerene) using a two-step process [12]. In a quartz tube, and using excess copper oxide (CuO), the sample's biological carbon is oxidized to CO2. The CO2 is then reduced to solid carbon by both reduction with titanium hydride and zinc powder and catalyzation with either iron or cobalt. Because this process is inde- pendent of the chemical nature of the sample, it elimi- nates interference or suppression from other sample components. Therefore, AMS provides one piece of infor- mation about the sample of carbon measured: the precise 12C:14C ratio. In AMS, one measures the isotope ratio with respect to that of a well-known (external) standard in order to produce an absolute isotope concentration for the combusted sample [13,14]. With AMS, experimenters only need the fractional elemental abundance of the sam- ple and the specific activity of the tracer compound in order to obtain, in the units most useful for interpretation, the concentration of the tracer in the sampled material. The mechanics of an AMS instrument, the mathematical conversions of the measured values to meaningful "Mod- ern" values, and the comparisons with LSC are well reviewed in the literature [3,11,15-17]. The most recent innovation using AMS technology is the so-called "microdosing" concept [10]. Choosing a drug for clinical trials from numerous candidates is very much a hit-and-miss business. Data are gathered from in vitro, in vivo, and in silico models in order to predict the drug's behavior in humans but such methods are probably only about 60% predictive. Presented with a choice of good candidates, it would be better to take them all into human subjects. This would, however, be prohibitively expensive, as each compound would require a significant package of toxicological safety testing. Alternatively, each candidate drug could be given to human volunteers at very low lev- els of a few tens, or at most a hundred μg. At these levels, only a limited toxicology package is required and in vivo human data can be acquired for candidate selection [22]. The development of biomedical AMS p f The use of radioisotopes has a long history in biomedical science. Isotopic enrichment of xenobiotics with 14C is routinely used as a method of following their metabolic fate in both animals and humans, and a drug is typically synthesized such that the natural abundance of 14C is increased from the background level of 1.2 × 10-10% to 20% or even higher depending upon the compound. The low energy β-radioactivity is then used to track the radi- olabeled compound and its metabolites in biological samples derived from laboratory animal or human stud- ies. LSC has been generally used for a long time to detect, follow and quantitate levels of radiotracer in such studies. There are occasions, however, when the low sensitivity of The most conventional method for the measurement of radioactivity for biomedical applications is liquid scintil- lation counting (LSC), a process known as decay count- ing. LSC, however, suffers from an innate insensitivity. In fact, it takes over a billion atoms of 14C to generate an average of only one disintegration per minute (dpm). AMS, on the other hand, allows direct measurement of 12C and 14C atoms by physically separating them in an ion beam [3]. Since the atoms are measured directly, without a necessity to wait for a disintegration event to occur, AMS is about six orders of magnitude more sensitive than LSC. Nowadays, AMS is the method of choice for carbon dating Page 2 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 Toxicant/drug metabolism study using AMS After the first biomedical application of sensitive and pre- cise quantitation of 14C by AMS in 1990 [18], the tech- nique has been explored for using animals and for fractionating tissues, cells and molecules in the study of metabolism, covalent macromolecule-ligand interactions, and non-covalent macromolecule-ligand interactions with amol sensitivity [8-10]. For example, Phillips et al. reported in 2000 a good application of AMS for these pur- poses by performing metabolism and macromolecular binding studies, primarily of environmental toxicants and toxins, as well as of vitamins in humans at physiological concentrations [23], which combined two general areas into an ongoing study of cancer chemoprevention by die- tary agents. They investigated covalent interactions of metabolic products with DNA and proteins, both in ani- mal hosts and in humans. The work with heterocyclic amine carcinogen has been expanded to the development of chemopreventive strate- gies for reducing the amount of DNA damage following carcinogen exposure and to the quantitation of the ability of certain dietary agents to reduce the levels of DNA adducts from two heterocyclic amines, PhIP and 2-amino- 3-methylimidazo [4,5-f]quinoline (IQ) [30]. It was found that chlorophyllin (a stable form of chlorophyll that is found in green leafy vegetables) and the isothiocyanate (found in broccoli) caused the greatest adduct reductions in prostate, liver and colon of rodents, presumably by modifying the metabolic processes leading to the final reactive metabolites. Turesky et al. performed a study to explore the use of coffee as a cancer chemopreventive agent as well [31]. They studied not only the effect of cof- fee on the enzymes that metabolize PhIP, but also its util- ity in reducing PhIP-DNA adduct levels. A 50% decrease in adduct formation was observed after 24-hour exposure in the liver of rats on the 5% coffee diet vs. the control group. An induction of glutathoine S-transferases, which are involved in the detoxication of HONH-PhIP and its reactive N-acetoxy intermediate might contribute to this protective effect of coffee in liver, not in extrahepatic tis- sues, based upon PhIP-DNA adduct formation. Various animal models were also used to study covalent binding levels of toxic compounds, including a quantitation of chromatin adducts of acrylamide in male germ cells of mice that was related to pre-implantation abnormalities in embryos [32]. The sample material available for analy- sis was so small that AMS quantitation could be the only reasonable route for obtaining very pure chromatin. http://www.jbiomedsci.com/content/16/1/54 Only AMS has the required sensitivity to conduct such studies at the low μg level. For the first time in 1990, sensitive and precise quantita- tion of 14C was applied to the analysis of biological sam- ples containing enriched 14C-labeled carcinogens for toxicology and cancer studies by Turteltaub et al. [18]. Their research quantified chemical binding of the 14C- labeled carcinogens to DNA at the level of 1 binding in 1011 bases. The benefits of using AMS for the analysis of In this review, the recent development of AMS methods to the present day in biomedical/bioanalytical research where it is being strategically used with high precision (see Figure 2 for the major applications of AMS discussed here) will be followed. Page 3 of 14 (page number not for citation purposes) Page 3 of 14 (page number not for citation purposes) Journal of Biomedical Science 2009, 16:54 http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 http://www.jbiomedsci.com/content/16 Major applications of AMS in various biomedical investigations discussed in the review Figure 2 Major applications of AMS in various biomedical investigations discussed in the review. Major applications of AMS in various biomedical investigations discussed in the review Figure 2 Major applications of AMS in various biomedical investigations discussed in the review. Major applications of AMS in various biomedical investigations discussed in the review Figure 2 Major applications of AMS in various biomedical investigations discussed in the review. led to a follow-on research to establish an AMS-based assay for quantifying PhIP-protein adducts in humans and the use of urinary PhIP-metabolites as biomarkers of PhIP exposure [28,29]. http://www.jbiomedsci.com/content/16/1/54 http://www.jbiomedsci.com/content/16/1/54 http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 Very recently, AMS was successfully used to measure the kinetics and repair of DNA adducts formed by two chem- otherapeutic compounds, carboplatin and oxaliplatin [33,34]. For carboplatin, AMS was used to measure carbo- platin-DNA binding in purified genomic DNA and in T24 human bladder cancer cells [34]. The kinetics observed for the first time for reaction with genomic DNA revealed that the rate of carboplatin-DNA adduct formation was approximately 100-fold slower than that reported for the more potent analog cisplatin, which may explain the lower toxicity of the compound. In human bladder cancer cells exposed to carboplatin, AMS allowed a measurement sensitivity of 1 amol per 10 μg of DNA. In addition, the rate of oxaliplatin adduction to salmon sperm DNA was measured, and oxaliplatin-DNA adduct distribution was further investigated at the nucleoside level by high-per- formance liquid chromatography (HPLC)-AMS following enzymatic digestion [33]. Importantly, rates of cellular drug influx, efflux, DNA damage and DNA repair in cul- tured platinum-sensitive testicular (833K) and platinum- resistant breast and bladder (MDA-MB-231 and T24, respectively) cancer cells incubated with a subpharmaco- logical dose of oxaliplatin (0.2 μM) were quantified and differentiated by AMS. quantitations. It should be noted that no specificity toward individual histone species was found though pro- tein adducts of benzene or its metabolites were indicated by elevated levels of 14C and that these studies used realis- tic inhalation doses. The incorporation of 14C was largely proportional to the density of gel staining, giving little evi- dence that the proteins were specific targets for selective labeling, implying high reactivity of benzene toward pro- teins which enables such attack to occur at multiple sites within multiple targets. In addition, Goldman et al. used postlabeling to eliminate the need for using a 14C-labeled compound directly in the biological subject in a study of benzo-pyrene adduction to DNA [39], where 14C-labeled acetic anhydride was used to recognize and label benzo[a]pyrene adducts on DNA. Liu's group applied AMS to the study of the DNA adduc- tion of several common 14C-labeled chemicals. One study shows that the adduction of nitrobenzene is suppressed by vitamin C, vitamin E, tea polyphenols, and other die- tary substances [40]. A high-dose level of sodium ben- zoate (500 mg/kg of body weight) in mice resulted in higher adduction in the kidney than in the liver. http://www.jbiomedsci.com/content/16/1/54 The levels of DNA-benzoate adducts decayed quite rapidly initially but persisted at a low level which is relevant to chronic use of sodium benzoate [41]. Radiolabeled adriamycin, also known as doxorubicin, was introduced to MCF-7 human breast cancer cells by Coldwell et al. [35]. Although adriamycin is an anti-cancer agent with the widest spectrum of anti-tumor activity, especially in the treatment of breast cancer and the domi- nant mechanism of action appears to involve impairment of topoisomerase IIα activity, the exact mechanism by which adriamycin exerts its anti-tumor activity is still uncertain. With many potential alternative mechanisms of action cited and reviewed, the technology of AMS has provided the first direct evidence of adriamycin-DNA adducts at clinically-relevant adriamycin concentrations. The dangers of certain dietary compounds to genetic material was then expanded to include aflatoxin B1 (AFB1) adducts as measured in the colon DNA of rats and humans [42], in which the levels of AFB1-DNA and AFB1- albumin adducts were investigated by AMS, indicating that there is a linear relationship between the exposure to AFB1 and AFB1-albumin adduct formation in rats in a dose range of 0.16 ng/kg–12.3 μg/kg, and that the protein adduct levels in the rats are similar to humans. The human metabolism of atrazine herbicide was traced in urine as a function of time for one week after a dermal exposure [43]. A highly polar metabolite, previously undiscussed, formed the largest single fraction of the excreted metabolites by the second day and continued at the same level for one week. These measurements consti- tuted a "rescue" effort, because the 14C level in the urine was too low for LSC of the chromatography fractions. The fractions were easily quantified by AMS, even for a total of 1.7 fmol of 14C loaded on the column in the 7-day post dose sample. Kwok et al. found dose-dependent binding of orthophe- nylphenol (OPP) fungicide to proteins in the rat urinary bladder, but found no significant covalent binding to the DNA [36]. The observed carcinogenic effect of OPP in the bladder might be due to the interference of critical cellular functions through quinone initiated oxidative stress and/ or an interaction between quinones and critical sulfhy- dryl-containing protein targets resulting in genetic altera- tions or cell death. The high sensitivity of AMS helps discard the hypothesis of DNA binding, even at the low doses administered. Boocock et al. Toxicant/drug metabolism study using AMS The heterocyclic amines are compounds that are found in cooked meat and are potent carcinogens in rodent mod- els. However, their role in human cancer remains largely unknown. Using AMS, Felton's group at the Lawrence Livermore National Laboratory has identified the metab- olites of [2-14C]2-amino-1-methyl-6-phenylimidazo [4,5- b]pyridine (PhIP) in humans and the relationships between the activities of key enzymes involved in PhIP metabolism and metabolite profiles [24,25]. In addition, the levels of PhIP adducts on the DNA and blood proteins of humans and rodents were quantified by AMS [26,27]. Through these works, it has been possible to establish the scaling factors between animal hosts and humans for DNA and protein adduct formation, as well as to establish plasma and urinary biomarkers of PhIP exposure. The sensitivity of AMS is required to keep both the chemical and the radiation doses to human volunteers to levels that do not exceed commonly accepted risks. These works have Page 4 of 14 (page number not for citation purposes) Page 4 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 Neuroscience study using AMS AMS has been used to study long-term pharmacokinetics, to identify biomolecular interactions, to determine chronic and low-dose effects or molecular targets of neu- rotoxic substances, to quantify transport across the blood- brain barrier (BBB) and to resolve molecular turnover rates in the human brain on the time-scale of decades. It is estimated that more than 90% of degenerate demen- tias are proteinopathies. i.e. caused by abnormal protein aggregation [50]. In Alzheimer's disease (AD), these are primarily different amyloid β (Aβ) peptides and a hyper- phosphorylated form of the tau protein [51], whereas α- synuclein is implicated in Parkinson's disease, dementia with Lewi bodies and other forms of dementia [52,53]. Although numerous contributing factors have been iden- tified, the etiology of these diseases is generally poorly understood. The bomb pulse of 14C (Figure 3) was used to determine the average date of formation of the major his- topathological features in AD brain: extracellular senile plaques (SP), composed primarily of Aβ peptide, and intracellular neurofibrillary tangles (NFT), composed of paired helical filaments containing hyperphosphorylated tau proteins [51]. The changing 14C level of contemporary carbon was also used to determine the carbon 'age' of nor- mal brain tissue (1.4 years). The SP and NFT structures have a much slower carbon turnover rate than normal tis- sue and are not in a formation/degradation equilibrium. The study showed that the average age of isolated SP and NFT was significantly greater than normal tissue from the same subjects (SP by 9.8 ± 4.9 years and NFT by 9.4 ± 3.8 years). Although a clear and consistent pattern of forma- tion of NFT and SP could not be formulated from the small number of analyzed subjects, in four out of six cases, average SP and NFT or both predated the onset of symp- toms by as long as 9 years. It is expected that more effi- cient isolation techniques that can accommodate smaller specimens from specific brain regions will produce more consistent patterns of information and such studies could provide valuable information on the etiology and pro- gression of AD and other neurodegenerative proteinopa- thies. Organophosphates, such as diisopropyl fluorophosphates (DFP), are frequently used as insecticides. DFP has been previously used as an experimental agent in neuroscience for its ability to inhibit cholinesterases and induce delayed peripheral neuropathy [46] and as an ophthalmic cholinesterase inhibitor in glaucoma treatment [47,48]. http://www.jbiomedsci.com/content/16/1/54 identified the human cytochrome enzyme involved in metabolizing the cancer therapeutic tamoxifen to reactive states that can lead to DNA adduc- tion [44]. They also quantified the level of binding tamoxifen to endometrial and colon DNA in an attempt to understand the role, if any, of this drug in endometrial Two studies [37,38] looked at the genotoxicity potential of benzene by quantifying species and strain differences of the protein and DNA binding at very low doses and by testing for histone-specific binding of benzene metabo- lites using gel separations and MS analysis with AMS Page 5 of 14 (page number not for citation purposes) Page 5 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 http://www.jbiomedsci.com/content/16/1/54 After the emergence of Gulf War Syndrome in veterans of the 1991 Gulf War, synergistic exposures to combinations of esterase inhibitors were a hypothesized contributor, and AMS was used to examine the effect of chronic expo- sure to PYB (7.75 mg/kg per day in chow) on acute doses of 14C-labeled PER (4.75 μg/kg). At 1 h after dosing, the amount of PER in brain and spinal cord was reduced by 30% for animals receiving PYB. At 24 h, there was no dif- ference in PER in the brain but the spinal cord had 70% less PER with PYB exposure. The levels of PER in the plasma was the same for each dose group. The sensitivity of the measurement was pg/g per equivalents in dissected tissue. Since PER and PYB are not direct competitors for enzyme binding, and the qualified effect is too large for competitive inhibition at these doses, a physiological effect such as decreased bioavailability is suggested. and colon cancer. They expanded the study to establishing if tamoxifen binds irreversibly to uterine DNA when given to women patients who were given a single therapeutic dose of 14C-labeled tamoxifen citrate orally ~18 hours prior to hysterectomy or breast surgery [45]. They demon- strated that after oral administration, tamoxifen forms adducts in human uterine DNA but at low numbers rela- tive to those previously reported in women after long- term tamoxifen treatment where levels, when detected, ranged from 15,000 to 130,000 adducts/1012 nucleotides. Bomb curv Figure 3 g g g Bomb curve used for dating recent biological materials. The levels of 14C in the atmosphere have been relatively stable over long time periods, with the exception that atmospheric nuclear weapons tests in 1955–1963 added significant amounts of 14C to the environment. The age of the biological material is calculated based on an assumption that the organism's biosynthe- sis is in isotopic equilibrium with its carbon sources. Choosing between the two dates requires additional data. [54,55], dendrite [56], and spine formation [57]. Although very sensitive fluorescent methods for quantify- ing [Ca2+] in vivo and in real time are well established [58,59], they require careful calibration and cannot directly distinguish between different sources of Ca2+. Fur- thermore, the Ca2+-sensitive dyes add a significant exoge- nous buffer capacity and distort the amplitude, time course and spread of [Ca2+] signals. Lin et al. in 2004 improved the sample chemistry required to extract cal- cium quantitatively from plasma, urine, and saliva using schemes that greatly increased the sample throughput as well as quality of the samples [60]. They studied 41Ca quantitation with AMS to access bone health for humans and dietary protein effects on bone resorption. It is expected to quantify changes in bone resorption using 41Ca arising from 3H- or 14C-labeled pharmaceuticals. The value of 41Ca quantitation for cancer, aging, and nutritive research is becoming better recognized, and AMS tech- nique will maintain its present dominance in high throughput measurement of this important biomedical tracer isotope. studies with humans. The maximum radioactive dose that can be administered to humans depends on the residence time in the body and whether it is accumulated in specific tissues. Under certain circumstances, i.e. when there is a long pharmacokinetic half-life, the amount of radioactiv- ity that can be administered is below the capabilities of LSC. Since the advent of biomedical AMS, it has been pos- sible to conduct radiotracer studies in humans, with the administration of such low levels of 14C that they are, from a regulatory point of view, considered non-radioac- tive. Furthermore, in the development of a new drug, there may be a number of candidate compounds available to go forward into clinical trials. The classical selection process involves conducting a series of modeling experiments using in vitro, cell-based or in silico techniques and some experiments with small numbers of laboratory animals. Bomb curv Figure 3 Through a process known as allometric scaling, a predic- tion is made of the pharmacokinetics of the candidate drug in humans. However, allometric scaling has occa- sionally failed to adequately predict the behavior of the drug in humans. In an ideal case, all the candidate drugs would be dosed to humans and selection made on the basis of true in vivo data. Practically, however, such an Neuroscience study using AMS The sensitivity of AMS permitted the study of low-level (sub-toxic) exposure to acutely toxic compounds in vivo (DFP has an oral LD50 in rat of 1.3 mg/kg). Vogel's group quantitated low-dose binding of the nerve agent analog, DFP, to the plasma and brain proteins of mice as a quan- tifiable biomarker of multiple chemical effects from pre- exposures of parathion (PTN), permethrin (PER), and pyridostigmine bromide (PYB) [49]. They found that brain DFP binding increased by 25–40% under various pesticide pre-exposure in food, although the plasma bind- ing concentrations did not change. A cholinergic-derived induction of NO was hypothesized to increase brain blood flow, resulting in higher delivery of DFP to the brain prior to its metabolism by copious plasma and liver estrases. Pyridostigmine produced a general 15% decrease in binding, presumably due to lower bioavailability of the food-delivered toxins arising from increased intestinal peristalsis. The effect on the permeability of the BBB to low doses of pesticide mixtures was also investigated using 14C-labeled DFP as a quantifiable probe of effects due to unlabeled PTN, PER and PYB separately and in conjunction [49]. The study concluded that if the increase in brain DFP level were due to increased permeability of the BBB, other toxins or pathogens might also induce increased BBB permeability with low pesticide exposure, and that the sensitivity of AMS allowed the probing of specific biochemical pathways using physiological doses, which did not perturb the natural system of the model animal. Calcium concentration and its spatial localization and dynamics are important in many neuronal processes, such as signaling, long-term potentiation and depression Page 6 of 14 (page number not for citation purposes) Page 6 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 Bomb curve used for dating recent biological materials Figure 3 Bomb curve used for dating recent biological materials. The levels of 14C in the atmosphere have been relatively stable over long time periods, with the exception that atmospheric nuclear weapons tests in 1955–1963 added significant amounts of 14C to the environment. The age of the biological material is calculated based on an assumption that the organism's biosynthe- sis is in isotopic equilibrium with its carbon sources. Choosing between the two dates requires additional data. Page 7 of 14 (page number not for citation purposes) Nutritional study using AMS It is becoming apparent that all humans do not identically respond to either diets or medicines, so their needs differ according to differences in their genetic information and physiological status. AMS may be particularly useful in obtaining accurate spatial information and low-level detection of essential and nonessential bioactive food components (nutrients) and their metabolites, and in enhancing the understanding of the impact of nutrient/ metabolite and biomolecular interactions. The fate and distribution of vitamins at physiological concentrations within healthy humans of all ages, for instance, had not been quantified prior to the use of AMS [63]. Vitamin research was performed including mathematical statistics and modeling using the high density human kinetic data to explore the parameter space of kinetic modeling [64,65]. AMS, an innovation technology for this purpose, has made it possible to administer such low amounts of 14C- labeled candidate drugs to humans and still retain suffi- cient levels of analytical sensitivity to determine its metab- olism and pharmacokinetics, even though the amount of radioactivity that can be administered to humans is lim- ited owing to the radiation exposure, not surprisingly. This technique is, however, very much in its infancy and it is not known, for the majority of drugs, whether the phar- macokinetics will be sufficiently linear so that the phar- macokinetics observed at the microdose will be predictive of those at the therapeutic dose. It is important, therefore, to study comparative pharmacokinetics of a drug candi- date at high and low doses to establish the validity of the "microdosing" concept, that is, a human microdosing study comprises the administration of a sub-pharmaco- logical/sub-therapeutic dose of novel drug candidate(s) in order to gain essential pharmacodynamic and pharma- cokinetic information [10,11]. To date, a major trial is underway financed by a group of pharmaceutical compa- nies to test this theory. The initial study involved sub-physiological doses of folic acid, which is especially important to the health of young mothers, but which had been studied only in elderly ill human subjects. AMS tracing doses contain a few hundred nanoCurie of 14C or less, even for highly recirculated nutrient chemicals, exposing the volunteer subject to less radiation damage than is obtained within 10 minutes in a commercial air flight. This is a commonly accepted level of radiation exposure, even among pregnant women, to whom a better understanding of their true folate needs is important. Pharmacokinetic study using AMS The benefits of using AMS for the analysis of samples derived from radiotracer studies include pharmacokinetic Page 7 of 14 (page number not for citation purposes) Page 7 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 approach would be prohibitively expensive due to the enormous amount of preclinical toxicology safety testing required. designed a new drug delivery microelectromechanical sys- tems (MEMS) to deliver tracer molecules as well as a ther- apeutic agent in vivo and evaluated their spatial and temporal release profiles using AMS [62]. An alternative approach, therefore, has been suggested [10] where very small amounts of drugs, in the microgram range, are administered. With such trace doses, the toxico- logical safety tests required are vastly reduced [22]. Hence, it is possible to dose a range of candidate drugs to humans and select the one with optimum pharmacokinetics to take forward for further clinical evaluation. The major bar- rier to this approach is the extremely sensitive methods of detection are required in order to quantitate the drug and its metabolites in plasma and excreta following the administration of such a small microdose. Page 8 of 14 (page number not for citation purposes) Nutritional study using AMS These studies were performed to determine if 8-oxodG can be phosphorylated and incorporated into DNA from oxida- tion of the nucleotide pool. The composition of the radi- olabeled nucleotides incorporated into DNA from the 14C-labeled 8-oxodG dosed cells was determined by nucl- eoside digestion followed by HPLC. Digestion of the DNA to nucleosides followed by separation with HPLC allowed determination of the composition of the radiolabeled nucleosides in the purified DNA. Each chromatogram shows a single peak that coelutes with an authentic stand- ard of 8-oxodG. The single peak observed is also confir- mation of the quality of the DNA digest and purification conditions, which were optimized to avoid artifactual 8- oxodG oxidation. The data collectively indicate that in the cells studied the nucleotide pool can be a significant source of 8-oxodG for incorporation into genomic DNA. Importantly, it was found that the rate of incorporation of 8-oxodG is approximately equal to that of dG and that the maximum concentration achieved was ~2 per 107 normal nucleotides, a level approaching that of background 8- oxodG levels in most cell types. Interestingly, it was unex- pectedly found that radiocarbon from 14C-labeled 8- oxodG was also incorporated into RNA, which was fol- lowed by a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA [81], allowing to propose an 8-oxodG metabolic mechanism in MCF-7 human breast cancer cells, as illustrated in Figure 4. A new approach is now opened to the mechanistic study of measuring the kinetics of small molecule fates at a very low level of detection with high precision and observing the initiating events in the nucleobase modification that lead to carcinogenesis or other diseases. apparently unrelated low folate intake [71], or common polymorphisms within the Hispanic population [72]. Relations between cirrhosis and homocystenemia, a risk factor in heart disease, also derived from the large recy- cling of demethylated folate through bile and back into the liver for remethylation [73,74]. The small samples used in AMS can provide high data density. AMS has the high sensitivity for long-term kinetic analysis to give detailed elimination information. Thus, only AMS could result in a model sufficiently detailed to reveal the hidden variables possibly responsible for these health effects. Dueker et al. Nutritional study using AMS An isotopic form of folate was required to dis- tinguish the dosed material from the greater amount of endogenous folate, but stable isotopic approaches have not been able to follow single physiologic doses for more than a few days in human volunteers [66]. Other studies used chronic isotopic dosing to obtain turnover and kinetic elimination measures, but do not provide detailed kinetic profiles [67]. The initial data quickly showed that folate was an effective label for the study of red blood cell production, lifetime and elimination [43], which has been approached by hematologists about the possibility of using this pulse-chase labeling mode to study the red blood cell lifetimes in disease states such as sickle-cell ane- mia or malaria. Lin et al. summarized the 6-months phar- macokinetic data from 13 human subjects with a median age of 24, including 7 women and suggested a connection between liver disease and folate deficiency, as revealed by detailed compartmental modeling of the entire high-den- sity data sets from all 13 subjects [60,68]. These works may explain the world's highest incidence of neural tube birth defects along the southern Rio Grande [69] as arising from the endemic levels of hepatitis [70], rather than the In 2004, Sandhu et al. reported [61] the first description of the full pharmacokinetic profile of a drug candidate assessed and of the comparisons of the kinetics of a phar- maceutical compound at pharmacological versus sub- pharmacological doses employing microdosing strategies, in order to address the unresolved issue of whether the pharmacokinetics determined following a microdose are representative of those following a conventional (phar- macological) dose. They successfully validated and uti- lized the technique of AMS to study the pharmacokinetics and disposition in dogs of a preclinical drug candidate after oral and intravenous administration. They empha- sized in the paper that only the exceptional sensitivity of AMS can provide a pharmacokinetic profile of the drug candidate, even following a microdose, which reveals aspects of the disposition of the agent that are inaccessible by conventional techniques. Li et al., on the other hand, Page 8 of 14 (page number not for citation purposes) Page 8 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 oped for rates of in vivo incorporation and repair of an 8- oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a well known DNA oxidative damage biomarker [81-83]. Nutritional study using AMS showed that naturally pro- duced β-carotene could be used at low doses to judge the vitamin A potential of carotene [75]. Reverse-phase HPLC was used with AMS to quantify plasma metabolites of the carotene in 99 hours after dosing. An unidentified acidic metabolite, possibly an epoxide, was found to comprise 15% of the circulating acidic fraction, and the metabolism of β-carotene to retinol and retinyl esters was surprisingly enhanced by a vitamin A supplementation that should decrease the need for carotene-derived retinal [76,77]. An increase in carotene absorption at the expense of intesti- nally produced retinyl esters was also found. Other important research using AMS An AMS method was devel- Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 Proposed mechanism of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG, a well known DNA oxidative damage biomarker) metabolism in MCF-7 human breast cancer cells, where PNP stands for purine nucleoside phosphorylase, HGPRT for hypoxan- thine-guanine phosphoribosyltransferase, RNA Pol for RNA polymerase, DNA Pol for DNA polymerase, RR for ribonucle- otide reductase, and BER for base excision repair, respectively Figure 4 Proposed mechanism of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG, a well known DNA oxidative damage biomarker) metabolism in MCF-7 human breast cancer cells, where PNP stands for purine nucleoside phos- phorylase, HGPRT for hypoxanthine-guanine phosphoribosyltransferase, RNA Pol for RNA polymerase, DNA Pol for DNA polymerase, RR for ribonucleotide reductase, and BER for base excision repair, respectively. MTH1 is a pyrophosphatase capable of cleaving the pyrophosphate either from 8-oxodGTP or 8-oxoGTP, thus preventing the accumulation of a potentially mutagenic species. p , y yg ( , g ) , p p p y , yp g p p y , p y , p y , , p , p y g Proposed mechanism of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG, a well known DNA oxidative damage biomarker) metabolism in MCF-7 human breast cancer cells, where PNP stands for purine nucleoside phos- phorylase, HGPRT for hypoxanthine-guanine phosphoribosyltransferase, RNA Pol for RNA polymerase, DNA Pol for DNA polymerase, RR for ribonucleotide reductase, and BER for base excision repair, respectively. MTH1 is a pyrophosphatase capable of cleaving the pyrophosphate either from 8-oxodGTP or 8-oxoGTP, thus preventing the accumulation of a potentially mutagenic species. the 14C content of tooth enamel and related it to the known concentrations in the atmosphere in different years to establish the year of tooth formation. The date was then related to the known age for enamel deposition of individual teeth to establish the person's year of birth, resulting in a remarkably precise estimate of age for 22 individuals (R2 = 0.99). The average systematic deviation from the correct value was +0.2 years, and the average absolute error for individual measurements was 1.6 ± 1.3 years, indicating that the precision is substantially higher than that obtained by other available methods. tions [85]. Other important research using AMS Non-covalent equilibrium binding was quantified by AMS in two studies seeking to develop more sensitive immunoassays (IA) [78,79]. Lu et al. developed an AMS- IA to one species of the parathyroid hormone related pro- tein that is a biomarker of prostate cancer [78], and Shan et al. showed that AMS increased sensitivity and quantita- tion over already sensitive ELISA-IA's for atrazine and dioxin without resulting in a waste stream that exceeded the government's definitions of radioactive materials [79]. These studies set the stage for further development of non- covalent labeling strategies that are not as straightforward as the previously listed covalent binding work, and may serve as models for the development of sensitive and "nonradioactive" IA for peptides, including polypeptide tumor markers. AMS was also used as a core technique to verify that 5'- methylthioinosine is an active nucleic acid precursor in Plasmodium falciparum which is unable to synthesize purine bases and relies on purine salvage and purine recy- cling to meet its purine needs [84]. To understand the purine pathways of malaria, they characterized the activi- ties of adenosine deaminase and purine nucleoside phos- phorylase from P. falciparum which have catalytic specificities that allow them to use methylthiopurines and therefore to function in both purine salvage and methyl- thiopurine recycling. By using AMS, they showed this pathway is active in P. falciparum cultured in human eryth- rocytes. The two IA efforts above are also forms of postlabeling recognition of specific protein moieties and chemicals in biological solutions. Miyashita et al., on the other hand, demonstrated highly sensitive protein sequencing by Edman degradation, which is an impetus for developing methods of analyzing extremely small amounts of biolog- ical systems [80]. Their method is expected to be applica- ble to the sequencing of proteins from cell culture and illustrates a path to more general methods for determin- ing N-terminal sequences with high sensitivity. Retrospective birth dating of cells in humans was achieved based on the AMS measurement, which is a generally applicable strategy that can be used to measure cell turno- ver in man under physiological and pathological condi- Biomedical applications of AMS were expanded to the study of DNA damage/repair. Other important research using AMS They took advantage of the fact that testing of nuclear weapons resulted in a dramatic global increase in the levels of the isotope 14C in the atmosphere, followed by an exponential decrease after 1963, as shown in Figure 3[86-88], and they showed that the level of 14C in genomic DNA closely parallels atmospheric levels and can be used to establish the time point when the DNA was synthesized and cells were born. They also found that the strategy can be used to determine the age of cells in the cortex of the adult human brain leading to the conclusion that whereas nonneuronal cells are exchanged, occipital neurons are as old as the individual, supporting the view that postnatal neurogenesis does not take place in this region. The technology of AMS has been utilized to establish the dynamics within the stable population of adipocytes in adults, by measuring adipocyte turnover and analyzing the integration of 14C derived from nuclear bomb tests in genomic DNA [85,90]. Spalding et al. demonstrated using AMS that approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index, although the number of adipocyte is set during childhood and adolescence. Their results suggest that neither adi- pocyte death nor generation rate is altered in early onset obesity, and that a tight regulation of fat cell number in this condition during adulthood. In addition to the novel forensic application of AMS above, the age at death of individuals, an important step in their identification, was also established with high pre- cision by AMS analysis of dentition [89]. It was demon- strated that the amount of radiocarbon present in tooth enamel as a result of nuclear bomb testing is a remarkably accurate indicator of when a person was born, since the enamel of individual teeth contains 0.4% carbon and there is no turnover of enamel after it has been laid down, i.e. the 14C concentration reflects that in the atmosphere at the time of enamel formation. They, therefore, measured Page 10 of 14 (page number not for citation purposes) http://www.jbiomedsci.com/content/16/1/54 http://www.jbiomedsci.com/content/16/1/54 Journal of Biomedical Science 2009, 16:54 Journal of Biomedical Science 2009, 16:54 For practical purposes, rapid methods have been devel- oped to convert organic species into forms compatible with direct introduction to the spectrometer for 14C anal- ysis to perform the AMS measurements more efficiently [12]. Ognibene et al. Conclusions and future prospects While the initial themes of biomedical research with AMS involved primarily the kinetics and binding of carcino- genic toxins and focused on toxicokinetics and toxin metabolism with new initiatives in nutrition and immu- noassays, scientists have now expanded the study of kinet- ics and dynamics directly in humans for disease [91,92], nutritional [75], and pharmaceutical [16] research, since AMS is now a proven sensitive and robust method for quantifying rare isotopes in biological systems. Moreover, AMS has been utilized for the detection of biomarkers or molecular targets of relevance to nutrition and cancer and other chronic diseases. This opens up the whole area of biomarker studies where currently only changes to the size of a metabolic pool are measured. For example, a decrease in a metabolic biomarker might be due to increased catabolism or decreased anabolism. Using a trace dose of 14C-labeled precursor, the turnover rates of the biomarker could be determined without unduly exposing the volunteers to adverse levels of radioactivity. In theory, any and all endogenous components of a bio- logical system can be quantified by versatile AMS coupled with amol radiolabeled-isotope detection capability if that system is uniformly labeled by an isotope. Under this condition, all structural, signaling, and nourishing com- ponents become quantifiable at amol levels by AMS. Such an approach depends on quantitative isolation of the cho- sen components from other isotopically labeled materi- als. Cultures can be commonly grown on isotopically enriched substrates, usually to produce specific proteins or lipids for further tracing experiments. More frequently, specifically labeled precursors can be added to cultures to enhance the isotopic signal of chosen components. These isotopic enrichments are akin to tracing specific com- pounds that cannot reveal the entire biochemical balance of a system. Biomolecules that are stable over those 20 to 50 years can also be quantified as being retained from a uniformly labeled system. Any biochemical pathway can be virtually quantified by AMS if a sufficiently specific labeling procedure can be found. It is also expected quan- titative postlabeling strategies can be developed for oxida- tive and other molecular modifications or functions as AMS has been called an enabling technology, and espe- cially AMS for 14C analysis has become more accessible and inexpensive, making the biomedical application of AMS no more difficult than other tracing and quantifying methods now used in routine biomedical research. Competing interests The author declares that he has no competing interests. Acknowledgements This research was supported partly by the Kyung Hee University Research Fund in 2008 (KHU-20081559) and by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea Government (MEST) (No. 2009-0064333). Conclusions and future prospects There are likely to be many more applications to biomedical sci- ence for this technology in the future, so far never- thought-of. Other important research using AMS developed a high throughput modi- fication of the reduction stage for carbon sample prepara- tions. The technique uses custom-made septa-sealed reaction vessels for the trapping, purification, and reduc- tion of combustion gases to the desired elemental carbon on an iron-group catalyst. The combustion gases can come from sealed combustion tubes that are the most effi- cient process for large numbers of mg-sized samples. examples of AMS application to a very broad field of bio- medical research. The unique analytical methods are expected to provide the scientific proof-of-principle framework that will proceed through increasing levels of complexity, to broaden the biomedical applications of AMS to problems in biochemistry, cell biology, develop- mental biology, pharmacology, immunology and others. The knowledge obtained is also expected to be combined with other biological studies to achieve more complete pictures of several important biological processes. Although use of these techniques was not widespread because of the high instrumentation costs of commer- cially available systems and the need for qualified physi- cists to operate the instrument (at the beginning of the new millennium there were, world-wide, approximately 50 labs engaged in AMS research), the situation is now far improving. At present, there are several companies for the commercial exploitation/analysis of AMS and it is also expected that the instrumentation costs will be less than $ 2 M. 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W2020271973.txt	https://www.scielo.br/j/ln/a/mpCY74ZtgjyK6GQyQHryN8F/?lang=pt&format=pdf	pt		Não multiplicar o indivíduo inutilmente	Lua nova	2,009	cc-by	7,067	Não multiplicar o indivíduo inutilmente* Louis Pinto** Introdução Concebido como um exercício de esclarecimento, este texto visa apresentar certas razões para ficar perplexo a propósito da utilização de noções como aquela de indivíduo. Um sociólogo deveria sentir uma desconfiança espontânea diante da obrigação de ter qualquer coisa a dizer e pensar sobre temas cuja origem e pertinência não lhe parecem muito claras, desconfiança que poderia ser ainda reforçada se levarmos em conta um contexto ideológico favorável à apologia polifônica da singularidade e àquilo que a acompanha, a denúncia das “rigidezes”, da “uniformidade”, obstáculos à inovação e à originalidade... Pode-se espantar ao ver a que ponto os intelectuais, presumíveis amigos da troca e da argumentação, se comprazem não nas causas difíceis, mas nas batalhas que, quando não ganhas de antemão, não deixam em todo caso muita escolha. O atual encontro entre pós-modernismo Publicado originalmente em Revue Interrogations, no 2, 1/6/2006. Disponível em: http://www.revue-interrogations.org/article.php?article=39. Acesso em: set. 2009. * ** Tradução de Carolina Pulici e Marcia Consolim. Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 205 17/11/09 4:15:29 PM Não multiplicar o indivíduo inutilmente 206 (a “diferença”) e espiritualismo personalista (o “Eu”), que determina tão largamente o horizonte do pensável, tem tudo de uma irresistível aliança, de tal forma que cada um pode dela tirar proveito, contribuindo ao bem comum marcado pelo gosto aristocrático do inclassificável e pela aversão para com a massa, a classe, o coletivo. Nessa paisagem, os sociólogos se encontram como que transplantados fora do domínio da pesquisa empírica. Donde um leque de discursos: os temerários se passam por filósofos, sem cautela nem objeto, enquanto os prudentes, com um dos pés no chão, se contentam com alusões na boa direção. Analisar o conteúdo das ideias concernentes ao indivíduo não é uma tarefa fácil devido a, pelo menos, duas razões. A primeira é que muitos dos discursos envolvidos são muito hábeis em cultivar a confusão e a aproximação. Poucas pessoas parecem verdadeiramente falar da mesma coisa, mas a acumulação dos discursos acaba por validar a existência de um terreno comum (a grande querela sobre o indivíduo) e, ao mesmo tempo, convidar a uma ultrapassagem radical das visões antigas. A segunda razão é que os benefícios científicos desses debates são bastante incertos. Os pensadores do indivíduo deveriam conseguir mostrar concretamente em que um programa de pesquisa e um estilo de análise dependem de sua contribuição. Eles deveriam, em todo caso, não ignorar as distinções que se podem fazer entre a análise global de um conceito (o que é o indivíduo?), a elucidação de um problema filosófico preciso (o individual é distinto, e em que, do coletivo?), e a exploração de questões sociológicas testáveis (onde encontrar pessoas que se dizem indivíduos ou que não se podem pensar senão como indivíduos?). O indivíduo epistêmico Que tipo de entidade é o indivíduo? Em que ele pode ser conhecido? A evocação de uma questão de lógica servirá de ponto de partida. O indivíduo é primeiramente um termo Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 206 17/11/09 2:15:32 PM Louis Pinto abstrato que designa qualquer objeto (árvore, homem...) graças a um predicado que permite identificá-lo: esse objeto pertence à classe daqueles que detêm o predicado (por exemplo: vizinho do quatrième,1 que se encontra às 13 horas na estação de Lyon, amigo de Paul, nascido sob o signo de libra etc.), já que vários indivíduos podem ter o mesmo predicado ou o mesmo conjunto de predicados. Ou, então, à maneira de Quine, que desconfia da terminologia das classes, pode-se dizer que “ser é ser o valor de uma variável” (e de uma variável “dependente”) (Quine, 1993, p. 51). O indivíduo é, se podemos dizer, imanente a uma linguagem e aos sistemas de classificação próprios a essa linguagem. Um indivíduo pode ter, como se sabe, uma constelação única de predicados e, reciprocamente, a uma constelação única de predicados não contraditórios pode corresponder seja uma multiplicidade de indivíduos, seja um indivíduo, seja nenhum indivíduo. Mas não há indivíduo sem predicado.2 “A ideia de um indivíduo é a ideia de uma ocorrência individual de alguma coisa geral. Não existe particular puro”, escreve Strawson (1977, p. 47). Evidentemente, o fato de ser um indivíduo não prejulga o número, a natureza dos predicados e de seu modo de coesão. Ao fazermos a referência, nos reportamos a um indivíduo na medida em que ele é distinto de um outro do qual ele pode no limite não diferir senão solo numero por sua posição espacial (uma das duas gotas está à esquerda da outra). O mito do indivíduo “puro” consiste em fazer de um par lógico uma antinomia, ao hipostasiar distinções nocionais do tipo singular/universal (concreto/abstrato). O argumento nominalista, fundado na desconfiança dos universais, sustenta que a árvore é mais “real” do que a floresta... O 207 Habitante do quarto arrondissement de Paris. Isso não significa necessariamente que o indivíduo seja simplesmente a soma dos predicados atribuídos por um observador (erudito ou leigo), e pode-se sublinhar, à maneira de Hilary Putnam, que a referência a um objeto não é inteiramente determinada pelo estado, flutuante e limitado, de nossas crenças relativas a esse objeto. 1 2 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 207 17/11/09 2:15:33 PM Não multiplicar o indivíduo inutilmente 208 argumento existencialista considera o indivíduo como um fato “puro” para além do conhecimento conceitual, um x radicalmente ininteligível (existente), um dom, uma dádiva. O argumento antiobjetivista, fundado na inadequação dos predicados em relação a uma determinada ordem da realidade, sublinha a distância entre a informação oferecida pelas propriedades objetivas de um indivíduo e a constatação do que ele é, do que ele faz concretamente, daquilo que ele é para ele mesmo. Deixarei de lado os dois primeiros argumentos para me dedicar especialmente ao terceiro. Não há que se maravilhar ante a descoberta de que a classe é menos rica do que os indivíduos. Se a intenção é se engajar na via do conhecimento objetivo, trata-se não de opor predicados e indivíduos, o que é um impasse, mas de partir em busca dos predicados os mais ricos possíveis, dotados de um forte valor descritivo, explicativo e, eventualmente, preditivo. Para retomar a terminologia de Pierre Bourdieu em Homo Academicus, pode-se dizer que o indivíduo epistêmico construído pela ciência através das operações de seleção e de construção não é a reprodução do indivíduo empírico percebido na experiência ordinária. Por que a variável profissão adquiriu um valor privilegiado na maioria das análises sociológicas? De maneira alguma porque ela conteria, tal como uma essência, a totalidade das propriedades que poderíamos manifestar, mas somente porque ela é, entre todas as variáveis objetivadas pelas instituições, aquela que, malgrado suas imperfeições, pode aparecer como a mais densa: de um lado, ela cristaliza relações sistemáticas com outras variáveis (renda, nível escolar...), e de outro, ela delimita de forma bastante ampla um espaço de possíveis sociais parcialmente redundantes (estratégias matrimoniais, escolares, práticas culturais...). Nada impede, aliás, de submeter as nomenclaturas de profissão a uma análise crítica, como fizeram vários autores. Seria preciso sublinhar, enfim, que o valor analítico da variável não decorre de considerações sobre a relação Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 208 17/11/09 2:15:33 PM Louis Pinto com o trabalho ou a identidade no trabalho, às vezes utilizadas para sublinhar o declínio da variável profissão como princípio explicativo e/ou princípio de mobilização? O ponto precedente nos conduz à questão propriamente epistêmica. Os indivíduos dos quais dizem se ocupar certos sociólogos respeitosos da complexidade do real seriam, em primeiro lugar, simplesmente aqueles que, por oposição aos indivíduos “genéricos” tratados pela sociologia dos grupos sociais, seriam difíceis de classificar, senão talvez até inclassificáveis: eles são médicos, comerciantes..., mas não apenas, tendo traços aparentemente contraditórios. Bom desafio à análise: esse médico, comerciante... neutraliza, contradiz os princípios de inteligibilidade da classe, de toda classe, já que realiza uma combinação única de predicados que não permite qualquer descrição definida. “Inclassificável” designa uma complicação das relações entre predicados resultante do encontro entre várias classes comumente separadas. Esse ponto já havia sido sublinhado por Gerhardt Lenski quando evocava a cristalização/descristalização dos atributos estatuários: se o dominante modal nos Estados Unidos é um wasp (branco, anglo-saxão, protestante), como pensar indivíduos que não detêm o conjunto de atributos (por exemplo, na burguesia judia ou negra)? O sociólogo se vê aqui convidado a renunciar a certos estereótipos, não para se abandonar ao êxtase da complexidade, mas para compreender as modalidades diversas de posse de um atributo. Essa configuração de atributos parcialmente contraditórios tem, certamente, efeitos sobre as representações (a começar pela relação consigo mesmo) e sobre as práticas: basta pensar nos detentores ilegítimos de posições, nos miraculados de todo tipo, nos dominantes em parte dominados, nos khâgneux à vie 3 (como diz Jean-Pierre Faguer) que nunca 209 Khâgneux são os alunos dos cursos preparatórios para as Escolas Normais (Khâgnes). “Khâgneux à vie”, no contexto em questão, se refere aos “eternos aprendizes”. 3 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 209 17/11/09 2:15:33 PM Não multiplicar o indivíduo inutilmente 210 se redimiram. Pierre Bourdieu insistia na pluralidade das vias de acesso a uma posição, isto é, nos efeitos da trajetória, recusando, assim, precisamente, todo nominalismo (de profissão, de família, de nacionalidade...). Ele propunha analisar a posição num campo, espaço social estruturado segundo polos, regiões, zonas fronteiriças, mas também o conjunto das posições simultâneas ou sucessivas ocupadas em diferentes campos a propósito dos quais se pode perguntar pelos efeitos de compatibilidade, de acumulação, de discordância etc. Os sociólogos que invocam a inesgotável diversidade das variáveis (profissão, diploma, religião...) para exprimir a dificuldade de dar conta das práticas e de sua evolução no tempo parecem ter renunciado à busca de inteligibilidade que implica o esforço por definir, de outra forma que não pela justaposição, as relações entre essas variáveis. Restaria analisar as lentes do sociólogo para se perguntar se elas não seriam geradoras dos paradoxos que seu portador pretende constatar. O que dizer dessa “dissonância” destinada a dar conta do que é apresentado como uma anomalia de paradigma (Lahire, 2004)?4 Dois traços que não combinam segundo o observador podem ser julgados como perfeitamente compatíveis pelo observado. O mesmo ocorre com a dimensão da legitimidade. Sociólogos acreditaram poder sensatamente afirmar que a probabilidade de encontrar práticas legítimas cresce com o nível escolar e a posição no espaço social. Mas se olharmos para o conjunto de práticas legítimas, a probabilidade de se conformar em todos os domínios ao mesmo tempo, e a cada hora do dia, aos modelos mais exigentes ou aos mais nobres não pode senão decrescer para o conjunto da população, aí inclusas as frações consideradas cultivadas. Tomemos o exemplo de 4 Eu apresentei uma análise crítica desse livro em “Comment négocier un tournant?”. Espaces Temps.net, 21/11/2004. Disponível em: http://espacetemps.net/ document778.html. Acesso em: set. 2009. Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 210 17/11/09 2:15:34 PM Louis Pinto uma mulher PDG5 que frequenta um karaokê. Três soluções se apresentam a nós. A primeira consiste em sublinhar o simplismo dos teóricos (e maus observadores) da “distinção” e a parar por aí esperando tempos melhores. A segunda consiste em propor uma melhor teoria das práticas culturais, uma teoria que, sem recorrer às propriedades de trajetória, estabelece um pequeno número de princípios a fim de dar conta da coerência dissimulada por uma aparente desordem. Mas, se a empreitada fosse viável, o risco seria então de novo o de fazer desaparecer a singularidade tão sublinhada de nosso indivíduo. Enfim, a terceira solução, a de Bourdieu, consiste em reconstituir a lógica prática dos agentes. Essa repousa sobre alguns princípios. Em primeiro lugar, a identidade social está engajada muito diferentemente segundo os domínios da prática (amador refinado em música, mas indiferente em pintura...): só o pequeno-burguês ansioso imagina, no modelo Bouvard-et-Pécuchet,6 que a norma cultural impõe a excelência universal e um enciclopedismo pesado. A única máxima dos agentes aqui é aquela do bom senso que os preserva de se envolver em domínios pouco familiares, nos quais não se tem certeza de resistir a juízes sem indulgência. Em segundo lugar, a segurança atestada e sustentada através de um conjunto de signos de autoridade é o que permite tomar distância com relação às fronteiras da legitimidade cultural: é suficiente pensar nessas falsas confidências nas quais os dominantes deixam que se saiba que eles “adoram” essa ou aquela atividade que eles sabem ser bem “fácil”, senão “vulgar”. Não é isso que deveria suscitar o espanto do sociólogo, mas sim o gosto (ou o tato) que evita, ao menos nas situações públicas, de se perder nas transgres- 211 5 A sigla PDG designa, em francês, o cargo de “presidente-diretor geral” de uma empresa. 6 Romance inacabado de Flaubert, publicado postumamente, em que os protagonistas anseiam a tudo conhecer (medicina, geologia, química, política etc.) e com resultados desastrosos. Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 211 17/11/09 2:15:34 PM Não multiplicar o indivíduo inutilmente 212 sões as mais infames ou ridículas (a Foire du Trône7 é talvez “divertida” para rir uma vez, mas bom...). Um agente determinado não é nem monolítico nem plural, nem transparente nem inclassificável, ele não é nem um bloco uniforme que age de forma monótona nem uma pura rapsódia de fenômenos justapostos. Tendo interiorizado esquemas de ação diversificados que funcionam em espaços diversos, ele está fadado, tanto objetiva quanto subjetivamente, a uma relativa ambiguidade. O que não significa que o “determinismo” seria desmentido, já que a conduta seguida, mesmo se não fosse inelutável, se inscreve de fato num espaço de possíveis ligado a um indivíduo através do conjunto de predicados do qual ele é portador. O fato de não haver um cenário único não implica que aquele que foi adotado o foi em virtude de uma decisão irracional, imotivada etc. Chega-se ao curioso argumento funcional que consiste em dizer: a realidade tornou-se de tal maneira “complexa”, “plural”, que não há nada senão o indivíduo para ocupar o lugar que outrora cabia às determinações objetivas (classe...), que tornavam possível a coerência das ações de um agente, de preferência sob a forma do automatismo. Ora, mesmo admitindo que se possa descrever um caso singular, como a forma em que um indivíduo combina “identidades” múltiplas, a dificuldade seria apenas adiada: restaria tomar por objeto, a não ser que se o considere não analisável, a lógica da instância de coordenação, espécie de superego que seleciona e combina a multiplicidade dos pertencimentos. Mas para que então todas essas considerações metateóricas sobre o indivíduo? Eis que finalmente retornamos a essa sociologia laboriosa que, recusando as seduções dos paradoxos e das dissonâncias, não teria nada mais a nos propor que A Foire du Trône é uma tradicional festa popular da França, que ocorre de março a maio no Bois de Vincennes, nos arredores de Paris, onde são instalados um parque de diversões e barracas de jogos. 7 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 212 17/11/09 2:15:34 PM Louis Pinto a necessidade de colocar em evidência as causas e as razões das práticas, analisando as distinções entre classes, frações de classes, universo da prática, conjunturas. Buscando discernir a “fórmula geradora” de um indivíduo determinado, somos impulsionados por uma preocupação científica de simplicidade que consiste em identificar o pequeno número de atributos de rendimento cognitivo elevado (em vista de pesquisas passadas) e dotado de relações regradas. Tranquilizemos aqueles que teriam medo de se entediar: se há regras de análise, as possibilidades combinatórias são muito vastas, as surpresas numerosas e, contanto que se aceite o preço de esforços e tateamentos, a engenhosidade pode perfeitamente se manifestar para dar conta desse médico marginal e desse comerciante fora do comum. A originalidade é, em todo caso, perfeitamente analisável: a sociologia não está fadada a estudar a média e os indivíduos “medíocres”. Um social opcional Para que um agente determinado seja acessível à ordem do conhecimento objetivo, exige-se ainda que ele lhe seja homogêneo. Pelo menos era esse o pressuposto da discussão precedente: os paradoxos exigiam implicitamente a invenção de novos instrumentos. Ora, o sociólogo não deveria também mudar seus instrumentos se ele considera que o mundo mudou de modo radical? A partir do momento em que a modernidade (ou pós-modernidade) dá a ver de forma incontestável a instabilidade criadora que separa as identidades fixas até sua dispersão, a intenção de conhecer o indivíduo parece confrontada à obrigação de renunciar aos pressupostos os mais arraigados. Afirma-se que uma nova inteligibilidade se anuncia. A primeira restrição feita pelo sociólogo à antiga seria sublinhar que esse tipo de raciocínio é incapaz de reconhecer a distinção, contudo elementar, entre o indivíduo e o individualismo: o primeiro é supostamente uma realidade 213 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 213 17/11/09 2:15:35 PM Não multiplicar o indivíduo inutilmente ao passo que o segundo não é senão um sistema de representações. A função de fundamento é atribuída tanto a um quanto ao outro termo, mas, num enunciado desse gênero, tem-se dificuldade de decidir: “É então, escreve François Dubet, quando a sociedade não pode mais ser descrita totalmente como um sistema organizado e coerente que o indivíduo emerge porque ele deve, pessoalmente, produzir uma coerência e uma série de ajustamentos que não pode mais garantir o sistema. O indivíduo existe porque ele regula problemas de identidade e de coerência, porque ele se constrói no arranjo de papéis, de habitus, de aspirações que se solidificam na sua personalidade” (Dubet, 2005, p. 6). 214 O individualismo, que se pode, aliás, explicar de mil maneiras plausíveis, não deve ser considerado como uma prova incontestável a favor do indivíduo. Dito de outra forma, não basta reivindicar o estatuto de indivíduo para ser um. Em primeiro lugar, essa reivindicação é tudo menos original na medida em que faz parte, em nossas sociedades, dos valores largamente experimentados sob formas diversas (eu ainda não encontrei o verdadeiro holista em matéria de ética). Durkheim, em seu texto famoso sobre essa questão, não dizia nada além disso: o individualismo, celebração do indivíduo, é um produto não do indivíduo, mas da sociedade. Bourdieu atribuía a reivindicação da “opinião pessoal” à escolarização que inculca em cada agente a ideia de que ele deve ter opiniões que lhe sejam próprias. Quanto ao conteúdo desse individualismo, poder-se-ia mostrar também que ele reproduz representações sociais (para não falar estereótipos) retiradas não das profundezas da pessoa, mas do anonimato de um conjunto de sistemas simbólicos. O verdadeiro indivíduo, sugere Vincent Descombes, bem poderia ser aquele solitário, esse “virtuose” (asceta, eremita...), que teria chegado a extirLua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 214 17/11/09 2:15:35 PM Louis Pinto par o “mundo” de si mesmo (Descombes, 2004, p. 269). Lá, onde alguns veem o reino dos indivíduos, outros puderam, ao contrário, discernir um nivelamento das diferenças e, então, remeter ao passado a originalidade e a grandeza. Não falemos da dificuldade de fixar a data de nascimento do conceito de indivíduo, que pode oscilar entre referências separadas por milênios (a Grécia, a Renascença, a queda do Muro...). Mas esse gênero de investigação é, afinal de contas, solucionável? Segunda reserva: seria preciso determinar em que medida os dados empíricos alegados evidenciam um princípio único contido na noção de individualismo. Pode-se considerar que se está lidando com uma mesma lógica, por exemplo, quando nos encontramos diante de um aluno procurando escapar aos efeitos das classificações escolares, de um estudante de longa data que inventa para si uma arte de viver, do assalariado desamparado na sua atividade profissional e fechado em si mesmo, de um casal que negocia espaços privados, dos jovens profissionais (cadres) da moda que buscam lazeres novos fora das vias comuns etc.? O que é proposto como prova do aumento do individualismo parece uma maneira de utilizar todos os meios disponíveis, amalgamando lógicas sociais no mínimo heterogêneas (oportunismo, hedonismo, apatia, desprendimento...), como não deveriam fazer os sociólogos animados pelo senso do campo, da complexidade... e da complexidade do campo. A mesma pergunta poderia ser retomada, da outra ponta da cadeia, a fim de determinar se os coletivos evocados num modo uniformizante (família, escola, partidos políticos...) não deveriam ser considerados em função da lógica específica que é a deles (a menos que, evidentemente, a tarefa exclusiva da sociologia consistisse em tomar por objeto as dimensões mais formais da relação subjetiva com os grupos de pertencimento). Que a relação consigo (com o outro, a cultura, as instituições...) possa tomar formas extremamente diversas, de adesão ingênua ou distância crítica, não é algo específico de uma época particular. 215 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 215 17/11/09 2:15:35 PM Não multiplicar o indivíduo inutilmente 216 Terceira reserva: o recurso à noção de subjetividade. O indivíduo, diz-se, não somente é inclassificável (objetivamente) ou avesso (subjetivamente) à ordem e aos constrangimentos, mas ele mantém uma relação privilegiada consigo ou, mais ainda, ele se define por essa relação: o que ele é não vem de forças “exteriores”, mas resulta de um processo engendrado na intimidade de uma consciência de si. Descombes, estudando o conceito de subjetividade dos filósofos modernos (na linha de Descartes), sublinhava o deslizamento imperceptível que faz passar do cuidado de si (souci de soi), noção ética comum elementar (o que vou fazer?) ao cuidado do eu (souci du soi), do eu que seria ao mesmo tempo o sujeito e o objeto do cuidado. O primeiro termo, tirado da linguagem ordinária, não contém nenhuma tese particular: ele designa a dimensão ética da escolha, ao sugerir a parte de esforço a ser feita (se você não fizer ninguém fará em seu lugar). O segundo pertence a uma terminologia erudita (metafísica). Passemos às aberrações “gramaticais” engendradas por esse uso inabitual do termo (eu tenho ou eu sou um eu). O problema que nos concerne aqui é a indeterminação da injunção de ser um eu (Descombes, 2004, p. 236). O sociólogo pode se permitir divergir sobre esse ponto? Seria preciso que ele pudesse nos dizer a partir de que se pode distinguir uma prática que depende da observância de convenções impessoais e uma prática surgida da autenticidade do eu. E aí as coisas ainda correm o risco de se complicar um pouco mais. Deve-se recorrer à autoridade do metafísico, à do sociólogo ou à autoridade do indivíduo que seria finalmente o único juiz a determinar aquilo que vem dele e aquilo que vem do exterior? Ou, então, deve-se remeter isso, simplesmente, a um critério negativo, à ausência de constrangimento visível? Chego a uma quarta reserva: a referência a essa noção de subjetividade implica uma mitologia do social. Com efeito, o argumento da autonomia (o “eu”) supõe uma dualidade dos princípios de ação: o eu desse médico, desse comerLua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 216 17/11/09 2:15:36 PM Louis Pinto ciante... se determina em função de uma singularidade profunda que escapa a toda determinação “externa”. O individualista consente que uma parte de nossos comportamentos provenha do exterior, mas ele reivindica que não seja assim para uma outra parte. O social aparece assim de maneira estranha, seja (versão fraca) como uma questão de grau, seja (versão forte) como uma opção revogável: ou bem eu me libero mais ou menos da sociedade, em função das circunstâncias, ou bem chega um momento em que, tendo cessado de pertencer à ordem das aparências sociais, eu me encontro assimilado, à maneira do sábio schopenhaueriano, a uma força eterna (um eu?) que se engendra a si mesmo. Quinta reserva: o que pode fomentar essa ilusão de revogabilidade do social é a identificação do social ao constrangimento. A palavra “constrangimento” é, por excelência, uma dessas que geram mal-entendidos. Pode-se primeiro pensar no poder de certas regras que se impõem aos agentes através das injunções, dos códigos, das chamadas à ordem e, finalmente, das sanções executadas pelos detentores de uma forma específica de autoridade. “O controle social”, escrevem François Dubet e Danilo Martuccelli, “é cada vez mais subjetivo, cada um se sentindo mestre de suas escolhas e de sua vida. Os códigos sociais são substituídos por regras morais interiorizadas, por obrigações subjetivas...” (Dubet e Martuccelli, 1998, p. 44). Reconhece-se aqui um dualismo de tipo ascription/achievment que reflete a dualidade das sociedades. As sociedades tradicionais (denominadas holísticas, segundo Louis Dumont), zelosamente conformistas, se veem eliminadas pelas sociedades modernas ou pós-modernas abertas à criação e à fluidez. A propósito das primeiras, Dubet nota com razão que 217 “o indivíduo nela está talvez menos ausente do que supõem os relatos correntes da modernidade e que o holismo é mais uma alteridade teórica cômoda do que uma realidade antropológica” (Dubet, 2005, p. 12). Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 217 17/11/09 2:15:36 PM Não multiplicar o indivíduo inutilmente 218 A respeito das segundas, o mesmo autor, evocando a escola, a família e a religião atribui a capacidade de se subtrair à autoridade das normas, papéis e instituições. Reencontra-se assim o argumento funcional já mencionado. A atenuação das formas mais coercitivas de autoridade não deve levar a afirmar que os indivíduos são, de agora em diante, livres para as escolhas das quais eles são as fontes exclusivas. Durkheim, teórico da integração, abordou efetivamente a questão dos modos diferenciais de constrangimento exercidos pelo grupo sobre seus membros, problema sociológico de fato não fora de moda. Mas, como teórico do social, ele também fez do constrangimento o critério de reconhecimento do social. Ocorre que a palavra assume nesse caso um sentido diferente, puramente epistemológico: o social não é uma criação dos indivíduos, ele é aquilo que se impõe a eles como alguma coisa de exterior. Essa exterioridade comporta algumas dificuldades, mas o que é incontestável é que, para trabalhar, o sociólogo deve postular a inteligibilidade do real, a qual implica, assim como para as “coisas”, a possibilidade de classificar, comparar, ordenar, hierarquizar, extrair relações de invariância. A ciência não tem de escolher os grupos contra os indivíduos, ou vice-versa. Basta-lhe descrever e explicar propondo os melhores princípios de generalização. Esses princípios podem ser mais ou menos satisfatórios, mas é preciso acabar com a ideia romântica segundo a qual o indivíduo seria um desafio à totalidade, seja a da sociedade, seja a da ciência. Sexta reserva. Pode-se perguntar se a concepção opcional do social não é inspirada, sobretudo, pela intenção de dar ao conceito de liberdade uma revanche sobre o que o social comporta de determinismo. Mas isso é mesmo necessário? O sociólogo trabalha com o objetivo prioritário não de colaborar com uma teoria da liberdade graças aos seus meios próprios, mas de dar conta das regularidades observáveis que pôde colocar em evidência por operações de construção de Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 218 17/11/09 2:15:36 PM Louis Pinto objeto. E mesmo se a liberdade se encontrasse justificada por uma infinidade de argumentos convincentes, ele não poderia usá-los, e, em primeiro lugar, porque uma construção especulativa não pode ser mobilizada na pesquisa empírica da mesma maneira que uma observação ou uma hipótese. A liberdade não é da ordem das coisas que se poderia constatar ou desmentir.8 Assim, os sociólogos não têm de estar divididos entre aqueles que são a favor e aqueles que são contra as faculdades criadoras do indivíduo, mas sim entre aqueles que, dizia Durkheim, assumem os “dois sentimentos contraditórios que podem ser vistos como os motores por excelência do desenvolvimento intelectual: o sentimento do obscuro e a fé na eficácia do espírito humano” (Durkheim, 1975, p. 173), e aqueles que oscilam entre um lado e outro. Paradoxalmente, os sociólogos que, à maneira de Bourdieu, tentam ir o mais longe possível no empreendimento de objetivação se veem suspeitos de se apegar de alguma forma demais ao jogo. Ora, acreditar nos poderes de compreensão e de explicação próprios ao conhecimento sociológico, pressuposto de preferência recomendável do ofício do sociólogo, não é reduzir os indivíduos ao estatuto de autômatos que não fariam senão seguir um programa fixado de antemão (o conceito de habitus evita o perigo). Como sublinha Bouveresse a propósito de Wittgenstein, nem a regra age “à maneira de uma força motriz que constrange o utilizador a ir numa direção determinada”, nem as “leis”, invocadas na ciência da natureza, como nas ciências do homem, podem ser encaradas “como regras às quais os fenômenos naturais são constrangidos a se conformar” (Bouveresse, 2004, p. 143). O modo científico de representação que tende, a partir de agora, a fazer parte de nossa imagem do mundo não 219 8 Durkheim sublinha que a sociologia “não deve tomar partido entre as grandes hipóteses que dividem os metafísicos. Ela não tem que afirmar mais a liberdade do que o determinismo” (Durkheim, 1968, p. 139). Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 219 17/11/09 2:15:37 PM Não multiplicar o indivíduo inutilmente deve ser fetichizado sob a forma de um sistema de constrangimentos imanente às coisas. Se, segundo Wittgenstein, “não há nada na regularidade que torne o que quer que seja livre ou não livre” (Bouveresse, 2004, p. 144) é porque o fato de conceber trilhas escondidas como modelo de conduta regrada (ele segue tal itinerário) não implica recorrer a um mecanismo constrangedor, “a procurar uma espécie de mecânica do não mecânico em si mesmo” (Bouveresse, 2004, p. 162). Desvelar as regularidades e explicá-las não consiste em opor um “mecanismo” à espontaneidade aparente. É recusar simplesmente escolher, renunciando a subordinar o conhecimento objetivo à ideia de que se estaria livre de seu trabalho uma vez identificado o mecanismo escondido: 220 “Uma boa parte da resistência que se opõe às ideias de Bourdieu provém não, como se poderia crer, da hostilidade ao mecanismo, mas da tendência a crer que nós compreenderíamos a sociedade se conseguíssemos de alguma forma ver a maquinaria social em ação” (Bouveresse, 2004, p. 162). A apologia da liberdade criadora dissimularia assim um ideal mecanicista que não é aquele de Bourdieu, mas precisamente o de uma boa parte de seus adversários. A escatologia pós-moderna No momento em que os pares de oposição filosóficos são projetados no tempo, os termos negativos remetidos ao passado e os positivos em direção ao futuro, tem-se aparentemente a maior parte dos ingredientes daquilo que os pensadores pós-modernos, sublinhando seu caráter mitológico, senão religioso, chamaram de “um grande relato”. Há então um grande relato dos pós-modernos que comporta alguns traços notáveis. Em primeiro lugar, uma orientação do tempo histórico que, se implica o abandono das antigas certeLua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 220 17/11/09 2:15:37 PM Louis Pinto zas dogmáticas e de uma busca ingênua da verdade (sob os auspícios inesgotáveis do “desencantamento do mundo”), garante em troca todos os prazeres, ao menos intelectuais, da liberação. Por outro lado, a chegada dos novos tempos, deixando de dever ser remetida às forças sociais passíveis de uma análise racional, pode ser considerada como o resultado de uma multiplicidade inesgotável (como era de se prever) de causas econômicas, tecnológicas, culturais, trabalhando providencialmente numa mesma direção: a época arcaica das identidades fechadas e dos coletivos devoradores deve dar lugar a uma época de diferenças, de singularidades e de crenças maleáveis. Último aspecto: o relato é estruturado segundo a oposição secular comunidade-sociedade. As ilustrações propostas fariam rir os historiadores de profissão se eles delas tomassem conhecimento. Durante milênios, a humanidade viveu sob o jugo daquilo que um filósofo não hesita em chamar... as “identidades naturais” (Hardt, 2004): contida por uma família repressora, uma nação exaltada, uma usina fordista disciplinar e aparelhos político-sindicais monolíticos, o indivíduo (aliás, ele existia?) não tinha outro horizonte senão a morna conformidade imposta pelos coletivos de todo tipo. Ao contrário, numa “sociedade pósmoderna”, caracterizada pela “dissolução dos corpos sociais tradicionais”,9 o indivíduo novo não recebe mais sua identidade do alto, ele é produtor de sua “diferença” (ele não é igual a nenhum outro, mas sem arrogância e gentilmente), imaginativo e feliz por ser acolhido no seio da “multidão” na qual há lugar para todos. Nessa forma de escatologia que nos propõem os autores do livro Multitude, o que deve acontecer não fará senão realizar a essência eterna da humanidade (seu desejo de liberdade), o advento da multidão “ontológica” tornado possível 221 Em seu simplismo, a palavra “tradicional” permite escamotear a análise precisa ao evocar desordenadamente a rotina, o passado, o indiscutido etc. 9 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 221 17/11/09 2:15:37 PM Não multiplicar o indivíduo inutilmente 222 pela multidão “histórica” ou “política” (2004, p. 259). “A multidão obedece, em consequência, a uma estranha temporalidade desdobrada: sempre aí e nunca ainda” (2004, p. 260). Para explicar a sobrevivência surpreendente de uma postura de devoção sob ares tão liberados, só se pode fazer referência à dupla propriedade de um discurso que deve se esforçar simultaneamente para propor as perspectivas de uma estratégia política e substituir o marxismo, grande escatologia de juventude com a qual ele não cessa de (se) confrontar, por uma linha aberta, criadora, em suma, “inteligente”. Todas as aquisições dos filósofos da “singularidade” (ou da “diferença”, mas no sentido deleuziano e não hegeliano) podem ser recuperadas no terreno da política: “Em termos conceituais, a multidão substitui o binômio comum/ singularidade pelo par identidade/diferença” (2004, p. 256). Passa-se do terreno da natureza àquele da graça. A viabilidade política dessa escatologia não será examinada aqui. O que, ao contrário, pode perfeitamente sê-lo é o presumível aporte conceitual do par indivíduo-multidão. Dizem-nos que as novas lutas não reproduziriam as imperfeições das lutas “tradicionais”, uma vez que o indivíduo conseguiria preservar sua singularidade. O obstáculo da classe trabalhadora poderia ser eliminado, já que a multidão seria de natureza “inclusiva” (e não “exclusiva”), como testemunham, ao que parece, movimentos como Act Up, Queer Nation e o antiglobalização. Com forças sociais mais diversificadas que o solitário proletariado, encontramo-nos finalmente diante de um problema relativamente clássico de sociologia política, aquele das condições e das modalidades de mobilização. A esse problema clássico, os autores de Multitude não têm uma resposta particularmente original. Quais as dimensões da mobilização? A primeira seria de ordem “intensiva”. Seguindo o adágio de que é forjando que se torna ferreiro, eles nos ensinam que a experiência do conflito reforça a determinação a lutar: “o odor acre dos Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 222 17/11/09 2:15:38 PM Louis Pinto gases lacrimogêneos aguça o sentido e os choques com a polícia fazem ferver o sangue de raiva e essa intensidade culmina na explosão” (2004, p. 251). É simples! A segunda, de ordem “extensiva”, consiste numa “comunicação de lutas locais” que se realiza segundo uma lógica de “rede”: “cada luta permanece, portanto, singular e ligada a condições locais, estando imersa numa rede comum” (2004, p. 255). É harmonioso! A rede supõe a diversidade das forças contestatórias coordenadas e um pluralismo proclamado, elementos que, desde sempre, estiveram no coração de noções como aquelas de front, de união, de agrupamento, de aliança. Equilíbrio instável no qual se vê mal o que, no futuro, poderia preservá-lo dos jogos de relações de força, a menos que se adotem as ideologias descentralizadoras e libertárias ao pé da letra. Fazer da “multidão” um instrumento de análise é algo supérfluo porque a maior parte dos movimentos sociais de alguma amplitude foi inicialmente heterogênea (E. P. Thompson dizia algo diferente disso?), tendo engendrado ou não uma linguagem comum. De fato, o uso principal do termo parece, sobretudo, performativo: ele impõe que se rejeite o centralismo democrático. Inventar uma terminologia bizarra (o “comum”) é uma coisa. Mostrar concretamente em que as lutas descentralizadas ou em rede vão “mudar o mundo” é outra, pois seria preciso elucidar o que está em questão aqui, a saber, o que essas lutas têm em comum afora o fato de que elas não são impulsionadas por militantes operários à antiga. Ocupar-se desse problema obrigaria os pensadores da multidão a confrontar suas ideias de “singularidade” e de “local” à hipótese inadmissível que possa existir uma hierarquia (ao menos estratégica) das causas e das urgências. E é ainda outra questão mostrar como os indivíduos em luta (que se hesita até agora em chamar de militantes) conseguiriam preservar sua indomável “singularidade”. Seria desejável que pensadores tão sequiosos de tirar ensinamentos da modernidade não cometessem 223 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 223 17/11/09 2:15:38 PM Não multiplicar o indivíduo inutilmente 224 o erro elementar que consiste em confundir as declarações dos boletins militantes e as práticas efetivas. Um grupo não é uma caixa-preta: pode-se perfeitamente analisá-lo (há dezenas de estudos de caso excelentes sobre isso).10 Mesmo nos círculos de estudos spinozianos ou nietzschianos, lugares de trocas entre singularidades pensantes, existem oposições mais ou menos patentes entre eruditos e amadores, comentadores e pensadores, oradores legítimos e aprendizes balbuciantes. A luta coletiva, aí compreendida a das causas célebres, não põe em jogo puras “subjetividades”, mas agentes portadores de uma história determinada, de interesses e de projetos que os incitam a se confrontar pela definição legítima da causa. O resultado dessas lutas não depende do prazer de fazer rede junto, mas de fatores objetivos que não são nem modernos nem pós-modernos, como os efeitos externos de conjuntura, a composição da base militante, os procedimentos internos de decisão etc. Sentimos certo incômodo em ser estraga-prazeres, mas, enfim, não vamos continuar a alimentar belos contos infantis sob o pretexto de terminar com grandes relatos. Conclusão Depois de ter sublinhado quão diferentes eram os problemas associados à palavra indivíduo, seria estéril querer reunir as teorias da individualidade numa mesma classe. Pode-se, pelo menos, observar que a maioria delas se aproxima em razão do mesmo adversário, cientista ou objetivista, que tem em mente. Um outro traço comum é aquele que poderíamos chamar um humor catastrofista simpático à ideia de que uma profunda mutação intelectual é requerida pela crise dos instrumentos tradicionais do conhecimento. Ora, se há uma coisa bem pouco nova é o fato de que a sociologia há muito tem de lidar, em seu próprio meio, com a tentação de ultrapassar as exigênComo desculpa a seu turismo filosófico, os autores de Multitude poderiam alegar que eles não puderam tomar conhecimento de trabalhos precisos sobre a antiglobalização. Aconselhamo-lhes Sommier e Agrikolianski (2005). 10 Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 224 17/11/09 2:15:38 PM Louis Pinto cias de cientificidade julgadas rígidas demais (a Durkheim se opõem pensadores mais abertos, como Tarde e Simmel). O adágio nominalista convidava a não multiplicar as entidades quando não fosse necessário.11 Poder-se-ia perguntar se, por uma inversão de papéis, o indivíduo não estaria hoje em meio a essas noções supérfluas. Louis Pinto é diretor de pesquisa no CNRS Referências bibliográficas BOUVERESSE, J. 2004. Pierre Bourdieu, savant et politique. Marselha: Agone. DESCOMBES, V. 1991. “Le pouvoir d’être soi”. Critique, no 529-553. __________. 2004. Le complément de sujet. Enquête sur le fait d’agir de soimême. Paris: Gallimard. DUBET, F. 2005. “Pour une conception dialogique de l’individu”. Espaces Temps. net, 21/6/2005. Disponível em: http://espacestemps.net/document1515.html,p. 6. Acesso em: set. 2009. __________; MARTUCCELLI, D. 1998. Dans quelle société vivons-nous? Paris: Seuil. DURKHEIM, É. 1968. Règles de la méthode sociologique. Paris: PUF. __________.1975. “L’empirisme rationaliste de Taine”. Textes. Paris: Minuit. tomo 1. HARDT, M.; NEGRI, A. 2004. Multitude: guerre et démocratie à l’âge de l’empire. Paris: La Découverte. LAHIRE, B. 2004. La culture des individus. Dissonances culturelles et distinction de soi. Paris: La Découverte. PINTO, L. 2004. “Comment négocier un tournant?”, EspacesTemps.net, 11/11/2004. Disponível em: http://espacestemps.net/document778. html. Acesso em: set. 2009. SOMMIER, I.; AGRIKOLIANSKI, E. (eds.). 2005. Radiographie du mouvement altermondialiste: Second Fórum Social Européen. Paris: La Dispute. STRAWSON, P. F. 1977. Études de logique et de linguistique. Trad. J. Milner. Paris: Seuil. QUINE, W. Van O. 1993. La poursuite de la vérité. Trad. M. Clevencin. Paris: Seuil. 225 11 Ou, mais precisamente, não postular, inadvertidamente, a existência de ficções verbais. Lua Nova, São Paulo, 77: 205-225, 2009 09066-LN77_meio_AF3c.indd 225 17/11/09 2:15:39 PM Resumos / Abstracts as a central dimension and a prerequisite of the philosopher’s thinking about political community. In that sense, it covers three aspects of that problematic. The firs aspect, the most visible one, which discovery is merit of Arendtian hermeneutics, attends specifically to the problem of bad chrematistic as a deconstruction of the politics’s object, in so far as communitarian life is related to the good life. The second aspect allows the philosopher to relate, for the first time in Western history, the political regimes to the social structure of the polis. The third aspect implies a vision of political regimes in economic terms and is the core of Aristotle’s profound criticism of oligarchy. In that way, it makes explicit another assumptiom of this paper: that Aristotle’s reflections are concentrated on the notion of public sphere and, for that reason, they privilege theoretically aristocracy, politeia, and even democracy. Keywords: Economics; Politics; Chrematistic; Political regimes. 235 Não multiplicar o indivíduo inutilmente Louis Pinto O artigo tenta distinguir os problemas que dizem respeito à noção de indivíduo. Partindo da ideia de que não se podem dissociar, num plano lógico, indivíduo e predicado, o texto sublinha que a sociologia tem a ver com uma construção, o “indivíduo epistêmico” (P. Bourdieu), e recusa o argumento antiobjetivista que consiste em erigir indivíduos inclassificáveis que seriam portadores de paradoxos. Além disso, opor o indivíduo moderno aos determinismos de outrora é um impasse, no mínimo porque ele repousa sobre a ideia indefensável de que o social seria uma opção revogável da qual poderíamos nos liberar. Enfim, a filosofia dos teóricos pós-modernos do individualismo e das redes é uma forma de escatologia, contestável ao mesmo tempo por seus pressupostos nocionais e pelas suas implicações sociológicas. Na medida em que os benefícios teóricos e empíricos da noção Lua Nova, São Paulo, 77: 229-236, 2009 09066-LN77_fim_AF3a.indd 235 17/11/09 4:17:26 PM Resumos / Abstracts de indivíduo são pequenos, ela merece ser colocada de lado até nova ordem. Palavras-chave: Indivíduo; Predicado; Classe; Liberdade; Social. Do not multiplicate the individuals if not necessary 236 The article is an effort to distinguish the problems linked to the notion of individual. Arguing that we cannot sever, on the logical level, individual from predicate, the author underlines that sociology has to do with a construct, the “epistemic individual” (P. Bourdieu) and rejects the anti-objectivist point asserting that individuals are necessary to clarify some paradoxes. Moreover, to oppose modern individuals to formerly determinisms is a deadlock, at least because it implies the idea that social would be a reversible option from which we could get free. Finally, the philosophy of postmodern theoricians about individualism and networks is a kind of eschatologism whose conceptual presuppositions as well as sociological implications are questionable. Since the benefits of the notion of individual, theoretically or empirically, are weak, that notion itself deserves to be put aside, until further notice. Keywords: Individual; Predicate; Class; Freedom; Social. Lua Nova, São Paulo, 77: 229-236, 2009 09066-LN77_fim_AF3a.indd 236 17/11/09 4:17:26 PM
https://openalex.org/W750377203	http://gospodarkanarodowa.sgh.waw.pl/pdf-113737-43522?filename=Integration with the.pdf	Polish	null	Integration with the European Union and the Labour Market in Poland	Gospodarka Narodowa	2,004	cc-by	6,455	81 81 Eugeniusz Kwiatkowski, niegacja + Unią Europejskąa rynek pracy w Polsce Autor jest pracownikiem Katedry Makroekonomii Uniwersytetu Łódzkiego. Artykuł wpłynął do redakcji w lutym 2004 r. Eugeniusz KWIATKOWSKI" Eugeniusz KWIATKOWSKI" Cechy charakterystyczne i zasady integracji krajów Unii Europejskiej Cechy charakterystyczne i zasady integracji krajów Unii Europejskiej Cechy charakterystyczne i zasady integracji krajów Unii Europejskiej Rozważając problem konsekwencji integracji z UE dla polskiego rynku pracy warto uświadomić sobie cechy i charakter ugrupowania integracyjnego, którego członkiemstanie się niebawem Polska. W szczególności istotne są tu- taj niektóre założenia i zasady polityki gospodarczej, będące podstawą funk- cjonowania gospodarekczłonkowskich, znajdujące wyraz w dokumentach UE i rozwiązaniach wynegocjowanych przez stronę polską w latach 1998-2002, przyjętych na szczycie w Kopenhadze, w grudniu 2002 roku. Należy przede wszystkim podkreślić, że UE jest ugrupowaniem integracyj- Należy przede wszystkim podkreślić, że UE jest ugrupowaniem integracyj- nym składającym się w większości z krajów najbardziej rozwiniętych gospo- darczo. Znajduje to wyraz między innymi we wskaźnikach produkcji i kon- sumpcji na \| mieszkańca w tych krajach, które są znacznie wyższe niż w Polsce. Wskaźnik produktu krajowego brutto na 1 mieszkańca jest w krajach UE średnio 2,6 razy wyższyniż w Polsce. Nawet w mniej rozwiniętych krajach UE, takich jak Grecja, Portugalia i Hisz- Nawet w mniej rozwiniętych krajach UE, takich jak Grecja, Portugalia i Hisz- pania wskaźniki te znacznie przewyższają wielkości charakterystyczne dla Pol- ski. Dystans dzielący Polskę od dotychczasowych krajów członkowskich UE wzakresie PKB per capita wiążesię przede wszystkim z różnicami wydajno- ści pracy, co rzutuje oczywiście na pozycję konkurencyjną integrujących się gospodarek. Istnienie dużego dystansu rozwojowego między Polską a większo- ścią krajów UE jest faktem o wielorakich konsekwencjach dla wpływu człon- kostwa na polski rynek pracy. Z początkiem maja 2004 r. Polska wchodzi do ugrupowania, które jest Z początkiem maja 2004 r. Polska wchodzi do ugrupowania, które jest strefą wolnego handlu. Zniesione będą wszelkie bariery (także natury pozata- ryfowej) w handlu towarami przemysłowymimiędzy krajami członkowskimi. Wprowadzonyzostanie również wolny handel artykułami rolnymi, aczkolwiek będą one musiały spełniać szereg warunkówsanitarnych oraz podlegać regu- łom wspólnej polityki rolneji rozwiązaniom wynegocjowanym i przyjętym w Ko- penhadze. Producenci krajowi staną więc w obliczu pełnej konkurencji ze strony producentów z innych krajów członkowskich. Zmiana w tym zakresie nie powinna mieć jednakże istotnych konsekwencji, gdyż zasadnicza liberali- zacja w zakresie handlu towarami przemysłowymi dokonała się w okresie przed- akcesyjnym, w rezultacie podpisanego wcześniej układu stowarzyszeniowego. Wchodzimy ponadto do ugrupowania, będącego unią celną, co oznacza Wchodzimy ponadto do ugrupowania, będącego unią celną, co oznacza stosowanie jednolitych przez kraje członkowskie stawek celnych wobec kra- jów trzecich. Wstęp Temat zawarty w tytule można rozumieć co najmniej dwojako.Z jednej stro- ny można go rozumiećjako analizę porównawczą rynku pracy w Polscei kra- jach członkowskich UE. Takie ujęcie tematu akcentuje różnice i dystans mię dzy tymi rynkami oraz niezbędne dostosowania polskiego rynku pracy do standardów unijnych. Z drugiej strony można go rozumieć jako wpływ proce- su integracji Polski z UE na polski rynek pracy. To ujęcie kieruje uwagę na prawdopodobne zmiany zachodzące na rynku pracy w Polsce w związku z ak- cesją do UE. W artykule odnoszę się do tematu przez pryzmat obu ujęć, choć w dalece W artykule odnoszę się do tematu przez pryzmat obu ujęć, choć w dalece niewyczerpujący sposób. W płaszczyźnie analizy porównawczej ograniczam roz- ważania do niektórychtylko aspektów, zwłaszcza tych o istotnym znaczeniu dla wpływu integracji na sytuację, na rynku pracy w Polsce. Jeszcze istotniej- sze ograniczenia dotyczą drugiego ujęcia tematu. Nie dokonuję interesujące go i ważnego skądinąd, choć niezmiernie skomplikowanego, szacunku wpły- wu akcesji na sytuację na rynku pracy w Polsce. Rozważania ograniczam natomiast do ukazania kierunków i mechanizmów wpływu integracji z UE na polski rynek pracy, zwracając przy tym uwagę na wiele dylematów i wyzwań stojących przed polską gospodarką. Struktura artykułu jest następująca. W części drugiej ukazane są cechy Struktura artykułu jest następująca. W części drugiej ukazane są cechy i zasady integracji krajów UE,które mogą mieć znaczenie dla wpływu człon- kostwa Polski na nasz rynek pracy. Część trzecia poświęcona jest charaktery- styce polskiego rynku pracy przed akcesją w porównaniu z rynkami pracy UE. W części czwartej akcent spoczywa na ukazaniu tych determinantów sytuacji na rynku pracy, które mogą mieć znaczenie przy rozważaniu wpływu integra- cji z UE na rynek pracy. Przedmiotem rozważańczęści piątej są kierunki i mechanizmy wpływu integracji z UE na polski rynek pracy. Część szósta za- wiera podsumowanie i wnioski. GOSPODARKA NARODOWANr4/2004 82 Cechy charakterystyczne i zasady integracji krajów Unii Europejskiej Dostosowania w tym zakresie będą oznaczały zmiany ceł na nie- które towary w stosunkach z krajami trzecimi i możliwe w związku z tym zmiany cen krajowych. Spadną cła na artykuły przemysłowe, a także na alko- hol i tytoń, nasilając konkurencję na tych rynkach, wzrosną natomiast cła m. in. na ryby, mleko i zboże. W literaturze przedmiotu podkreśla że skutki zmian ceł dla krajowej produkcji nie powinny być duże [Kawecka-Wyrzykow- ska, 2003, s. 158-159]. UEjest ponadto ugrupowaniem integracyjnym o charakterze wspólnego UEjest ponadto ugrupowaniem integracyjnym o charakterze wspólnego rynku,oznaczającego występowanie tzw. czterech swobód i wolnościtj. swo- 83 Eugeniusz Kwiatkowski, Integracja Unią Europejską rynek pracy w Polce bodnego przepływu towarów, kapjtałów, usług i osób na rynku wewnętrznym ugrupowania. Co prawda pełna swoboda przepływu pracowników zostanie wprowadzona w okresie 7 lat od daty członkostwa, ale już w pierwszym ro- ku członkostwa zostaną zastosowane znaczne koncesje, zaś w dalszych latach są zapowiedziane skrócenia okresu przejściowego przez kolejne kraje człon- kowskie. Należy podkreślić, że UE staje się w coraz większej mierze ugrupowaniem Należy podkreślić, że UE staje się w coraz większej mierze ugrupowaniem integracyjnym opartym na zasadzie unii gospodarczej. Począwszy od Traktatu Rzymskiegocoraz więcej obszarów polityki gospodarczej podlega koordynacji bądź wręcz unifikacji na szczeblu wspólnotowym. Wysoki stopień wspólnoto- wegocharakteru polityki gospodarczej dotyczy zwłaszcza polityki handlowej, rolnej, podatkowej w zakresie podatków pośrednich, regionalnej czy też poli- tyki pieniężnej w przypadku krajów należących do unii gospodarczej i walu- towej. Oznacza to, że kraje członkowskie znaczne obszary polityki gospodar- czej opierają na zasadach ustalonych na szczeblu Wspólnoty. Członkostwo Polski w UE będzie oczywiście wymagało respektowania tych ustaleń i niezbędnych dostosowań polityki gospodarczej. Jest godne podkreślenia, że w niedużym stop- niu dotyczy to polityki rynku pracy. Polityka rynku pracy zaczęła nabierać wspólnotowego charakteru dopiero Polityka rynku pracy zaczęła nabierać wspólnotowego charakteru dopiero w ostatnich latach. W zasadzie aż do początku lat dziewięćdziesiątych trudno dostrzec oznaki integracji polityki rynku pracy krajówczłonkowskich. W Bia- łej Księdze „Growth, Competitiveness and Employment” z 1993 r. podkreślo- no wzrost zatrudnienia jako istotny filar (obok wzrostu gospodarczego i kon- kurencyjności) strategii gospodarczej UE. W 1994r. w Essen, Rada Unii Europejskiej sformułowała strategię gospodarczą zawierającą elementy polity- ki rynku pracy (koniecznośćrozwoju szkoleń zawodowych,elastycznychform zatrudnienia, konieczność redukcji pozapłacowych kosztów pracy i poprawy efektywności programów rynku pracy) oraz zalecenie ich stosowania przez pań- stwa członkowskie. W Traktacie Amsterdamskim z 1997 r. GOSPODARKA NARODOWANr 4/2004 84 Europejski Fundusz Orientacji i Gwarancji Rolnej oraz fundusze finansujące sektorowe programy w zakresie rolnictwa, jak i dopłaty bezpośrednie do pro- dukcji rolnej. Wszystkie te fundusze staną do dyspozycji polskich wnioskodaw- ców, oczywiście w rozmiarach i na warunkach przyjętych w procesie negocja- cyjnym. Cechy charakterystyczne i zasady integracji krajów Unii Europejskiej wymieniono wyso- kie zatrudnienie wśród celów makroekonomicznych, a ponadto kraje członkow- skie zobowiązały się do koordynowania swych polityk rynku pracy. Polityka rynku pracy zaczęła nabierać wspólnotowego charakteru w rezultacie ustaleń szczytu w Luksemburgu w 1997 r., gdzie przyjęto wytyczne dla polityki za- trudnienia oraz kryteria jej oceny. Ponadto kraje członkowskie zostały zobo- wiązane do opracowywania Narodowych Planów Działania na rzecz Zatrud- nienia oraz ich monitoringu. Również na szczycie Rady Europejskiej w Lizbonie w 2000 r. podkreślonoistotną rolę wzrostu zatrudnienia w gospodarce opar- tej na wiedzy. Przytoczone wyżej ustalenia zawarte w dokumentach UE wska- zują, że następuje stopniowa unifikacja polityki rynku pracy w krajach człon- kowskich, choć nadal zasadnicze elementy tej polityki podlegają narodowej legisacji Dla realizacji celów i zadań wspólnotowych polityk gospodarczych tworzo- Dla realizacji celów i zadań wspólnotowych polityk gospodarczych tworzo- ne są w UEróżnorodnefundusze stanowiącefinansową podstawęich realiza- cji. Wymienić tutaj można fundusze strukturalne, takie jak: Europejski Fun- dusz Socjalny, Europejski Fundusz Rozwoju Regionalnego, Fundusz Spójności, GOSPODARKA NARODOWANr 4/2004 Rynek pracy w Polsce na tle krajów Unii Europejskiej Członkostwo Polski w UE może spowodowaćróżnorodne zmiany na kra- jowym rynku pracy. W kontekście możliwych zmian warto przyjrzeć się naj- pierw charakterystycznym cechom polskiego rynku pracy w przededniu człon- kostwa w UE. Ich konfrontacja z rynkami pracy w krajach UE pokazuje nie tylko różnice i dystans między nimi, ale sugeruje również możliwą drogę i kie- runki zmian na polskim rynku pracy, jeśli wierzyć w teorię ekonomicznej kon- wergencji. Charakterystycznącechą polskiego rynku pracy w przededniu członkostwa Charakterystycznącechą polskiego rynku pracy w przededniu członkostwa jest bardzo wysoka stopa bezrobocia. W 2002 r. wskaźnikten (obliczany me- todą BAEL) przekraczał 20%, podczas gdy średni wskaźnik dla UE wynosił 7,7%, zaś w wielu krajach członkowskichbył on niższy niż 5% (por. tabl. 1). Tablica 1 lata Tablica 1 Stopy zatrudnienia,bezrobocia i aktywności zawodowej w grupie wiekowej 15-64 lata w krajach UE i Polsce w 2002 roku (w %) Kaj Stopa zatrudnienia Stopa aktywności zawodowej Belgia. 597 04,1 Dania. 764 799 Niemcy 554 TS Grecja 569 83,1 "Hiszpania 584 653 Francja 52.9 530 Jniandia- EN GEJ [wiochy 554 610. Laksembur, 83,6 65.3 "Holandia 745 765. „Austria 58.2 TI Portugalia 68,6 221 Finlandia. 69,1 Ti2. Szwecja 740 780 Wielka Brytania 115 EJ UE-15 542 636 Polska 517 649 Źródło: [Employment in Europe 2003,s. 35] Stopy zatrudnienia,bezrobocia i aktywności zawodowej w grupie wiekowej 15-64 lata w krajach UE i Polsce w 2002 roku (w %) Stopy zatrudnienia,bezrobocia i aktywności zawodowej w grupie wiekowej 15-64 lata w krajach UE i Polsce w 2002 roku (w %) Kaj Stopa zatrudnienia Stopa aktywności zawodowej Belgia. 597 04,1 Dania. 764 799 Niemcy 554 TS Grecja 569 83,1 "Hiszpania 584 653 Francja 52.9 530 Jniandia- EN GEJ [wiochy 554 610. Laksembur, 83,6 65.3 "Holandia 745 765. „Austria 58.2 TI Portugalia 68,6 221 Finlandia. 69,1 Ti2. Szwecja 740 780 Wielka Brytania 115 EJ UE-15 542 636 Polska 517 649 Źródło: [Employment in Europe 2003,s. 35] Porównanie to wypadłobyjeszcze gorzej, gdyby uwzględnić znaczne roz- miary ukrytego bezrobocia w polskiej gospodarce, przejawiające się wistnie- niu nadmiernego zatrudnienia. Dotyczy to przede wszystkim rolnictwa, gdyż w dziedzinach pozarolniczych nastąpiła w okresie transformacji znaczna re- 85 Eugeniusz Kwiatkowski, Integracja Unią Europejską a rynek pracy w Polsce dukcja tego zjawiska. Takie różnice w sytuacji na rynku pracy w Polsce i UE mają wielorakie konsekwencje. Z jednej strony, znaczna część polskiego spo- łeczeństwa upatruje w członkostwie szansę na poprawęsytuacji na rynku pra- cy. Rynek pracy w Polsce na tle krajów Unii Europejskiej Z drugiej zaś, duże różnice w stopach bezrobocia są często wykorzystywa- ne dla argumentacji o dużym potencjale emigracyjnym w Polsce (zwłaszcza przez autorów z krajów UE). O trudnej sytuacji na polskim rynku pracy świadczy nie tylko wysoka sto- O trudnej sytuacji na polskim rynku pracy świadczy nie tylko wysoka sto- pa bezrobocia, ale również relatywnie niskie wskaźniki zatrudnienia (relacja liczby pracujących do liczby ludności w grupie wiekowej 15-64 lata). O ile w niektórych krajach UE wskaźniki te w 2002r. przewyższały 70%, zaś wskaź- nik średni przekraczał 64%, to w Polsce nie osiągnął on nawet 52% (tabl. 1). Tak niski wskaźnik (wraz z relatywnie niską stopą aktywności zawodowej — tabl. 1) świadczy o dużym obciążeniu ekonomicznym pracujących, co nie jest bez znaczenia dla stanu finansów publicznych i możliwościfinansowego wspie- rania programów UE. Charakterystyczną cechą polskiego bezrobociajest jego stagnacyjny cha- Charakterystyczną cechą polskiego bezrobociajest jego stagnacyjny cha- rakter, polegający na niskiej rotacji osób w zasobie bezrobocia. Wzrost bezro- bocia w latach 1998-2002był związany nie ze wzrostem liczby osób napływa- jących do bezrobocia, lecz z wydłużeniem się przeciętnego okresu trwania bezrobocia. Stagnacyjny charakter bezrobocia przejawia się właśnie w stosun- kowodużej liczbie osób bezrobotnych długotrwale. Stopa bezrobocia długo- okresowego (stosunek liczny bezrobotnych dłuższy niż 12 miesięcy do liczby aktywnych zawodowo)jest w Polsce bardzo wysoka i w 2002 roku zbliżała się do 11%, podczas gdy Średnia stopa w UE wynosiła 3% (por. tabl. 2) Wysoka stopa bezrobocia długookresowego ma liczne negatywne reperku- Wysoka stopa bezrobocia długookresowego ma liczne negatywne reperku- sje społecznei ekonomiczne, przyczyniając się do zjawiska histerezy bezrobo- cia. Negatywne konsekwencje bezrobocia długookresowego zostały dobrze rozpoznane w krajach UE, stąd też poprawastopy zatrudnienia bezrobotnych długotrwale znalazła się wśród wytycznych dla realizacji Europejskiej Strate- gii Zatrudnienia przyjętej w Luksemburgu w 1997r. Ze względu na wysoki poziom bezrobocia długookresowego w Polsce nie będzie łatwo zrealizować tych wytycznych. Inną charakterystyczną cechą polskiego bezrobocia jest bardzo wysoka Inną charakterystyczną cechą polskiego bezrobocia jest bardzo wysoka stopa bezrobocia wśród młodzieży. W grupie wiekowej 15-24 lata wskaźnik ten w 2002 r. wyniósł 41,7%, przy średnim wskaźniku w UE wynoszącym 15,1% (zob. tabl. 2). Tak wysoki wskaźnik bezrobocia wśród polskiej młodzieży ma wielorakie negatywne skutki społeczne i ekonomiczne. Jest również czynni- kiem, który będzie sprzyjał procesom emigracji zarobkowej z Polski po wpro- wadzeniu swobody przepływu osób. Ważnącechą polskiego bezrobocia jest stosunkowo wysoki poziom tzw. Ważnącechą polskiego bezrobocia jest stosunkowo wysoki poziom tzw. bezrobocia równowagi, którego podstawowym elementem jest bezrobocie struk- turalne. GOSPODARKA NARODOWANr 4/2004 36 ską mobilnośćsiły roboczej w przekrojach przestrzennych, zawodowych i kwa- lifikacyjnych. Wysoki poziom bezrobocia równowagi w polskiej gospodarce ma ważneimplikacje, także w kontekście integracji z UE. Z jednej strony, su- geruje on stosunkowo niską wrażliwość koniunkturalną polskiego bezrobocia (co jest istotne przy możliwym wzroście produkcji po akcesji), z drugiej zaś wskazuje, że rozwój programów rynku pracy zaadresowanych do grup proble- mowych (także programów UE) może być dosyć skutecznym instrumentem ograniczania bezrobocia. Tablica 2 Stopy bezrobociadługookresowego i wśród młodzieży (w grupie 15-24 lata) w krajach UE 1 Polsce w 2002 roku (w siły roboczej) Stopa bezrobocia Stopa bezrobocia wśród mi Bel 36 182 Dania. 05. 31 Niet 30 57 G EM 264 35 222 Franć 27 200 13 EG 'Wiochy 53 Zi2. Lukset 03 83 Holandia 0 52 „Austria 08 68 Poż 18 115 Finlandia 23 210. Sza 10 115 Wielka Bi LI 121 UE-I5 50 15 „Polska. 109. 417 Źródło: jak do tablicy 1, s. 209-234 Stopy bezrobociadługookresowego i wśród młodzieży (w grupie 15-24 lata) w krajach UE 1 Polsce w 2002 roku (w siły roboczej) w 2002 roku (w siły roboczej) Stopa bezrobocia Stopa bezrobocia wśród mi Bel 36 182 Dania. 05. 31 Niet 30 57 G EM 264 35 222 Franć 27 200 13 EG 'Wiochy 53 Zi2. Lukset 03 83 Holandia 0 52 „Austria 08 68 Poż 18 115 Finlandia 23 210. Sza 10 115 Wielka Bi LI 121 UE-I5 50 15 „Polska. 109. 417 Źródło: jak do tablicy 1, s. 209-234 Stopa bezrobocia Stopa bezrobocia wśród mi Bel 36 182 Dania. 05. 31 Niet 30 57 G EM 264 35 222 Franć 27 200 13 EG 'Wiochy 53 Zi2. Lukset 03 83 Holandia 0 52 „Austria 08 68 Poż 18 115 Finlandia 23 210. Sza 10 115 Wielka Bi LI 121 UE-I5 50 15 „Polska. 109. 417 Źródło: jak do tablicy 1, s. 209-234 Istotne różnice między Polską a krajami UE dotyczą aktywnej polityki pań- stwa na rynku pracy, skierowanej głównie na redukcję bezrobocia struktural- nego.Po pierwsze, wydatki na aktywne programy rynku pracy są w Polsce znacznieniższe niż w krajach UE,i to nie tyko w wymiarze absolutnym, ale i względnym. W krajach UE średni wskaźnik udziału wydatków na aktywną politykę rynku pracy w PKBkształtował się na poziomie 1,1% w 1997r., zaś w Polsce tylko na poziomie 0,3% (por. [Kryńska, 2001, s. 61]), by spaść jeszcze bardziej w dalszych latach (0,1% w 2000 r.). Rynek pracy w Polsce na tle krajów Unii Europejskiej Z badań wynika, że pod koniec lat dziewięćdziesiątych stopa bezro- bocia równowagistanowiła ok. 80% rzeczywistej stopy bezrobocia (zob. [So- cha, Sztanderska, 2000, s. 155)). Trudno się temu dziwić, jeśli wziąć pod uwagę duże niedopasowania strukturalne na rynku pracy oraz stosunkowoni- GOSPODARKA NARODOWANr 4/2004 GOSPODARKA NARODOWANr 4/2004 Po drugie, w Polsce mamy od- mienną strukturę wydatków na aktywne programy rynku pracy niż w UE. Znajduje to wyraz w niskim udziale wydatkówna szkolenia (7%, przy średniej w UE 25% — zob. [Kryńska, 2001, s. 60]). Po trzecie, w aktywnych progra- mach rynku pracy bierze udział w Polsce znacznie mniejszy odsetek bezrobot- nych(ok. 4%) niż w UE(ok. 10%). Trzeba więc stwierdzić, że w Polsce przy- wiązuje się znacznie mniejszą wagę do aktywnych programów rynku pracy niż w UE, pomimoznacznie wyższego bezrobocia wPolsce. Różnice te poka- 87 Eugeniusz Kwiatkowski, Integracja z Unią Europejską a rynek pracy w Polce zują zmiany, jakie muszą zajść w polityce rynku pracy w Polsce po wejściu do UE,aby możnabyło realizować wytyczne Europejskiej Strategii Zatrudnienia. Na zakończenie analizy porównawczej rynków pracy w Polsce i krajach zują zmiany, jakie muszą zajść w polityce rynku pracy w Polsce po wejściu do UE,aby możnabyło realizować wytyczne Europejskiej Strategii Zatrudnienia. Na zakończenie analizy porównawczej rynków pracy w Polsce i krajach Na zakończenie analizy porównawczej rynków pracy w Polsce i krajach UE warto zwrócić uwagęna strukturę zatrudnienia. Z analizy porównawczej struktury zatrudnienia w Polsce i krajach UE wynika, że zasadnicze różnice dotyczą udziałów sektora rolniczego i usługowego. Odsetek zatrudnionych w sek- torze rolniczym przewyższał w 2002 r. 18%, przy średniej w UE — 3,7%; nato- miast udział sektora usługowego był w Polsce znacznie niższy niż w Unii Eu- ropejskiej (52,5% wobec 67,9% w UE - zob.tabl. 4). Takie różnice w strukturze zatrudnienia są niewątpliwie w istotnej mierze wynikiem różnie w poziomie PKB percapita, choć pewną rolę w ukształtowaniu się wysokiego wskaźnika udziału sektora rolniczego w Polscetrzeba przypisaćistniejącej strukturze agrar- nej, a także pełnionej przez rolnictwo w okresie transformacji funkcji amor- tyzatora napięć na rynku pracy. Pożądanekierunki zmian strukturalnych w za- trudnieniu sąw Polsce oczywiste. Członkostwo Polski w UE powinno przyspieszyć te zmiany (m.in. poprzez fundusze strukturalne i wspólną politykę rolną) Determinanty sytuacji na rynku pracy Sytuacja na rynku pracy zależy od wielu czynników. Nie jest celem tych rozważań wyczerpujące ujęcie wszystkich. Uwaga zostanie zwrócona nato- miast na te, które mogą mieć znaczenie przy analizie wpływu procesów inte- gracyjnych na sytuację na rynku pracy wPolsce. Czynniki determinujące sytuację na rynku pracy można podzielić na trzy Czynniki determinujące sytuację na rynku pracy można podzielić na trzy grupy (zob.rys.1): związane z podażą pracy, związane z podażą pracy, — związane z popytem na pr — związane z popytem na pracę, - związanez niedopasowaniami s - związanez niedopasowaniami strukturalnymi na rynku pracyi efektywno- ścią funkcjonowania rynku pracy. Spośród czynników kształtujących stronę podażową rynku pracy istotną - związanez niedopasowaniami strukturalnymi na rynku pracyi efektywno- ścią funkcjonowania rynku pracy. Spośród czynników kształtujących stronę podażową rynku pracy istotną Spośród czynników kształtujących stronę podażową rynku pracy istotną rolę w zakresie wpływu procesów integracyjnych na sytuację mogą odegrać jgracje zagraniczne ludności. Jest oczywiste, że w przypadku ujemnego sal- da migracji napięcia na rynku pracy ulegają złagodzeniu. Warto zauważyć,iż przy analizie wpływu migracji zagranicznych na sytuację na rynku pracy waż- ne znaczenie manie tylko saldo migracji, ale również struktura emigrantów i imigrantów, zwłaszcza pod względem kwalifikacji zawodowychi wykształce- nia, gdyż ma to znaczenie dla produktywności pracy i dynamiki wzrostu go- spodarczego. Jeśli chodzi o czynniki związane z popytową stroną rynku pracy, to warto Jeśli chodzi o czynniki związane z popytową stroną rynku pracy, to warto podkreślić tutaj przede wszystkim trzy czynniki, za pośrednictwem których re- alizuje się wpływ akcesji na sytuację na rynku pracy: —_ bezpośrednie inwestycje zagraniczne, —_ bezpośrednie inwestycje zagraniczne, -- transfery funduszy z UE, -- transfery funduszy z UE, —- eksport netto. —- eksport netto. GOSPODARKA NARODOWANr 4/2004 88. Rysunek 1. Determinanty sytuacji na rynku pracy Sytacjaa rynku pracy fony Niedopaoania Poi pracę ielektywność - rynku pracy + Poziom produkcji * Ludność w wieku Ludęośćwwie iiwzrogospodarczy + Współęzynnik aktywności * Śraławalnych aa” zawodowej : Zagraniczne (BIZ) <w * Elastyczność rynku pracy. ligracje Mobilność siły roboczej * Fundusze strukturalne * Polityka państwa na » Eks netto Polkapań Eksport neti + Wydajność pracy Postęp techniczny Źródło: opracowanie własne Rysunek 1. Determinanty sytuacji na rynku pracy Źródło: opracowanie własne Źródło: opracowanie własne Bezpośrednie inwestycje zagraniczne mają wiele korzystnych skutków dla gospodarki i rynku pracy. Przede wszystkim zwiększają nakłady inwestycyjne i tworzą nowe miejsca pracy. Ponadto bezpośrednie inwestycje zagraniczne są ważnym nośnikiem innowacji, które korzystnie wpływają na dynamikę wzro- stu gospodarczego. Determinanty sytuacji na rynku pracy Przy większej ska- li zmian strukturalnych niedopasowania strukturalne na rynku pracy mo- gą być większe,a sytuacja na rynku pracy gorsza, -_ elastyczność rynku pracy, a zwłaszcza mobilność siły roboczej oraz ela- -_ elastyczność rynku pracy, a zwłaszcza mobilność siły roboczej oraz ela- styczność zatrudnienia i elastyczność czasu pracy. Przy wyższej mobilno- ści i elastyczności można liczyć na mniejsze niedopasowania strukturalne na rynku pracy i w rezultacie lepszą sytuację na tym rynku, — zasięg i skuteczność polityki państwa na rynku pracy. Im większy jest za- — zasięg i skuteczność polityki państwa na rynku pracy. Im większy jest za- sięg aktywnej polityki państwa na rynku pracy(im więcej uczestników pro- gramówrynkupracy) oraz im bardziej skuteczna jest polityka państwa na rynku pracy, tym mniejsze niedopasowania strukturalne na tym rynku i lepsza sytuacja na rynku pracy. — zasięg i skuteczność polityki państwa na rynku pracy. Im większy jest za- sięg aktywnej polityki państwa na rynku pracy(im więcej uczestników pro- gramówrynkupracy) oraz im bardziej skuteczna jest polityka państwa na rynku pracy, tym mniejsze niedopasowania strukturalne na tym rynku i lepsza sytuacja na rynku pracy. Determinanty sytuacji na rynku pracy Rozważając wpływ wymienionych wyżej trzech czynników na sytuację na Rozważając wpływ wymienionych wyżej trzech czynników na sytuację na rynku pracy warto zwrócić uwagę na dwie perspektywy, w ramach których ten wpływ się dokonuje: perspektywę krótkookresową, kiedy zmianytych czynników wpływają na perspektywę krótkookresową, kiedy zmianytych czynników wpływają na krajowezatrudnienie i bezrobocie poprzez oddziaływanie na efekty popy- towe i kształtowanie produkcji, —_ perspektywę długookresową, kiedy te czynniki mogą wpływać na zatrud- perspektywę krótkookresową, kiedy zmianytych czynników wpływają na krajowezatrudnienie i bezrobocie poprzez oddziaływanie na efekty popy- towe i kształtowanie produkcji, —_ perspektywę długookresową, kiedy te czynniki mogą wpływać na zatrud- —_ perspektywę długookresową, kiedy te czynniki mogą wpływać na zatrud- nienie i bezrobocie za pośrednictwem oddziaływania na efekty podażowe i kształtowania wzrostu gospodarczego(dotyczy to zwłaszcza bezpośred- nich inwestycji zagranicznych i transferów funduszy z UE). Biorąc pod uwagę perspektywę długookresową wpływu procesów integra- —_ perspektywę długookresową, kiedy te czynniki mogą wpływać na zatrud- nienie i bezrobocie za pośrednictwem oddziaływania na efekty podażowe i kształtowania wzrostu gospodarczego(dotyczy to zwłaszcza bezpośred- nich inwestycji zagranicznych i transferów funduszy z UE). Biorąc pod uwagę perspektywę długookresową wpływu procesów integra- Biorąc pod uwagę perspektywę długookresową wpływu procesów integra- cyjnych ma zatrudnienie i bezrobocie, w którym istotną rolę odgrywa oddzia- ływanie na dynamikę wzrostu gospodarczego, trzeba uwzględnić również dy- namikę wydajności pracy. Wiadomo, że przyniższej dynamice wzrostu wydajności pracy skutki dla zatrudnienia w warunkach założenia ceteris pa- ribus będą bardziej korzystne. Jeśli jednak procesyintegracji z UE podnoszą dynamikę wydajności pracy wkrajowej gospodarce, to pozytywny wpływna sytuację na rynku pracy byłby wówczas osłabiony. Sytuacja na rynku pracy zależy również od stopnia niedopasowań struktu- Sytuacja na rynku pracy zależy również od stopnia niedopasowań struktu- ralnych (niedopasowań między strukturą podaży pracya strukturą popytu na pracę pod względem kwalifikacji, zawodów, wykształcenia, miejsca zamiesz- kania i miejsca pracy) oraz efektywności funkcjonowania rynku pracy. W tej grupie warto zwrócić uwagęna trzy czynniki: —_ skalę zmianstrukturalnych wprodukcji i zatrudnieniu. Przy większej ska- —_ skalę zmianstrukturalnych wprodukcji i zatrudnieniu. Przy większej ska- li zmian strukturalnych niedopasowania strukturalne na rynku pracy mo- gą być większe,a sytuacja na rynku pracy gorsza, -_ elastyczność rynku pracy, a zwłaszcza mobilność siły roboczej oraz ela- —_ skalę zmianstrukturalnych wprodukcji i zatrudnieniu. Determinanty sytuacji na rynku pracy Co prawda bezpośrednie inwestycje zagraniczne mogą również w pewnym stopniu wypierać krajowąprodukcjęi zatrudnienie, a tak- że przyczyniać się do zwiększenia importu osłabiającego poziom i wzrost za- trudnienia,to jednak sumaryczny efekt wpływu bezpośrednich inwestycji za- granicznych na zatrudnienie, zwłaszcza na dłuższą metę, wydaje się dodatni. Jeśli chodzi o transfer funduszy z UE, to ich pozytywneskutki dla krajowe- Jeśli chodzi o transfer funduszy z UE, to ich pozytywneskutki dla krajowe- go zatrudnienia i bezrobocia są oczywiste. Mogą one bezpośrednio poprawiać sytuację na rynku pracy poprzez aktywizację bezrobotnychdzięki ich uczest- nictwu w programach rynku pracy, a także poprzez rozwój szkoleń zawodo- wych oraz rozwój obszarów wiejskich. Warto również zwrócić uwagę na po- pytotwórcze efektytransferu funduszy podnoszące poziom krajowej produkcji. Jeśli chodzi o wpływ eksportu netto na sytuację na rynku pracy, to w ra- mach analizy krótkookresowej należy założyć pozytywny wpływ w przypadku dodatniego eksportu netto oraz wpływ niekorzystny w przypadku ujemnego eksportu netto. Przy analizie tego wpływu istotną rolę odgrywa również struk- tura eksportu i importu, a w szczególności udział importu inwestycyjnego. Jeśli chodzi o transfer funduszy z UE, to ich pozytywneskutki dla krajowe- go zatrudnienia i bezrobocia są oczywiste. Mogą one bezpośrednio poprawiać sytuację na rynku pracy poprzez aktywizację bezrobotnychdzięki ich uczest- nictwu w programach rynku pracy, a także poprzez rozwój szkoleń zawodo- wych oraz rozwój obszarów wiejskich. Warto również zwrócić uwagę na po- pytotwórcze efektytransferu funduszy podnoszące poziom krajowej produkcji. Jeśli chodzi o wpływ eksportu netto na sytuację na rynku pracy, to w ra- Jeśli chodzi o wpływ eksportu netto na sytuację na rynku pracy, to w ra- mach analizy krótkookresowej należy założyć pozytywny wpływ w przypadku dodatniego eksportu netto oraz wpływ niekorzystny w przypadku ujemnego eksportu netto. Przy analizie tego wpływu istotną rolę odgrywa również struk- tura eksportu i importu, a w szczególności udział importu inwestycyjnego. 89 rpmek pracy w Polsce Eugeniusz Kwiatkowski,/niegracja z Unią Europej Wysokie wskaźniki tego udziału przynoszą na dłuższą metę znaczące korzyści w zakresie wzrostu gospodarczego i krajowegozatrudnienia. Rozważając wpływ wymienionych wyżej trzech czynników na sytuację na Wysokie wskaźniki tego udziału przynoszą na dłuższą metę znaczące korzyści w zakresie wzrostu gospodarczego i krajowegozatrudnienia. Kierunki wpływu integracji z Unią Europejską na rynek pracy w Polsce Rozważmyobecnie prawdopodobne zmiany na polskim rynku pracy w związ- ku z procesami integracyjnymi z UE. Trzeba jednak od razu zaznaczyć,iż nie chodzi tutaj o tendencje zmian w sytuacji na rynku pracy, jakie mogą wystą- GOSPODARKA NARODOWANr 4/2004 90 pić w najbliższych latach, lecz o to w jakim kierunku członkostwoPolski w UE może wpłynąć na zmiany tych tendencji. Jeśli chodzi o przewidywanetendencje w zakresie migracji zagranicznych, Jeśli chodzi o przewidywanetendencje w zakresie migracji zagranicznych, 10 trzeba zauważyć wyraźny rozwój badań w tym zakresie w ostatnich latach. Próby szacunku tendencji migracyjnych zostały podjęte zarównow literaturze polskiej, jak i w opracowaniach zagranicznych. W opracowaniach autorów zagranicznych podkreśla się duży potencjał W opracowaniach autorów zagranicznych podkreśla się duży potencjał emigracyjny w Polsce. Wychodząc z analizy czynników determinujących mi- gracje zagraniczne (stosunkowoniskiego poziomu PKB na głowę w Polsce re- latywnie wysokiej stopy bezrobocia) autorzy z krajów UE sugerują wysoki po- tencjał emigracyjny w polskiej gospodarce. Jednak w szacunkachtychnie bierze się pod uwagę wielu barier procesów emigracyjnych, zwłaszcza językowych, kulturowych,a także ekonomicznych. Biorąc pod uwagęte bariery trzebastwier- dzić, że szacunki potencjału emigracyjnego w Polsce dokonywaneprzez auto- rów zachodnich są znacznie przesadzone. Pogląd ten znajduje wsparcie za- równo w spadkowej tendencji rozmiarów emigracji w Polsce w latach 90. (zob.tabl. 3), jak również w doświadczeniach Hiszpanii, Portugalii, Irlandi i Grecji, w których nie zanotowanozasadniczego wzrostu fali emigracyjnej po uzyskaniu przez te kraje członkostwa w UE. Rozmiary emigracji w Polsce w latach 1981-2002 (w osobach) Taa Liczba emigrantów Taa Liczba emigrantów 1591585. 120 148. 1555 22ATT 1986-1950 146 820. 1959. 21536 1951-1995. 12 725 2000. 26999 1995 26371 2001 23.368 1556 21297 2002 24.532 1557 20210 Źródło: iglicka, 2001, s. 47]; (Mały Rocznik Staystyczny Polki, 2003, s. 133] Rozmiary emigracji w Polsce w latach 1981-2002 (w osobach) j wiarygodne wydają się bardziej umiarkowane szacunki przyszłych procesów emigracyjnych autorów polskich, uwzględniające istnie- nie barier społecznych procesów emigracji (zob. tabl. 4). Uwagi te nie ozna- czają, iż może wystąpić w Polsce dodatnie saldo migracji. Wysoce prawdopo- dobne jest ujemne saldo migracji zagranicznych, które będzie łagodzić napięcia w sytuacji na rynku pracy w Polsce. Wiele argumentów przemawia za korzystną strukturą z punktu widzenia Wiele argumentów przemawia za korzystną strukturą z punktu widzenia polskiej gospodarki strukturą migracji zagranicznych. Dotychczasowe do- świadczenia wskazują, iż wśród emigrantów dominują osoby o niższych kwa- lifikacjach zawodowych, posiadające wykształcenie zawodowei podstawowe. Stosunkowo nieduży odsetek wśród emigrantów stanowiły osoby o wyższych kwalifikacjach i wykształceniu. Kierunki wpływu integracji z Unią Europejską na rynek pracy w Polsce Co prawda wysokie stopy bezrobocia wśród młodzieży w Polsce sprzyjają emigracji osób z wyższym poziomem kwalifika- cji, to jednak tendencja ta nie powinna mieć zasadniczego znaczenia. Biorąc 91 Eugeniusz Kwiatkowski, Integracja : Unią Europejską rynek pracy w Polsce pod uwagę fakt, iż w krajach UE występują tendencje do wzrostu relatywne- go popytu na pracę w zakresie prac prostych, uciążliwych, nie wymagających wysokich kwalifikacji, należy zakładać że tendencje te utrzymają się również w najbliższej przyszłości, podtrzymując istniejącą strukturę emigrantów pod względem kwalifikacji i wykształcenia. Jeśli chodzi natomiasto strukturę imigrantów z UE do Polski, to wiele wska- pod uwagę fakt, iż w krajach UE występują tendencje do wzrostu relatywne- go popytu na pracę w zakresie prac prostych, uciążliwych, nie wymagających wysokich kwalifikacji, należy zakładać że tendencje te utrzymają się również w najbliższej przyszłości, podtrzymując istniejącą strukturę emigrantów pod względem kwalifikacji i wykształcenia. Jeśli chodzi natomiasto strukturę imigrantów z UE do Polski, to wiele wska- Jeśli chodzi natomiasto strukturę imigrantów z UE do Polski, to wiele wska- zuje na to, iż wśród imigrantów przeważać będą osoby o wysokich kwalifika- cjach i wykształceniu. Dotychczasowe doświadczenia pokazują, iż wraz z na- pływem kapitału zagranicznego dokonuje się transfer siły roboczej o wysokich kwalifikacjach. Należy sądzić, iż przyspieszenie napływu kapitału zagranicz- nego do Polski nasili te tendencje. Tablica 4 Przewidywane rozmiary emigracji z Polski do krajów UE w 2010 rok (w tys. osób) Kae "Wariant A Wariant B Wariant © ieżące relacje PKB) (wolny wzrost PKB w Polsce)\| szybki wzrost PKB w Polsce) 310 276 195 154 TI 35 123 5 35 106 8. ia 104 8 i5 10ż. 6 12 55 52 2T 51 50 Bi 50. 38 5 85 42 16 45 i 44 5 4 5 12 0 o 0 rar2 m Wariant A — realny wzrost PKB w Polsce 2-36% rocznie Wariant B - realny wzrost PKB w Polsce 4% rocznie Wariant C — realny wzrost PKB w Polsce 7% rocznie Źródło: [Zienkowski, 2001, s. 117) Przewidywane rozmiary emigracji z Polski do krajów UE w 2010 rok (w tys. osób) Pozytywnego wpływu na sytuację na rynku pracy w Polsce można oczeki- wać w związku z transferem płatności z budżetu UE. Po uzyskaniu członko- stwa wysokość transferów środków finansowych będzie wzrastać w kolejnych latach osiągając łączną kwotę prawie 20 mld euro w 2013r. (por. tabl. 5). GOSPODARKA NARODOWANr 4/2004 GOSPODARKA NARODOWANr 4/2004 92 Tablica 5 „Płatności z budżetu UE dla Polski (ceny bieżące) w latach 2004-2013 (w min euro) Wd lk ez \| Pomoc Pianości Lata Fundusze Fundusz Poliyka wewnętrzne poprawę przed. Płatności jako srukturalne spójności Rolna irozwój płynności akcesyjna razem procent ifratruknury budżetowej [PKB (w 2) 2004 12 2_\|_15 55 389 10 32 \|__14 2005 \|__1663 38 \|1770- 455 5 ST 515 24 2006 \|_1813 60 \|207 573 517 585 628 \|_24 2007 \|__6115. 946 2487 457 o 153 10157 37 2008\| 179 1555 2816 472 o 19 2661 43 2005 8975 2159 3283 545 o 0 14859 47 2010 8779 2821 3725 637 o 0—\| 5962 \|_46 2011 \|__8955 3526 4186. 513 o 01540 47 2012\| 9134 4145 4660 IB 0 0 mós2 46 \| 2013 9517 4630 5157 755 0 0159 45] Źródło: [Samecki, 2003,s. 45 i 47] Tab „Płatności z budżetu UE dla Polski (ceny bieżące) w latach 2004-2013 (w min euro) Wd lk ez \| Pomoc Pian Źródło: [Samecki, 2003,s. 45 i 47] wpływ integracji z UE na polski rynek pracy za pośrednictwem oddziały- wania na eksport netto nie zapowiada się optymistycznie, przynajmniej w krót- kim okresie. Dotychczasowe tendencje salda obrotów handlu zagranicznego były wysoceniekorzystne,aczkolwiek trzeba odnotowaćzmniejszenie się ujem- nego salda w ostatnich latach. Biorąc pod uwagę dosyć wysoką importochłon- ność polskiej gospodarki, trudno zakładać istotne zmiany w zakresie salda ob- rotów handlu zagranicznego wnajbliższych latach. Na poprawę w tym zakresie można jednak liczyć w dłuższym okresie, wraz z poprawą konkurencyjności polskiej gospodarki. Podsumowując, o ile zwiększony napływ bezpośrednich inwestycji zagra- Podsumowując, o ile zwiększony napływ bezpośrednich inwestycji zagra- nicznych oraz funduszy z UE podnosić będą krajową produkcję i zatrudnie- nie, to utrzymujący się ujemny eksport netto osłabiał będzie te tendencje. W sumie możnaliczyć na wzrost produkcji w rezultacie członkostwa, bardziej znaczący w średnim i długim okresie. Efekty tych procesów dla zatrudnienia będą jednakże słabsze z powodusilnych tendencji do bezzatrudnieniowego wzrostu gospodarczego w Polsce. Od połowy lat dziewięćdziesiątych tempo wzrostu wydajności pracy (mierzone wartością PKB na \| pracującego) jest w polskiej gospodarce bardzo wysokie i kształtuje się na poziomie 4-5% rocz- nie. Jeśli tempo to utrzyma się wprzyszłości (co jest prawdopodobne w wa- runkach silnej presji konkurencyjnej i otwarcia gospodarki), to prozatrudnie- nioweefekty podniesienia wzrostu gospodarczego będą początkowo znikome. Kierunki wpływu integracji z Unią Europejską na rynek pracy w Polsce Te znacząceśrodki (powyżej 4% PKB po 2007 1:) wspierać będą rozwój produk- cji i zatrudnienia w Polsce, początkowo za pośrednictwemefektów popytowych, później zaś również poprzez efekty podażowe. Warto podkreślić, że znaczna Część transferów środków finansowychskierowana będzie na rozwójinfrastruk- tury gospodarczeji zasobów ludzkich, przyczyniając się w ten sposób do po- prawy efektywności gospodarowania. GOSPODARKA NARODOWANr 4/2004 Trudnojednoznacznie ocenić wpływ członkostwa na rynek pracy dokonu- Trudnojednoznacznie ocenić wpływ członkostwa na rynek pracy dokonu- jący się za pośrednictwem niedopasowań strukturalnych i efektywności funk- cjonowania rynku pracy. Z jednej strony należy założyć zwiększoną pod wpły- wem członkostwa skalę zmian strukturalnych w zatrudnieniu, pomimo przyjęcia systemu uproszczonego opłat bezpośrednich w rolnictwie, hamującego odpływ siły roboczej z rolnictwa. Z drugiej zaś, transfer płatności z UE do Polski skie- rowanyjest w dużej mierze na poprawę mobilnościsiły roboczeji elastyczno- ści rynku pracy, co powinno zmniejszać niedopasowania strukturalne popytu 93 Eugeniusz Kwiatkowski, Znteracja : Unią Europejską a rynek w Polsce i podaży pracy. Ze względu na to, że poprawa elastyczności rynku pracy mo- że nastąpić w dłuższym okresie, trzeba liczyć się początkowo z negatywnymi dla rynku pracy konsekwencjami przyspieszenia zmian strukturalnych i relo- kacji siły roboczej. i podaży pracy. Ze względu na to, że poprawa elastyczności rynku pracy mo- że nastąpić w dłuższym okresie, trzeba liczyć się początkowo z negatywnymi dla rynku pracy konsekwencjami przyspieszenia zmian strukturalnych i relo- kacji siły roboczej. Zakończenie Integracja z UE jest wielkim wyzwaniem dla polskiej gospodarki. Integru- jemy się z ugrupowaniem o wysokim poziomie rozwoju gospodarczego, nowo- czesnej i wysoce konkurencyjnej produkcji. Sprostanie konkurencji z takimi partnerami wymaga wielu dostosowań w polskiej gospodarce, w tym również w sferze rynku pracy. Dostosowania te (m.in. wytyczne dla polityki rynku pra- cy), choć niełatwe do urzeczywistnienia i wywierające na krótką metę wiele niekorzystnych skutków dla rynku pracy, są w perspektywie dłuższego okresu bardzo pożądanei przyczyniają się do poprawy konkurencyjności polskiej go- spodarki. Akcesja do UE przypada na okres charakteryzujący się trudnąsytuacją na Akcesja do UE przypada na okres charakteryzujący się trudnąsytuacją na polskim rynku pracy (stosunkowo wysokie bezrobocie, przestarzała struktura zatrudnienia). Perspektywy polskiego rynku pracy zależą w dużym stopniu od wewnętrznychi zewnętrznych czynnikówrozwojowych oraz polityki państwa w zakresie zatrudnienia i bezrobocia. Członkostwo w Unii Europejskiej win- no być wykorzystane dla wsparcia krajowej polityki w sferze zatrudnienia i bezrobocia. Wpływ członkostwa w UE na sytuację na polskim rynku pracyzależy w du- Wpływ członkostwa w UE na sytuację na polskim rynku pracyzależy w du- żej mierze od horyzontu czasowegoanalizy. W okresie krótkim (2-3 lata) efek- ty czynników pogarszających sytuację na rynku pracy (ujemny eksport netto, przyspieszone zmiany strukturalnei realokacja siły roboczej, wdrażanie postę- pu technicznego i wzrost wydajności pracy) mogą przeważaćnad efektami dzia- łania czynników poprawiających sytuację na tym rynku (ujemnesaldo migra- cji zagranicznych, transfer środków finansowych z UE do Polski, programy poprawy mobilnościsiły roboczej). W okresach średnim i długim siła działa- nia obu grup czynników winna ulec odwróceniu, wrezultacie czego można oczekiwać korzystnego wpływu akcesji na sytuację na rynku pracy w Polsce. "Kawecka-Wyrzykowska E., [2003], Koszty i korzyści wynikające 2 przyjęcia współnej polityki han. dowej oraz z wejścia we wspólnotowy system ekonomicznystosunkówzewnętrznych, w: Ko- rzyści i koszty członkostwa Polski w Unii Europejskiej. Raport z badań, Centrum Europej. skie Natolin, Warszawa. Employment in Europe, [2003], Europcan Commission. Iglicka K., [2001], Obecne tendencje wmigracjach zatrud Iglicka K., [2001], Obecne tendencje wmigracjach zatrudnieniowych w Polsce, [w:] A. Stępniak (red), Swobodne przepływy pracowników w kontekście wejścia Polski do Unii Europejskiej, „pełnomocnik rządu do spraw negocjacji o członkostwo w Unii Europejskiej, Kancelaria Pre- esa Rady Ministrów, Warszawa. "Kawecka-Wyrzykowska E., [2003], Koszty i korzyści wynikające 2 przyjęcia współnej polityki han. GOSPODARKA NARODOWANr 4/2004 94 yńska E., [2001], Dylematy Polskiego rynku pracy, IPS, Warszawa. mecki P, [2003], Przepływy finansowe między UE a Polską w latach Kryńska E., [2001], Dylematy Polskiego rynku pracy, IPS, Warszawa. Samecki P, [2003], Przepływy finansowe między UE a Polską w latach Samecki P, [2003], Przepływy finansowe między UE a Polską w latach 2004-2013, w: Korzyści i koszty członkostwa Polski w Unii Europejskiej, Centrum Europejskie Natolin, Warszawa. Socha M., Sztanderska U., [2000], Strukturalne podstawy bezrobocia w Polsce, Wydawnictwo Na- Samecki P, [2003], Przepływy finansowe między UE a Polską w latach 2004-2013, w: Korzyści i koszty członkostwa Polski w Unii Europejskiej, Centrum Europejskie Natolin, Warszawa. Socha M., Sztanderska U., [2000], Strukturalne podstawy bezrobocia w Polsce, Wydawnictwo Na- Samecki P, [2003], Przepływy finansowe między UE a Polską w latach 2004-2013, w: Korzyści i koszty członkostwa Polski w Unii Europejskiej, Centrum Europejskie Natolin, Warszawa. Socha M., Sztanderska U., [2000], Strukturalne podstawy bezrobocia w Polsce, Wydawnictwo Na- ukowe PWN, Warszawa 2002. Zienkowski L., Ekonomiczne aspekty swobodnego przepływu pracowników w rozszerzonej Unii Eu- i koszty członkostwa Polski w Unii Europejskiej, Centrum Europejskie Natolin, Warszawa. Socha M., Sztanderska U., [2000], Strukturalne podstawy bezrobocia w Polsce, Wydawnictwo Na- ukowe PWN, Warszawa 2002. Zienkowski L., Ekonomiczne aspekty swobodnego przepływu pracowników w rozszerzonej Unii Eu- Socha M., Sztanderska U., [2000], Strukturalne podstawy bezrobocia w Polsce, Wydawnictwo Na- ukowe PWN, Warszawa 2002. Zienkowski L., Ekonomiczne aspekty swobodnego przepływu pracowników w rozszerzonej Unii Eu- Zienkowski L., Ekonomiczne aspekty swobodnego przepływu pracowników w rozszerzonej Unii Eu- ropejskij, w: A. Stępniak (red.) Swobodny przepływ pracowników w kontekście wejścia Pol- ski do Unii Europejskiej Zienkowski L., Ekonomiczne aspekty swobodnego przepływu pracowników w rozszerzonej Unii Eu- ropejskij, w: A. Stępniak (red.) Swobodny przepływ pracowników w kontekście wejścia Pol- ski do Unii Europejskiej Summary tive analysis o The article takes up a comparative analysis of labour markets in Poland and in ihe EU MemberStates,and presents the expected impact of EU membership on the Polish labour market. Poland's accession to the EU comes in a period characterised by a difficult situation on the Polish labour marke (high and sustained unemployment, obsolete structure of employment). EU membership should be used to support the national policy in the field of employment and unemployment. The impact of EU membership on situation on the Polish labour market depends on the time-span of the analysis. In a short-term perspective (two-three years) the effecis of factors adversely affecting the labour market situation (trade defici, accelerated structural change and re.allocation of labourforce, implementation of technological progress and labour produciivity growih) may dominate overtheeffects of factors acting toward improvement of the situation on that market (negative balance of foreign migrations, increased inflow offoreign direct investment, transfer offunds from the EU to Poland,programmes for improvement of labour force mobility). In the medium and long term the strength of impact of both groups offactors should be reversed, hence a favourable influence of accession on the labour marketsituation in Poland can be expected.
https://openalex.org/W2125707156	https://sajems.org/index.php/sajems/article/download/1053/350	English	null	Mentorship alliance between South African established and developing farmers for sustainable agriculture sector reform	Suid-Afrikaanse tydskrif vir ekonomiese en bestuurswetenskappe/South African journal of economic and management sciences	2,014	cc-by	7,782	SAJEMS NS 9 (2006) No 4 553 Introduction to section be robust and well motivated, and should be of relevance to scientific discourse and humanity. We therefore see this section as an opportunity for senior scholars to provide insight into specific issues or ideas that are unlikely, or not ready, to take the form of a full scientific manuscript. We also see this as an opportunity for students and young scholars to share their research results from a less daunting (and belligerent) platform. Also, as is the case in this issue, we would like to see interesting information and research results, which could spark further discussions, based on a small sample survey. In line with international trends, the SAJEMS editorial board decided to introduce a section in the journal called: Viewpoints, perspectives or letters to the editor. We solicit submissions to this section that comprise novel concepts, ideas, or even a dialogue with either the editor or an author of an earlier paper either in SAJEMS or elsewhere. Controversial perspectives are even welcome, as long as they are presented in a constructive manner. Submissions to this section are likely to be shorter than a conventional manuscript, varying from 1 page to approximately 2 500 words. Submissions, though not necessarily qualifying in terms of all the rigorous requirements of a fully fledged scientific contribution, will nonetheless have to I would therefore wish to extend an invitation to all our readers to submit shorter, focused, robust and well-articulated views, perspectives, comments, letters or papers to this new section. Yours truly, James Blignaut Editor: SAJEMS 554 SAJEMS NS 9 (2006) No 4 MENTORSHIP ALLIANCE BETWEEN SOUTH AFRICAN FARMERS: IMPLICATIONS FOR SUSTAINABLE AGRICULTURE SECTOR REFORM OO Olubode-Awosola Department of Agricultural Economics, University of the Free State Department of Agricultural Economics, Obafemi Awolowo University HD Van Schalkwyk Faculty of Natural and Agricultural Sciences, University of the Free State Faculty of Natural and Agricultural Sciences, University of the Free State Abstract The South African government provides access to agricultural land for people not adequately represented in the agricultural sector. However, the government lacks sufficient funds and institutional infrastructure to provide post-settlement support to the settled developing farmers. A farmer-to-farmer mentorship programme between established and developing farm types has been identified as an institutional arrangement that could complement the government’s efforts. However, at this stage government and other role-players lack frameworks for this type of mentorship programme.This study conceptualises a complementary mentorship alliance that is loosely structured, without the complicated legal and contractual processes involved in corporate business alliances. This alliance will hopefully lead to highly committed joint ventures in the industry in the near future. The study also provides frameworks within which the role-players could contribute to the success of mentorship programme. Q JEL Q43 1 Introduction (AgriBEE) and Land Redistribution for Agricultural Development (LRAD). Market deregulation and trade liberalisation are also being implemented to make the industry more competitive. The purpose of land redistribution is to settle blacks as commercial farmers. The policy thrust is to empower previously disadvantaged people in a competitive and sustainable environment (Jooste, Van Schalkwyk & Groenewald, 2003; NDA, 1995, 2004a). Introduction South Africa’s agricultural productivity and industry structure are the result of a long history with respect to having or not having access to farm resources and experience in farming. The structure has political antecedents, because the previous government favoured production by large-scale, capitalist white farmers who used wage labour, mostly provided by blacks (NDA, 1995, 2004a, 2004b). The structure was thus characterised by two sectors namely the white commercial farmers and black subsistence farmers. At best, the reforms have resulted in the emergence of another dimension of a dual economy in the sector relating to the characteristics of the new entrant farmers. Compared to established commercial farmers, the emerging commercial farmers lack managerial and financial skills, capital assets, etc. Furthermore, the deregulated and liberalised market poses a threat to both farm types. There is increasing rate of farm sequestration among established commercial farmers and lack of However, the new government is committed to redressing this imbalance by means of what it calls agriculture-led Growth, Employment and Redistribution (GEAR) strategies. The strategies, within the agricultural sector, include Agricultural Black Economic Empowerment SAJEMS NS 9 (2006) No 4 555 conceptualises a mentorship alliance based on the principles of business alliance. It also highlights probable frameworks within which role-players can contribute to the success of the conceptualised alliance. This study is based on the premise that speeding up the pace of land reform can help to avoid land seizure and violent expropriation. Also, sustainable equity or wealth redistribution measures can promote stable political and socio-economic environments conducive for national economic efficiency. sustainability among emerging farmers (NDA, 2004a; Statistics South Africa, 2005). This may be attributed to the new challenges posed by trade liberalisation and market deregulation, among other things, in the economy. For example, Swanepoel and Stroebel (2004), in a study that evaluates the empowerment policies, strategies and performance within the agricultural sector of the Free State province, report that the Free State province has an articulate and intentional framework for empowerment. However, they note a number of problems that characterise such projects. Firstly, implementation is often insufficient. Second, there is a widespread lack of experienced officials to assist the settled farmers; available officials often lack understanding of essential concepts such as commercialisation, coordination, beneficiaries, mainstream economy, small farmer development, etc. Thirdly, many projects are small in scope, which could limit their impact and adaptation to the competitive industry. Introduction Lastly, monitoring and post-settlement training for the developing farmers are lacking. 2 Commercial, large-scale, white-owned farms dominated South Africa’s agricultural industry under the previous dispensation. These farms contributed about 95 per cent of value added and utilised about 87 per cent of the agricultural land in the country. This politically inspired economy was characterised by an acute lack of markets, capital and education among black agricultural producers in the so-called homelands. Consequently, most blacks were involved in subsistence farming using the remaining 13 per cent of agricultural land (Lipton, 1989; Brand, 1992; Bromberger & Antonie, 1993; World Bank, 1994; Percival & Homer-Dixon, 1995; Kirsten, 1998). While the government lacks sufficient funds and institutional arrangements to provide post- settlement support to emerging farmers, some authors (Darroch & Mashatola, 2003; Louw, Madewu, Jordaan & Vermeulen, 2004; Vink, 2004) identify mentorship programmes between farms of the two types as a viable institutional arrangement that can complement government’s reform efforts. However, recent government efforts to redress the imbalances have led to a new dichotomy, namely, between established and developing commercial farmers. Umhlaba Wethu (2005), an update on land and agrarian reform in South Africa, reports that at the end of that year a total of 3.1 million ha had been transferred through the various land reform programmes, of which 1.3 million ha (about 43 per cent) were transferred through the LRAD programme. The Ministry of Agricultural and Land Affairs hopes to transfer land at the rate of 2.2 million ha per year from 2006 to 2015 in order to reach the 30 per cent transfer target by 2014. However, at this stage, government and other role-players lack a framework for this type of mentorship programme. For example, the general belief among role-players is that this type of programme should be voluntary (NDA, 2005). Therefore, this study aims to contribute to a discussion of mentorship between established and emerging farmers by addressing this identified knowledge gap, namely the ways in which mentorship objectives, implementation and rewards may affect the sustainability of land reform projects. The aim of this study is to provide information that could help in designing an effective mentorship programme. Developing farmers are expected to operate at commercial levels, but they lack a significant proportion of the resources that would enable them to operate competitively. 2.1 Characteristics of established commercial farmers Most established commercial farmers are reported to have high management aptitudes, which, in turn, are reported to correlate positively with their farms’ general characteristics and resources. These characteristics and levels of resources include long histories of financial success, high turnover and economic viability, good socio-economic standing, and capital- intensive agricultural production and marketing facilities (Burger, 1971; Jansen, Swanepoel & Groenewald, 1972; Callow, Van Zyl, Von Back & Groenewald, 1991; Nel, Botha & Groenewald, 1998; Van Schalkwyk, Groenewald & Jooste, 2003). In addition, most of these farmers lack knowledge relating to the implementation of 1) production strategies, such as forward pricing of outputs, diversification of enterprises and land rental, 2) marketing strategies, such as the development of new markets, timing of access to markets, hedging of future contracts, forward contracting and spread of sales throughout the year, and 3) financial strategies, such as maintaining costs and credit reserves to meet unexpected cash flow difficulties, maintaining financial stability, etc. (DBSA, 1997; Brown, 2000; De Villiers, 2004). Reports from Statistics South Africa (2005) of the 2002 Census of Commercial Agriculture show that (i) the number of active farm units declined by about 27 per cent from 57,980 in 1993 census to 45,818 in 2002 and (ii) the percentage contribution of field crops to total income increased from 25.5 per cent in 1993 to 30.9 per cent in 2002. Likewise, the percentage contribution of horticultural products to total income increased from 24 per cent in 1993 to 26.7 per cent in 2002. However, the percentage contribution from animals and animal products decreased from 49.8 per cent in 1993 to 39.8 per cent in 2002. Farming debt value of about R31 billion was reported at a debt ratio of about 31.4 per cent (i.e. farming debt as percentage of market value of assets). More generally, the Abstract of Agricultural Statistics (2006) shows a persistent increase in total farming debt from 1970 to 2005. Also, questionable ethics and values and low levels of management capacity reported among emerging farmers influence their business practices, making it impossible for them to establish agricultural cooperatives among themselves; such cooperatives are a viable means of sharing risk in an industry that is characterised by risk and uncertainty (DBSA, 1997; NDA, 1995). 2 On a national level, the established commercial white farmers The study involves an investigation into a proposed mentorship programme and SAJEMS NS 9 (2006) No 4 556 such as land, market access and credit and management abilities (Makhura, Goode & Coetzee, 1998). They operate below competitive levels, probably because they lack experience and were confined to subsistence operation for a long period. Their constraints include inadequate technology and lack of entrepreneurial skills, marketing infrastructure and information. For example, studies by Gouse, Pray, Kirsten and Schimmelpfennig (2005) and Raney (2006) show that these farmers are not competitive in the agricultural input market because the adoption of insect-resistant white maize varieties by these farmers is constrained because they cannot afford the cost of the seeds. are represented by the South African Agricultural Union (Agri-SA) while the smallholder developing black farmers are represented by the National African Farmers’ Union (NAFU). The two farm types are characterised briefly in the next two subsections. 2.1 Characteristics of established commercial farmers For example, Tapela (2005), in a study of joint ventures of the Hereford Irrigation Schemes comprising emerging small-scale irrigation farmers, private investors and government, observes the failure of such ventures with evidence of decreasing farm income and increasing debt among the farmers. The study suggests that such ventures, in the context of Integrated Sustainable Rural Development Programme (ISRDP) and LRAD, may be faulty in concept and implementation. Problem statement In South Africa, the present government is committed to redressing imbalances in the farming industry. However, a number of problems may hinder the sustainability of economic reform. Firstly, from the mid-90s the South African government has expended huge amounts of money on the acquisition of land for previously disadvantaged people. However, not only are funds for providing land at the target rate limited, but also proper institutional arrangements have not been made to deliver supports to land reform beneficiaries (DBSA, 1997). Where such services are delivered, they are provided on an ad hoc basis and focused on smallholder emerging farmers. The effect of this support is, at best, marginal for emerging farmers, while established commercial farmers feel marginalised in a more liberal market (World Bank, 1994; Makhura, 1994; NDA, 1995, 2004a). 2.2 Characteristics of developing farmers Compared to established commercial farmers, many emerging farmers lack farm resources SAJEMS NS 9 (2006) No 4 557 3 Problem statement satisfy the needs of developing farmers; how should the mentorship programme operate and should mentors’ efforts be rewarded? Answers to these questions could help each stakeholder to contribute in a more significant way to the success of South African economic reform. SAJEMS NS 9 (2006) No 4 government is positive towards reform and minority political parties have indicated their willingness to join the government in the reconstruction of the nation (NewsHour, 2004). The government has identified a skills shortage in the agricultural sector and is in the process of promoting mentorship programmes, specifically by means of the National Skills Development Strategy 2005-2010 and the AgriBEE framework (NDA, 2005). Partnerships are necessary because of the competitive global economy, rapid produce cycles, capital constraints and advances in technology, which prevent a single firm from maintaining market share or expanding markets (Stanek, 2004). Sarkar, Echambadi, Cavusgil and Aulakh (2001) postulate that an alliance is necessary when the partners: i. have similar characteristics in certain dimensions; Though the situation in South Africa is complex, there have been a few cases of black workers and white managers sharing property and jointly managing farms. Some black settlers have also retained white farmers as managers after land transfer (NewsHour, 2004). Some commercial banks are in the process of requiring mentorship between an experienced commercial farmer and emerging farmers as a prerequisite for crop insurance and credit services to emerging farmers, as a means of reducing risk. Specifically, the Land Bank’s Social Discount Product promises commercial farmers lower interest rates on borrowing if they become involved in mentorship programmes (Gerry, 2003; NewsHour, 2004). ii. have different characteristics in different dimensions; iii. have different resource and capability profiles, and iv. share similarities in their social insti- tutions. Sarkar et al. (2001) further stress that, for the alliance to succeed, partners must pursue the objective simultaneously. The empirical results of the study by Sarkar et al. (2001) into the performance of alliances suggest that complementary resources and compatible cultural and operational norms between partners help to create values in alliances. If each partner is to pursue the partnership’s shared aims, its objectives must be clearly identified and an operational process established for the mentorship alliance. Examination of the conditions that make alliances necessary, the basic elements of business alliances and the characteristics differentiating the two groups of South African farmers, all discussed above, clearly suggests that mentorship alliance between the farm types could be a viable institutional arrangement that would complement the current government’s economic reform efforts. These events can be seen as signs of future success, not only in land reform but also the success and sustainability of its impact in South Africa. SAJEMS NS 9 (2006) No 4 Trends and opportunities could be investigated and further promoted by support for new farmers from institutions, government and public-private-partnership (PPP). This could also hasten land reform. 5 4 Problems and prospects of a mentorship programme One feasible institutional arrangement by which the economic reform efforts of the South African government could be complemented is a mentorship programme. However, a number of problems may prevent such mentorship programmes from occurring voluntarily as envisaged by the role-players. Firstly, most of the government’s strategies as embedded in the BEE framework, especially the AgriBEE schemes, are perceived by civil society to exclude and discriminate against commercial white farmers (NDA, 1995). Secondly, the perception that agriculture is becoming less profitable, while debt and insecurity are increasing and transformation is slow, may discourage emerging farmers from exploiting the mentorship of experienced colleagues in the industry. Thirdly, the perception that established agriculture is dominated by a racial group is detrimental to the potential of mentorship alliances. Related to this is an exaggerated sense of the threat of marginalisation and neglect among established farmers. This problem is evident in the stereotype that certain racial groups may not make good farmers (NDA, 1995; Brown, 2000). To improve farming skills among emerging farmers who find it difficult to cope with evolving and challenging production and marketing environments, a voluntary farmer-to-farmer mentorship programme between the two types of farmers has been initiated, and is being promoted by concerned stakeholders and the government. At this stage, the government is requesting frameworks for voluntary mentorship programmes (NDA, 2005). However, at a recent Senwes-organised workshop on BEE (held in April 2005) where mentorship programmes were considered, some speakers referred to the need for not only moral and political, but also economic and business imperatives in the South African economic policy reform if programmes and projects are to succeed. Specifically, the need to reward mentorship efforts was mentioned. Some individual prospective mentors indicated that they and the people they were mentoring were uncertain of mentorship objectives under the voluntary framework. However, despite these threats to the potential of a successful mentorship programme among South African farmers, prospects for success abound. These can be seen in the strengths of South African commercial farmers. Their many years of experience are worth exploiting in developing the skills of emerging farmers. Concerted efforts have been made by private stakeholders, banks, NAFU and business groups to work towards a mentorship programme between the two types of farm (Sandstone Agriculture News, 2004). Conceptual framework Business alliances come in various forms of partnership, which often involve cooperative or mutual agreements between two or more firms (Hill, 2005). Most partnerships are formed between firms to achieve corporate objectives (Morgenson & Harvey, 2002). The most appealing definition in the context of this paper is that of Dibb, Simkin, Pride and Farrell (2001), namely, a partnership that will transform South African agriculture into a more efficient and competitive sector in the global economy. 4 Problems and prospects of a mentorship programme Furthermore, the To this end, the objective of this exploratory and concept development study is to suggest what prospective mentors have to offer to 558 SAJEMS NS 9 (2006) No 4 6.2 Case study Telephonic interviews were used to conduct case studies of Senwes’ enlisted mentors. Case studies are particularly useful in studies with relatively few respondents. The key researcher introduced himself and the other colleague in the study to the respondents. The purposes of the study were explained to the respondents, and they were told how they were selected for the case study. Efforts were made to make the interviews as neutral and void of leading questions as possible. Empirical investigation This study examines present efforts and proposed mentorship programmes, using case study analysis and interviews with key informants, including prospective mentors and key role-players in the mentorship programme. Both the interviews and case studies deal with the objectives, implementation and possible rewards of mentorship. SAJEMS NS 9 (2006) No 4 559 informant interview and discussion was carried out with a Senwes agricultural services manager to determine his opinion about mentorship practices and obtain an overview of the proposed mentorship programme. The interviews are summarised in Boxes 1 and 2. The Free State is one of South Africa’s nine provinces and one of the country’s leading agricultural producers, with considerable agricultural resources (Swanepoel & Stroebel, 2004; NDA, 2004a). An overview of the country shows evidence of mentorship in the Free State Province only; this mentorship is however limited and in the pilot stage at the time of this study. Box 1 Key Informant 1 6.1 Pilot and proposed mentorship programme The key informant interview is a useful tool for exploratory surveys and for conducting socio- economic inventories of resource availability and management in farming communities. The technique has the advantage of being relatively rapid and inexpensive (Dvorak & Izac, 1996). Key informant interviews and discussions were used in this study to gather information about clarity of objectives, implementation and possibility of reward in the mentorship programmes proposed by the government and private initiatives. One farmer interviewed claimed he had not officially assumed the responsibility of a mentor. He considered himself to be merely assisting neighbouring emerging farmers. Two farmers had given Senwes an indication of interest in the mentorship programme, but were not yet linked to any emerging farmer as mentor. The last farmer, however, is engaged with a group of emerging farmers and so his experience was thoroughly interviewed. This interview is summarised in Box 3. An experienced extension agent, who is also a project manager, was interviewed as a key informant to provide information on the government’s strategic plan for mentorship. Senwes is a private organization that acts as role-player and service provider in offering support to settled farmers, and so another key Box 1 Summary of the interview with a key informant from the government Key Informant 1 There was no government organised mentorship programme in place when this research was conducted. Instead, non-formal training programmes were operating, in which government subject-matter specialists and extension agents dispense their services to farmers in general. In the Province and at national level, however, government is in the process of developing a farmer-to- farmer mentorship programme package as part of the strategic plan for agriculture for the years 2004-2006. This plan has not been finalised, but the objective of the farmer-to-farmer mentorship programme is to establish a link between emerging commercial and established commercial farmers for the purpose of skills transfer from the latter to the former. The programme will be coordinated at district level within each province. Government will identify a group of emerging commercial farmers that can be linked to an established and experienced commercial farmer, with whom they can share technical experience. A study group will be organised by the farmers to share knowledge about the SAJEMS NS 9 (2006) No 4 560 economics of farming. The farmers will be expected to visit one another’s farms to share practical experience and farm demonstrations. However, the criteria for selecting a mentor farmer are still being debated among policy makers, as some believe that retired commercial farmers should be engaged in the process. economics of farming. The farmers will be expected to visit one another’s farms to share practical experience and farm demonstrations. However, the criteria for selecting a mentor farmer are still being debated among policy makers, as some believe that retired commercial farmers should be engaged in the process. The intention is to link about 27 emerging farmers to a mentor farmer. The means to evaluate progress have not yet been established in the government plan. However, it is believed that the rate of increase in the number of emerging farmers linked to mentors and their rates of success could be an indication of progress. The programme currently being designed is expected to be voluntary and reward is expected to be recognition of the mentors by the government at annual speeches by ministers or other government officials. However, for this arrangement to work, government hopes that mentors will cooperate by their willingness to share time and experience with emerging farmers. Emerging farmers are expected to respond to and avail themselves of the opportunity; the Departments of Agriculture are expected to update policy relevancy, implementation and dissemination of information. Key Informant 1 The banks are not yet considered in the plan. The Departments of Land Affairs are expected to disseminate information about new settlers as soon as possible for immediate linking to mentor farmers. Cooperatives are expected to develop among farmers, to help in the referral and linking of farmers to the mentorship programme. This key informant believes that farmers should be linked without undermining the roles of extension agents and that mentorship linking is only necessary when there are insufficient extension agents available to help emerging farmers. Source: Authors’ field survey, 2005 Source: Authors’ field survey, 2005 Box 2 Summary of the interview with a key informant from SENWES Case-Study: De Boer* Mr De Boer is a 32-year-old professional commercial farmer and the chairperson of the Young Farmers Association in a district of the Free State. He grew up in a farm household. He has about nine years of full–time experience in commercial farming on about 1500 ha, of which about 350 ha is grazing land. He has also spent about 11 years acquiring academic qualifications in agriculture, specifically agricultural economics. He is engaged as a volunteer in the Senwes organized mentorship programme. Presently he assists a group of 28 LRAD, commonage emerging farmers who have about 150 ha of farmland. He attends to these farmers on a weekly basis, either on his farm or in a designated centre, sharing with them his entrepreneurial, farming and risk management skills and practices. These emerging farmers have high expectations of De Boer as regards information relating to marketing opportunities and agronomy. De Boer enjoys the mentorship programme with these farmers, yet he expects a measure of reward for the time he spends helping them. He expects this reward in the form of a share of the profit accrued to these farmers’ projects, or in the form of an input subsidy from the government. De Boer mentions a number of problems that have prevented his mentorship efforts from yielding tangible fruit or encouraging other commercial farmers to become involved in the programme. One is that the emerging farmers do not have operating cash. They only keep a few cattle on their farmland. De Boer has helped them to develop business plans for growing maize, wheat, etc., with which they can secure loans from commercial banks. However, the banks could not approve these applications because the land was not registered in the farmers’ names but was sublet to them by the original LRAD beneficiaries. In spite of this experience, De Boer is positive about the mentorship programme and he feels that other commercial farmers would be very willing to become involved. However, the general problem is that the commonage arrangement does not encourage business attitudes in the settlers. In some cases, the land area is too small for the number of settlers, preventing each member of the commonage from having an economic unit of production. To ameliorate some of these problems, De Boer expects the government to develop a viable and business-oriented land transfer programme for effective mentorship. Note: De Boer is a pseudonym used to ensure anonymity and confidentiality Key Informant 2 Senwes is also in the process of developing a strategic plan for a mentorship programme. Senwes has what could be categorised as an experimental mentorship programme that is currently operating mainly in the Free State Province. In its plan, Senwes hopes to attach mentors to a group of emerging farmers for the purpose of transferring technical and management skills from the latter to the former. It is expected that, for cash crop enterprises, the group will meet on a seasonal basis. However, for livestock enterprises, the group is expected to meet annually as most livestock enterprises have an average life cycle of at least one year. g y y Mutual understanding between the farmers, especially regarding diverse cultural and business practices, is deemed a prerequisite for the success of the programme. A group of not more than ten farmers making up a household or commonage is expected to be attached to a mentor. However, where emerging farmers represent individual farm units, a group of at most eight emerging farmers to a mentor is ideal. Monitoring of progress and dispute resolution is expected to be carried out by a third party, namely Senwes. Stakeholders such as banks are expected to approve more of the applications for operating loans from emerging farmers who are linked to a mentor, as mentorship is expected to reduce the risk of the enterprise. It is also expected that the Provincial Department of Agriculture will clarify the role of mentors to the extension agents, to avoid misrepresentation or conflict of advice given by extension agents and mentors respectively. Reward for mentoring is at the discretion of the mentors and emerging farmers. However, it is expected that transportation costs associated with mentoring exercises will be covered, but who will pay them is an issue still to be resolved. This key informant SAJEMS NS 9 (2006) No 4 561 observes failures in the arrangement so far which could be related to difficulties associated with the identification of suitable mentors and the time that this involves. Presently there are about four mentors in the Senwes mentorship programme list. These are all experienced farmers who have experience and knowledge of mentorship. These farmers were interviewed and the results are reported in the next section. Source: Authors’ field survey, 2005 Source: Authors’ field survey, 2005 Note: De Boer is a pseudonym used to ensure anonymity and confidentiality 7.1 Complementary mentorship alliance Analysing the case study and key informant studies suggests that mentorship alliances between the farm types may not be as voluntary as government or other role-players propose. Rather, the mentorship conceptualised in this study is a complementary mentorship alliance, which is expected to be loosely structured without the legal and contractual processes involved in corporate business alliances. The alliance is expected to be simple but to involve three of the key elements of a successful partnership, namely identifying clear objectives, establishing an operational process and establishing a measure of reward. Prospective mentors might be persuaded to view mentorship as good neighbourliness; for example, a prospective mentor could tell his neighbouring farmer how to set his planter correctly. On the other hand, it may also be worthwhile conscientising the developing farmers about making use of such opportunities, should they arise. They should be persuaded to learn as much as possible from successful neighbouring farmers. Social events such as Farmers’ Day should be supported and organised more frequently by the government and other public service providers. At such events, prospective full-time mentors could be more easily identified, which would enable the government to implement a more formalised arrangement. The established commercial farms could provide complementary mentorship to the developing farms, in the form of addressing specific areas where both farms experience the same strengths and weaknesses. By doing this, the established farms would not only complement the developing farms’ contribution to the industry but also strengthen the industry’s productivity and thus the nation’s competitiveness in the global economy. The mentorship alliance will hopefully form a foundation for highly committed joint ventures in the industry in the future. While this framework may lack a strong economic base, it suffices to stress that sustained national economic efficiency depends on equity. If equity is not consistently pursued, successes may not be sustainable. Case-Study: De Boer* He believes this will encourage banks to grant operating capital to emerging farmers, especially if the farms are of commercial size and individual farmers can be identified, rather than the communal arrangement of land ownership. Note: *De Boer is a pseudonym used to ensure anonymity and confidentiality 562 SAJEMS NS 9 (2006) No 4 7 The conceptualised mentorship alliance and the frameworks In addition, the impact of BEE places further pressure on established commercial farmers who need moral persuasion not only to cooperate with government’s BEE initiatives but also to sacrifice some resources to complement the government’s economic reform efforts. Although the case studies conducted in this study are few in number, they do seem to indicate that, at farm level, a number of farmers may be willing to mentor developing farmers. 7.2.2 Changes in social structure and societal attitude Much more importantly, if an alliance is to be achieved, an enabling environment and forum must be created. Such environments are highlighted under different possible frameworks including that which allows spontaneous formation of partnerships. Inductive examination of the possible problems and prospects of the mentorship programme may suggest that more than policy reforms is required for restructuring the farm industry and achieving competitiveness, especially in the South African context. Changes are needed in social structure and societal attitudes because these concepts form ideological barriers to the successful implementation of policy reforms. 7.2.4 Mentorship within broad-based BEE 7.2.4 Mentorship within broad-based BEE As commercial farmers still struggle with BEE because of lack of knowledge about correct procedures, mentorship alliances with developing farmers could be a means of scoring in the broad-based BEE measurement. The broad-based BEE gives more flexibility to commercial farmers to contribute to BEE, not only by offering to sell pieces of land to settled developing farmers but also by empowering developing farmers in a number of ways such as preferential procurements, training and capacity development. Established commercial farmers can therefore become more aware of their lack of BEE status, which could motivate them to be involved in a mentorship alliance to improve their BEE status and scores. Such an enabling environment and forum for farmers’ identification and for fair play of market forces could be created by the three main role-players namely, Agri-SA, NAFU and the Department of Agriculture. Other stakeholders and groups, such as non-governmental organisations (NGOs), community-based organisations (CBOs), and the media, could also create such enabling environments. Forums could include enterprise- specific intra- and inter-cooperatives, unions and associations, farm exhibitions, seminars, workshops, etc which would bring South African farmers together. The forums should avoid discrimination on the base of political or racial class and differentiation according to size of business operation, because these would further widen the gap between prospective mentors and developing farmers. 7.2.1 Moral persuasion The prospects for mentorship among farmers must be encouraged and exploited. To match the extensive management skills and many years of experience among established commercial farmers with the latent demand for such experience among the developing farmers The South African economic reform measures, especially trade liberalisation and market deregulation, put all farmers in a position where each farmer has had to adapt in order to reach or maintain a strong position in the economy. SAJEMS NS 9 (2006) No 4 563 third-party intermediation or transfer payment is that, if the government or another public service provider is obliged to reward mentorship, developing farmers may demand too many services and mentors too many rewards, thereby increasing public transfer payments at a lower efficiency level. an environment and forum should be created to allow the two farm types to identify with each other. This identification will specifically address the needs of developing farmers by utilising appropriate mentors thereby making the objectives of the mentorship programme comprehensive. This should also eliminate problems for the government and role-players in identifying the right mentor for the right developing farmers. Identification between mentors and developing farmers could also give rise to a market- determined reward system for mentorship, which would encourage both types of farmer to commit themselves to the mentorship alliance. If there is a reward for mentors proportional to demand for and supply of such mentorship, this could lead to measurable progress in mentorship programmes, which could translate to progress in reform. Therefore, it is proposed that government or other public service providers should commit resources to the mentorship programme, but that the objective should be not only equity in terms of land acquisition but also in terms of efficiency and productivity improvement. Thus, mentors and developing farmers should be allowed to identify each other in a market-driven manner. It may be sufficient to simply promote open communication between commercial farmers, developing farmers and the government. 7.3 Conclusion and policy recommendations A mentorship programme between the two farm types has been identified as a means through which the stakeholders in the South African farm industry can complement the government’s economic reform efforts. Such a programme can also help to alleviate some of the problems and challenges created by reform, which affect not only farmers but also the nation as a whole. This study offers conceptual frameworks for effective mentorship programmes. 7.2.3 Mentorship alliance with limited public service 7.2.3 Mentorship alliance with limited public service A paradox in the theory of public investment is that projects, which most economists agree ought to be public, usually fail (Fisher, 1995). Therefore, the mentorship conceptualised in this study should have limited intermediary or third-party intervention such as government or other public service provider intermediation or transfer payment. The potential problem in SAJEMS NS 9 (2006) No 4 564 The study empirically examines pilot mentorship programmes and confirms that knowledge and consensus currently tend to be lacking among Free State farmers about the objectives, implementation and rewards for mentorship. A theoretical perspective for enhancing the mentorship programme between emerging and established farmers was developed. The mentorship is expected to be complementary and loosely structured, without the complicated legal and contractual processes involved in corporate business alliances. However, it is hoped that the alliance would be a precursor for highly committed joint ventures in the industry. and inter-cooperatives, unions and associations, farm exhibitions, seminars and workshops, as mentioned above, which could help bring South African farmers together. The forums should avoid discrimination, and producers or traders associations that are multi-racial and multi- cultural should be encouraged. Therefore, a successful mentorship programme needs not only social and moral imperatives but also an economic imperative. Farmers’ confidence in the South African farm industry must also be maintained. The government needs to work at maintaining this confidence, and established commercial farmers at finding a good position in this transformation process. To enhance this mentorship alliance, a number of frameworks could be explored to provide enabling environments and forums for this type of alliance. This could encourage relationships and collaboration between established and emerging farmers, thereby creating spontaneous and market-driven mentoring relationships. South Africa’s previous agricultural economy was characterised by high efficiency but a lack of equity. The present government’s efforts could lead to “equity of possession” i.e. land acquisition, which may reduce regional and national economic efficiencies. However, extending equity beyond this “equity of possession” to incorporate equity of efficiency development i.e. equity of productivity between farm types, will increase both regional and national economic efficiencies. This in turn may increase business rivalry and formalised contractual alliances, which will further improve efficiency. When this stage is reached, any shock to the national economic efficiency should hopefully not stem from equity or political issues but from macro-economic variables which will increase efficiency. 7.2.3 Mentorship alliance with limited public service This kind of environment and forum will enable emerging and established farmers to identify themselves and the need for mentorship. This identification will specifically address the needs of emerging farmers by utilising appropriate mentors, thereby making the objectives of the mentorship programme comprehensive. This will also eliminate problems for the government and role-players in identifying the right mentor for the right emerging farmer. Identification between mentors and emerging farmers could also give rise to a market-determined reward system for mentorship, encouraging both types of farmer to commit themselves to the mentorship alliance. 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https://openalex.org/W4286716110	https://www.frontiersin.org/articles/10.3389/fpls.2022.901782/pdf	English	null	Strigolactone alleviates the salinity-alkalinity stress of Malus hupehensis seedlings	Frontiers in plant science	2,022	cc-by	9,933	Strigolactone alleviates the salinity-alkalinity stress of Malus hupehensis seedlings OPEN ACCESS EDITED BY Pasqualina Woodrow, University of Campania Luigi Vanvitelli, Italy REVIEWED BY Xin Li, Tea Research Institute (CAAS), China Ayman EL Sabagh, Siirt University, Turkey CORRESPONDENCE Xiaodong Zheng zheng.xiao.d@163.com †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science RECEIVED 22 March 2022 ACCEPTED 04 July 2022 PUBLISHED 22 July 2022 CITATION Ma C, Bian C, Liu W, Sun Z, Xi X, Guo D, Liu X, Tian Y, Wang C and Zheng X (2022) Strigolactone alleviates the salinity-alkalinity stress of Malus hupehensis seedlings. Front. Plant Sci. 13:901782. doi: 10.3389/fpls.2022.901782 OPEN ACCESS EDITED BY Pasqualina Woodrow, University of Campania Luigi Vanvitelli, Italy REVIEWED BY Xin Li, Tea Research Institute (CAAS), China Ayman EL Sabagh, Siirt University, Turkey CORRESPONDENCE Xiaodong Zheng zheng.xiao.d@163.com †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science RECEIVED 22 March 2022 ACCEPTED 04 July 2022 PUBLISHED 22 July 2022 CITATION Ma C, Bian C, Liu W, Sun Z, Xi X, Guo D, Liu X, Tian Y, Wang C and Zheng X (2022) Strigolactone alleviates the salinity-alkalinity stress of Malus hupehensis seedlings. Front. Plant Sci. 13:901782. doi: 10.3389/fpls.2022.901782 OPEN ACCESS EDITED BY Pasqualina Woodrow, University of Campania Luigi Vanvitelli, Italy Changqing Ma1,2†, Chuanjie Bian1,2†, Wenjie Liu1,2, Zhijuan Sun3, Xiangli Xi1,2, Dianming Guo1,2, Xiaoli Liu1,2, Yike Tian1,2, Caihong Wang1,2 and Xiaodong Zheng1,2* 1College of Horticulture, Qingdao Agricultural University, Qingdao, China, 2Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province, Qingdao, China, 3College of Life Science, Qingdao Agricultural University, Qingdao, China Salinity-alkalinity stress can remarkably affect the growth and yield of apple. Strigolactone (SL) is a class of carotenoid-derived compounds that functions in stress tolerance. However, the effects and mechanism of exogenous SL on the salinity-alkalinity tolerance of apple seedlings remain unclear. Here, we assessed the effect of SL on the salinity-alkalinity stress response of Malus hupehensis seedlings. Results showed that treatment with 100 µM exogenous SL analog (GR24) could effectively alleviate salinity-alkalinity stress with higher chlorophyll content and photosynthetic rate than the apple seedlings without GR24 treatment. The mechanism was also explored: First, exogenous GR24 regulated the expression of Na+/K+ transporter genes and decreased the ratio of Na+/K+ in the cytoplasm to maintain ion homeostasis. Strigolactone alleviates the salinity-alkalinity stress of Malus hupehensis seedlings Second, exogenous GR24 increased the enzyme activities of superoxide, peroxidase and catalase, thereby eliminating reactive oxygen species production. Third, exogenous GR24 alleviated the high pH stress by regulating the expression of H+-ATPase genes and inducing the production of organic acid. Last, exogenous GR24 application increased endogenous acetic acid, abscisic acid, zeatin riboside, and GA3 contents for co-responding to salinity-alkalinity stress indirectly. This study will provide important theoretical basis for analyzing the mechanism of exogenous GR24 in improving salinity-alkalinity tolerance of apple. COPYRIGHT © 2022 Ma, Bian, Liu, Sun, Xi, Guo, Liu, Tian, Wang and Zheng. 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. Malus hupehensis, strigolactone, salinity-alkalinity stress, ion homeostasis, oxidative stress Malus hupehensis, strigolactone, salinity-alkalinity stress, ion homeostasis, oxidative stress KEYWORDS TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fpls.2022.901782 TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fpls.2022.901782 TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fpls.2022.901782 Introduction Soil salinization-alkalization is a severe environmental factor that inhibits plant growth and productivity for aggravating soil degradation (Jia et al., 2019). To date, 20% of agricultural land is aﬀected by salinity-alkalinity all over the world, and the trend is constantly expanding (Ye et al., 2019). Apple (Malus domestica) is a highly valued and widely cultivated fruit around the world (Ma et al., 2019). Apple trees are sensitive to saline-alkali conditions and negatively aﬀected by soil Frontiers in Plant Science Frontiers in Plant Science 01 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 salinization-alkalization. Thus, strategies for improving the salinity-alkalinity tolerance of apple trees should be explored. rice (Ling et al., 2020). Moreover, exogenous GR24 application protects the chlorophyll and maintains the photosynthetic rate of apple seedlings under KCl stress (Zheng et al., 2020). In addition, exogenous GR24 can improve the cold and drought resistance of rape seedlings by improving cell viability and inhibiting the production of reactive oxygen species (Zhang X. et al., 2020; Wang et al., 2021). Therefore, we hypothesized that SL might play positive roles on salinity- alkalinity stress in apple seedlings. However, the mechanisms and functions of GR24 under salinity-alkalinity stress in apple remain unknown. Salinity-alkalinity stress simultaneously induces oxidative, high pH, osmotic, and ionic stress (Guo et al., 2017; Jin et al., 2019). Reactive oxygen species (ROS) induced by saline-alkali stress, including superoxide anions (O2−), hydrogen peroxide (H2O2), and singlet oxygen, result in oxidative stress that can lead to plant cell membrane permeability increasing and ion leakage, which may impede plant development (Miller et al., 2010; Zhang et al., 2016; Xu et al., 2021). High pH aﬀects the availability of mineral elements and the absorption of inorganic anions, thus disrupting intracellular ion balance (Yang et al., 2009). Osmotic stress reduces the stomatal openings and decreases plant photosynthesis (Zhang et al., 2019). Moreover, the uptake of K, Mg, and Zn in apple leaves is inhibited, whereas the absorption of Fe, Cu, or Mn is increased under saline-alkali stress (Jia et al., 2019). In the present study, we explored the functions of exogenous GR24 in Malus hupehensis, one of the important rootstocks in apple culture, under salinity-alkalinity stress. Diﬀerent concentrations of exogenous GR24 were applied on M. hupehensis seedlings under salinity-alkalinity stress, the positive regulation of GR24 was evaluated in terms of the photosynthetic system, oxidative damage, osmotic balance, and ion homeostasis. Plant materials and growth conditions After low-temperature vernalization, the seeds of M. hupehensis (an apple rootstock with apomixis characteristics) were sown in nutrient soil and grown in a greenhouse under controlled temperature (25 ± 2◦C), photoperiod (16/8 h day/night), humidity (60–65%), and light intensity (100 µmol/m2/s). After one-month-old, when the seedlings developed into to four leaves, they were transplanted into a plastic pot and irrigated with Hoagland solution every 3 days. Ten days later, seedlings with similar growth status were selected for subsequent saline-alkali stress and exogenous GR24 treatment. Plant hormones play major roles in regulating plant growth and tolerance to abiotic stress. Strigolactone (SL), as a class of carotenoid-derived compounds, is essential in regulating numerous aspects of plant development (Duan et al., 2019). The enzymes involved in the SL signaling pathway include ubiquitin- related protein F-box leucine-rich repeat protein (D3/MAX2), SL receptor α/β hydrolyzyme (D14), and transcriptional repressor Clp ATPase family protein (D53/SMXL6/7/8) (Yao et al., 2016; Shabek et al., 2018). The exogenous application of GR24, a synthesized SL, signiﬁcantly increases the enzymatic activities of SOD and POD, decreases the malondialdehyde (MDA) content, and mitigate the adverse eﬀects of salt stress in Introduction The expression levels of ion transporter genes, key SL signaling pathway genes, and SL biosynthesis genes under GR24 treatments were also determined. This study helped clarify the regulatory mechanism of SL in apple plants under salinity-alkalinity stress and provided a new way to improve salinity-alkalinity tolerance in apple production. Plants resist external pressure via several biochemical reaction mechanisms, redox balance, and complex signal transduction pathways throughout their long-term evolutionary process (Jiang et al., 2016; Xu et al., 2021). Plants regulate the osmotic potential by increasing the concentrations of proline, soluble protein, and soluble sugar. Furthermore, multiple elements such as Ca, K, and Fe are involved in photosynthesis, carbon assimilation, and signal transduction in plants (Yang and Guo, 2018). Maintaining a low Na+/K+ ratio is an important mechanism for preventing cellular damage and nutrient deﬁciency in plant (Zhang et al., 2018). In addition, plant hormones, such as auxin (IAA), jasmonic acid (JA), cytokinin, and gibberellin (GA) are important for regulating plant development and tolerance to diverse stresses (Zwack and Rashotte, 2015). The application of plant growth regulators can eﬀectively improve plant salt tolerance (Shahzad et al., 2018; Jiang et al., 2021). Some metabolites such as spermidine and γ-aminobutyric acid improve plant salinity-alkalinity tolerance by scavenging ROS and regulating cellular osmotic pressure (Li et al., 2015; Jin et al., 2019). Plants can activate the antioxidant enzyme activities, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) cooperate together to scavenge ROS and protect plants from oxidative harm (Zheng et al., 2020). Frontiers in Plant Science Quantiﬁcation of mineral elements assay The apple seedlings were collected and washed with deionized water to remove the excess impurities after 15 days of saline-alkali stress and GR24 treatment. The leaves were dehydrated at 105◦C for 30 min and baked at 80◦C for 72 h. Afterward, 0.5 g of kiln-dried leaves was ground into powder and added with 12 ml of HNO3 and HClO4 with the ratio of 5:1. After digestion, the solution was diluted with deionized water to 25 ml. The concentrations of sodium (Na), potassium (K), calcium (Ca), iron (Fe), magnesium (Mg), and phosphorus (P) were determined through inductively coupled plasma-optical emission spectrometry (PerkinElmer, Waltham, United States) as described by Su et al. (2020). Saline-alkali stress and exogenous GR24 treatment assay A total of 200 M. hupehensis seedlings were randomly divided into ﬁve groups. The seedlings in group I were watered with a complete nutrient solution as the control, group II were treated by 100 mM NaHCO3 and NaCl with concentration ratio of 1:1. On the basic of group II, groups III-V were treated with the 10, 100, and 1,000 µM of exogenous GR24, respectively. Frontiers in Plant Science 02 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 Biotechnology, Suzhou, china), respectively. Each experiment was repeated thrice. GR24 (Solarbio, Beijing, China) was sprayed every 3 days. After 15 days of treatment, the seedlings were photographed, and the wilting rate, fresh weight, and dry weight were measured. Both the technical and biological duplications of each experiment were repeated thrice. Measurement of endogenous hormone After saline-alkali and exogenous GR24 treatment for 15 days, the endogenous acetic acid (IAA), gibberellin3 (GA3), zeatin riboside (ZR), and jasmonic acid (JA) concentrations were determined. Fresh leaves (0.5 g) were prepared for phytohormone extractions, and hormonal analysis and quantiﬁcation were performed via electrospray ionization- high-performance liquid chromatography-tandem mass spectrometry, as described by Min et al. (2018). Each experiment was repeated thrice. Measurement of chlorophyll content, photosynthetic parameters, and root activity After saline-alkali and exogenous GR24 treatment for 15 days, fresh leaves (0.5 g) from each group were used for the detection of electrolyte leakage and osmolytes. Electrolyte leakage was measured as described by Ahmad et al. (2016). Osmolytes including proline, soluble sugar, and soluble protein were detected. Proline content was measured as described by Wani et al. (2017). Soluble sugar and soluble protein contents were determined as described by Sharma et al. (2019) and Qiu et al. (2019) respectively. Each experiment was repeated thrice. After 15 days of saline-alkali stress and exogenous GR24 treatment, 20 apple seedlings from each group were randomly selected to determine the chlorophyll content and basic photosynthetic parameters. Four leaves of each seedlings were measured. Under light condition, the chlorophyll content was measured using SPAD-502 Plus (Konica Minolta, Tokyo, Japan). The photosynthesis rate, transpiration rate, and stomatal conductance were measured using the CIRAS-3 portable photosynthetic system (PP Systems, Amesbury, United States). The light intensity was controlled at 800 µmol/m2/s at an approximately 50% humidity, and the temperature was set at 22◦C. 2,3,5-triphenyltetrazolium chloride (TTC) method was applied for qualitatively and quantitatively assess the root activity according to Gong et al. (2017). Each experiment was repeated thrice. Determination of antioxidant enzyme activity and organic acid content Fresh leaves (0.5 g) were ground in 5 ml of extracted buﬀer after saline-alkali and exogenous GR24 treatment for 15 days. After centrifugation at 12,000 rpm for 10 min, the supernatants were immediately used for SOD, POD, and CAT content assay. SOD, POD, and CAT kits (Grace, Suzhou, China) were used to detect the activities of antioxidant enzymes according to the manufacturer’s instructions. The malic acid and citric acid content of apple leaves were measured using Malic acid assay Kit and Citric acid assay Kit (Suzhou Geruisi Determination of reactive oxygen species levels and malondialdehyde content Nitroblue tetrazolium and 3,3-diaminobenzidine were used to stain H2O2 and O2−, respectively. The H2O2 level was measured using H2O2 kits (Grace, Suzhou, China). The MDA content of the leaves was measured using a plant MDA extraction kit (Grace, Suzhou, China). Three biological duplications for each experiment were set. Frontiers in Plant Science Real-time quantitative PCR assay Total RNA was extracted from each group by using the RNA prep pure Plant Plus kit (Tiangen, Beijing, China), Frontiers in Plant Science 03 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 Effects of exogenous GR24 on the growth of apple seedlings under saline-alkali stress The apple seedlings were seriously damaged by saline- alkali stress, and the leaves became withered and chlorotic after 15 days. After the treatment of low (10 µM) and high (1 mM) concentrations of GR24, the growth vigor of the seedlings was much better than those without GR24 treatment, but the leaves remained withered and chlorotic. At the low (10 µM) and the high (1 mM) concentrations, the wilting rates of the seedlings substantially decreased from 73.3% to 45% and 48.3%, respectively (Supplementary Figure 1B), and the fresh weights remarkably increased to 0.34 and 0.36 g, respectively (Supplementary Figure 1C). However, when the middle concentration of GR24 (100 µM) was applied, the growth vigor of the seedlings under saline- alkali stress was similar to that of the control under normal conditions, and the wilting rate of the seedlings remarkably decreased to 13.3% compared with those without GR24 treatment under saline-alkali stress (Supplementary Figure 1B). In addition, under saline-alkali stress, the fresh and dry weights of the seedlings sprayed with 100 µM GR24 increased signiﬁcantly compared with that without exogenous GR24 (Figures 1C,D). The result suggested that exogenous GR24 could protect the apple seedlings from saline-alkali stress, and the concentration of 100 µM GR24 exhibited the best eﬀect, which was therefore selected for further research. Effects of exogenous GR24 on the chlorophyll content and photosynthetic parameters under saline-alkali stress which includes RNase-free DNase treatment. Total RNA was adjusted to the same concentration for cDNA synthesis by using 5 × All-In-One RT MasterMix (ABM, Sydney, Australia) according to the manufacturer’s instructions. LightCycler R⃝480 II system (Roche, Rotkreuz, Switzerland) was used for the qPCR assay, and the primers are listed in Supplementary Table 1. The M. hupehensis actin gene (GenBank accession number GQ339778.1) was used to normalize gene expression levels. Data were analyzed using the 2−11Ct method (Min et al., 2018). All qRT-PCR experiments were repeated thrice. Exogenous GR24 could prevent the chlorosis of the apple seedlings under saline-alkali stress (Figure 1). To explore the physiological mechanism, we determined the chlorophyll content and photosynthetic parameters after saline-alkali stress and GR24 treatment for 15 days. The chlorophyll content of the apple seedlings sharply decreased from 44.0 SPAD to 26.1 SPAD under saline-alkali stress. When exogenous GR24 was applied, the chlorophyll content of apple seedlings under saline-alkali stress remarkably increased to 34.5 SPAD (Figure 2A). The photosynthetic parameters, including photosynthesis rate, transpiration rate, and stomatic conductance, under saline-alkali stress and exogenous GR24 treatment followed a similar variation tendency as the chlorophyll content. All values were substantially inhibited under saline-alkali stress but increased after exogenous GR24 application (Figures 2B–D), especially the photosynthesis rate. Under saline-alkali stress, the photosynthesis rate decreased signiﬁcantly from 16 µmol/m2/s to 4 µmol/m2/s but recovered to 11.5 µmol/m2/s when exogenous GR24 was applied (Figure 2B). Therefore, exogenous GR24 could protect the chlorophyll level and photosynthetic system against saline- alkali stress. Statistical analysis Data were subjected to ANOVA followed by Fisher’s LSD or Student’s t-test analysis. Statistically signiﬁcant diﬀerences were indicated by P < 0.05. Statistical computations were conducted by using SPSS software (IBM, Armonk, NY, United States). Effects of exogenous GR24 on the oxidative damage and antioxidant enzyme activity of apple seedlings under saline-alkali stress Plants produce ROS under stress conditions. The staining results of superoxide (O2−) and H2O2 revealed that the leaves of apple seedlings were seriously damaged by saline-alkali stress (Figure 3A). When exogenous GR24 was sprayed, the O2− and H2O2 levels remarkably decreased (Figures 3A–C). The variation tendency of the MDA content was similar to that of O2−and H2O2. The MDA content under saline-alkali stress (2.6 nmol/g) was more than 1.6 times that of the control group (1.6 nmol/g), but was signiﬁcantly decreased to 2 nmol/g after exogenous GR24 was applied (Figure 3D). The activities of antioxidant enzymes were also measured. Under saline-alkali stress, the SOD activity decreased from 5.1 U/g to 3.8 U/g, but recovered to 4.4 U/g after exogenous GR24 was applied (Figure 3E). The POD activity under saline-alkali stress signiﬁcantly decreased from 259.0 U/g to 110.0 U/g. However, when exogenous GR24 was applied, the POD activity recovered to 203.7 U/g (Figure 3F). CAT activity was only 1572.0 U/g under saline-alkali stress, while Frontiers in Plant Science 04 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 FIGURE 1 Phenotypes of Malus hupehensis seedlings treated with salinity-alkalinity stress and exogenous 100 µM GR24 on day 0 and day 15 (A). Effect of GR24 on wilting rate (B), fresh weight (C), and dry weight (D) of apple seedlings after salinity-alkalinity stress for 15 days. The bar (A) represents 4.0 cm. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 1 Phenotypes of Malus hupehensis seedlings treated with salinity-alkalinity stress and exogenous 100 µM GR24 on day 0 and day 15 (A). Effect of GR24 on wilting rate (B), fresh weight (C), and dry weight (D) of apple seedlings after salinity-alkalinity stress for 15 days. The bar (A) represents 4.0 cm. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). 14.7 mg/g to 8.6 mg/g when exogenous GR24 was applied (Figure 4D). increased to 2515.7 U/g when exogenous GR24 was applied (Figure 3G). Frontiers in Plant Science Effects of exogenous GR24 on the root activity and organic acid contents of apple seedlings under saline-alkali stress increased from 4.4 mg/g to 5.6 mg/g under saline-alkali stress but that had no signiﬁcant changes when exogenous GR24 was applied (Figure 5D). The variation tendencies of Fe and Mg were similar. The Fe and Mg contents of apple seedlings with exogenous GR24 treatment increased to 0.305 mg/g and 1.472 mg/g respectively, compared with that without GR24 treatment under saline-alkali stress (Figures 5E,G). However, the P content did not change substantially under saline-alkali stress and exogenous GR24 treatment (Figure 5F). Triphenyl tetrazolium chloride is a REDOX compound, which is commonly used as the receptor of H+ for the alysis of the activity of diﬀerent enzymes. Deep red color reported the highest content of H+. After 15 days of saline-alkali stress, the root tips of apple seedlings with exogenous GR24 treatment were darker red than those without GR24 treatment under saline-alkali stress (Figure 7A). The TTC reductive intensity in apple roots decreased from 0.29 mg/g FW/h to 0.20 mg/g FW/h under saline-alkali stress. When exogenous GR24 was sprayed, the TTC reductive intensity in apple roots increased to 0.26 mg/g FW/h (Figure 7B). Moreover, the contents of citric and malic acid in apple leaves remarkably increased to 2.76 mg/g and 4.28 mg/g, respectively, under saline-alkali stress. When exogenous GR24 was applied, the citric acid content of plant leaves under saline-alkali stress decreased to 1.88 mg/g, but the malic acid content in leaves of apple seedlings increased to 5.1 mg/g (Figures 7C,D). Effects of exogenous GR24 application on the electrolyte leakage and osmolytes under saline-alkali stress Electrolyte leakage was detected after saline-alkali stress and exogenous GR24 treatment for 15 days. After saline- alkali stress, the electrolyte leakage remarkably increased from 24.1% to 47.4% but decreased to 38.7% when exogenous GR24 was applied (Figure 4A). Osmolyte content under saline-alkali stress and exogenous GR24 treatment was also detected. The proline, soluble sugar, and soluble protein contents increased under saline-alkali stress. When exogenous GR24 was applied, proline content notably increased from 151.2 µg/g to 253.0 µg/g (Figure 4B), while the soluble sugar content had no signiﬁcant changes (Figure 4C). However, the soluble protein content substantially decreased from The mineral elements of apple seedlings were measured after saline-alkali stress and exogenous GR24 treatment for 15 days. The Na content was signiﬁcantly increased from 3.3 mg/g to 14.1 mg/g under saline-alkali stress but decreased to 8.0 mg/g after exogenous GR24 treatment (Figure 5A). When exogenous GR24 was applied, the K level substantially increased from 14.6 mg/g to 15.9 mg/g under saline-alkali stress and increased to 19.4 mg/g (Figure 5B). As an important indicator of plant tolerance to abiotic stress, Na+/K+ ratio was also detected, the Na+/K+ ratio notably increased to 90.6% under saline- alkali stress but decreased to 41.4% by exogenous GR24 after 15 days treatment (Figure 5C). The Ca content substantially Frontiers in Plant Science 05 frontiersin.org Ma et al. Ma et al. 10.3389/fpls.2022.901782 FIGURE 2 Effects of exogenous GR24 application on the chlorophyll content (A), photosynthetic rate (B), transpiration rate (C) and stomatic conductance (D) of Malus hupehensis seedlings after salinity-alkalinity stress for 15 days. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 2 Effects of exogenous GR24 application on the chlorophyll content (A), photosynthetic rate (B), transpiration rate (C) and stomatic conductance (D) of Malus hupehensis seedlings after salinity-alkalinity stress for 15 days. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). Frontiers in Plant Science Effects of exogenous GR24 on endogenous hormone content under saline-alkali stress Plant hormone regulates the mechanisms of plant stress responses. Under saline-alkali stress, the IAA, GA3, ZR, and JA levels notably decreased. When exogenous GR24 was applied, all of them increased substantially (Figures 6A–D). The result indicated that exogenous GR24 increased the sensitivity of endogenous hormone to regulate the tolerance of apple seedlings to saline-alkali stress. 5.1 mg/g (Figures 7C,D). Frontiers in Plant Science 06 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 FIGURE 3 Effects of GR24 treatment on H2O2, O2- (A), and H2O2 content (B), O2·- content (C), malondialdehyde (MDA) content (D), superoxide dismutase (SOD) activity (E), peroxidase (POD) activity (F), and catalase (CAT) activity (G) under salinity-alkalinity stress. The bar (A) represents 1.0 cm. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 4 Effects of exogenous GR24 treatment on electrolyte leakage (A), proline content (B), soluble sugar content (C), and soluble protein content (D) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 3 Effects of GR24 treatment on H2O2, O2- (A), and H2O2 content (B), O2·- content (C), malondialdehyde (MDA) content (D), superoxide dismutase (SOD) activity (E), peroxidase (POD) activity (F), and catalase (CAT) activity (G) under salinity-alkalinity stress. The bar (A) represents 1.0 cm. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 3 Effects of GR24 treatment on H2O2, O2- (A), and H2O2 content (B), O2·- content (C), malondialdehyde (MDA) content (D), superoxide dismutase (SOD) activity (E), peroxidase (POD) activity (F), and catalase (CAT) activity (G) under salinity-alkalinity stress. The bar (A) represents 1.0 cm. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 4 Effects of exogenous GR24 treatment on electrolyte leakage (A), proline content (B), soluble sugar content (C), and soluble protein content (D) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). Effects of exogenous GR24 on the expression levels of salinity-alkalinity-related genes in apple seedlings under salinity-alkalinity stress To elucidate the mechanism of exogenous GR24 involvement in the salinity-alkalinity stress response, we performed qPCR to detect the expression levels of stress- related genes under salinity-alkalinity stress and exogenous GR24 treatment. As shown in Figure 8, exogenous GR24 remarkably upregulated the expression of MhCHX15, MhSOS1, and MhCAX5, as Na+ transporter genes, by 1.63, 1.81, and 1.58 times, respectively. The expression of two K+ transporter genes, namely, MhNHX1 and MhNHX2, remarkably increased under salinity-alkalinity stress. When exogenous GR24 was applied, the expression levels of them decreased to 1.58 and 1.41 times, respectively, whereas that of MhSKOR was substantially downregulated after saline-alkali stress and exogenous GR24 treatment. Moreover, exogenous GR24 signiﬁcantly upregulated the expression of MhAHA1, MhAHA3, and MhAHA9, as H+-ATPase (AHA) enzyme family genes to 2.57, 8.36, and 4.36 times, respectively. The expression levels of antioxidant enzyme genes MhGPX6, MhPER65, MhpOXN1, were signiﬁcantly induced by salinity-alkalinity stress and were substantially decreased after exogenous GR24 treatment. However, the expression levels of MhSOD, MhPOD and MhCAT were signiﬁcantly decreased under the salinity-alkalinity Effects of exogenous GR24 on endogenous hormone content under saline-alkali stress FIGURE 4 Effects of exogenous GR24 treatment on electrolyte leakage (A), proline content (B), soluble sugar content (C), and soluble protein content (D) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). 07 Frontiers in Plant Science frontiersin.org Ma et al. 10.3389/fpls.2022.901782 FIGURE 5 Effects of GR24 treatment on Na content (A), K content (B), and Na+/K+ ratio (C), Ca content (D), Fe content (E), P content (F), Mg content (G) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 5 Effects of GR24 treatment on Na content (A), K content (B), and Na+/K+ ratio (C), Ca content (D), Fe content (E), P content (F), Mg content (G) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). Effects of exogenous GR24 on the expression levels of salinity-alkalinity-related genes in apple seedlings under salinity-alkalinity stress stress, while exogenous GR24 signiﬁcantly upregulated the expression levels of MhSOD, MhPOD and MhCAT to 1.36, 1.93 and 2.47 times, respectively. Moreover, the expression of three kinases, namely, MhANP2, MhMAPKKK, and MhGK, and three selected transcription factors, namely, MhMYB39, MhERF019, and MhNAC56, remarkably changed under salinity- alkalinity stress and exogenous GR24 treatment (Figure 8). This ﬁnding indicates their potential important functions in plant response to salinity-alkalinity stress and SL signaling transduction pathway. Effects of exogenous GR24 on the expression levels of strigolactones biosynthesis and signal transduction pathway genes in apple seedlings under salinity-alkalinity stress To determine whether the SL biosynthesis and signal transduction pathway genes were involved in the response to salinity-alkalinity stress, we screened out eight genes, which are also involved in the response to salinity-alkalinity stress by analyzing RNA-Seq in apple. The four SL signal transduction pathway genes included a ubiquitin ligase component F-box protein gene (MhMAX2) and three DWARF14 genes (MhD14- 1, MhD14-2, and MhD53). The expression of MhD14-1 was decreased by salinity-alkalinity stress. The transcription levels of MhMAX2 and MhD53 were increased by salinity-alkalinity Frontiers in Plant Science 08 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 FIGURE 6 Effects of GR24 treatment on the contents of auixn (A), glbberellin (B), cytokinin (C) and jasmonic acid (D) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 6 Effects of GR24 treatment on the contents of auixn (A), glbberellin (B), cytokinin (C) and jasmonic acid (D) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). stress. However, all of the four genes were induced by exogenous GR24 treatment (Figure 9). The expression of the four SL biosynthetic enzyme genes, including a cytochrome gene (MhCYP711), two carotenoid cleavage dixoygenase genes (MhCCD7 and MhCCD8), and a 9-cis/all-trans-β-carotene isomerase gene (MhD27), were also quantiﬁed by qPCR. The results showed that the expression levels of these four genes were decreased by salinity-alkalinity stress but remarkably induced by exogenous GR24 treatment (Figure 9). various developmental and adaptation processes in plants (Wang et al., 2020). The external application of SL analog GR24 is a promising approach for stablishing various abiotic stress tolerances in plants (Bhoi et al., 2021). Notably, the exogenous application of GR24 could improve plant growth and photosynthesis under salinity and drought stress in plant (Zulfqar et al., 2020; Bidabadi and Sharif, 2021). However, the eﬀect of exogenous GR24 application on plants under salinity-alkalinity stress has not been reported. In the present study, we applied diﬀerent concentrations of GR24 to salinity- alkalinity-stressed apple seedlings and found that the eﬀects of 100 µM GR24 application was much better than that at 10 µM and 1 mM, and had the lowest wilting rate and the highest fresh weight (Supplementary Figure 1). Effects of exogenous GR24 on the expression levels of strigolactones biosynthesis and signal transduction pathway genes in apple seedlings under salinity-alkalinity stress This study ﬁrst reported the function of GR24 in apple tolerance to salinity- alkalinity stress. Frontiers in Plant Science Discussion Salinity-alkalinity stress is an important factor that limits apple production. Damage caused by alkaline salt stress is more severe than that only caused by neutral salt stress. Phytohormones are inherent signaling molecules, which regulate the growth and development of plants by producing complex responses under various stresses (Verma et al., 2016; Waadt et al., 2022). SL, as a group of carotenoid- derived plant hormones, play an important role in regulating Chlorophyll is essential for photosynthesis. Saline-alkali stress damages the chlorophyll metabolism and photosynthesis in plant (Hu et al., 2016; Feng et al., 2021). In the present study, salinity-alkalinity stress could signiﬁcantly inhibit the chlorophyll content and photosynthesis rate in apple Frontiers in Plant Science 09 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 FIGURE 7 Effects of GR24 treatment on root activity (A), triphenyltetrazolium chloride (TTC) reductive intensity of apple roots (B), citric acid content of apple leaves (C), and malic acid content of apple leaves (D) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 7 Effects of GR24 treatment on root activity (A), triphenyltetrazolium chloride (TTC) reductive intensity of apple roots (B), citric acid content of apple leaves (C), and malic acid content of apple leaves (D) under salinity-alkalinity stress. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). Zulfqar et al. (2020), in which GR24 treatment increased K+ content and reduced Na+/K+ ratio in sunﬂower (Helianthus annuus) under salt stress. Plants have evolved some important protein, which may protect themselves and reduce the poisoning of Na+, such as the cation/H+ exchangers, salt overly sensitive l (SOS1), and cation exchangers, which expel Na+ from cells. Our results showed that MhCHX15, MhSOS1, and MhCAX5 expression levels were increased by exogenous GR24 treatment under salinity-alkalinity stress (Figure 8). We assumed that these three genes could function to balance Na+ homeostasis in the cytoplasm under salinity-alkalinity stress. Stellar K+- outward rectiﬁer (SKOR) is responsible for K+ eﬄux from the cytoplasm to the outside of the cell (Xue et al., 2019). The vacuolar K+/H+ antiporters (NHX) in the tonoplast facilitate K+ inﬂux and eﬄux in the vacuoles (Xue et al., 2019; Xu et al., 2020). Discussion The expression levels of MhSKOR, MhNHX1, and MhNHX2 were substantially inhibited after GR24 treatment (Figure 8). Therefore, exogenous GR24 could decrease the expulsion of K+ out of the cells to ensure Na+/K+ homeostasis in the cytoplasm under salinity-alkalinity stress. Moreover, Ca regulates plant signal transduction pathways under salt stress (Hu et al., 2016). Therefore, the Ca content was remarkably induced by exogenous GR24 possibly as the salinity-alkalinity stress response of the apple seedlings to balance Na+/Ca2+ in the cytoplasm. Fe is essential for plant resistance to oxidative stress (Dai et al., 2018). Therefore, Fe content increases after (Ding et al., 2010; Guo et al., 2015). It was reported that GR24 treatment displayed greater tolerance to KCl stress by regulating chlorophyll components and photosynthetic rate in apple (Zheng et al., 2020). Our results showed that the application of exogenous GR24 could remarkably increase the stomatic conductance and transpiration rate of apple under salinity-alkalinity stress. Furthermore, the chlorophyll content and photosynthesis rate were much higher in GR24- treated than in non-GR24-treated salinity-alkalinity-stressed apple seedlings (Figure 2). Thus, exogenous GR24 could protect the photosynthetic system from salinity-alkalinity damage. Plants normally suﬀer ionic toxicity, high pH, oxidative damage, and osmotic stress from saline-alkaline conditions. The ionic toxicity caused by salinity-alkalinity stress can lead to the excessive accumulation of Na+ in the cytoplasm, thereby aﬀecting plant growth (Javid et al., 2012; Xu et al., 2020). In plant responses to salt stress, the ionic toxicity can lead to an imbalance in cytosolic Na+/K+ ratio and disrupt normal plant growth (Dai et al., 2018; Ma et al., 2021). In the present experiment, the contents of Na+ and K+ increased under salinity-alkalinity stress (Figure 5). The increase of K+ content was attributed to the achievement of balance for Na+ and K+ homeostasis under salinity-alkalinity stress. When exogenous GR24 was applied to the apple leaves, Na+ content decreased with increased K+ content, thus decreasing the leaf Na+/K+ ratio (Figure 5). This condition is similar to the ﬁndings of Frontiers in Plant Science 10 frontiersin.org 10.3389/fpls.2022.901782 Ma et al. The plasma membrane (PM) H+-ATPase extrudes protons from the plant cell, thus generating an electrochemical gradient across the plasma membrane and plays a pivotal role in abiotic stresses, such as salinity, drought, and temperature (Palmgren and Nissen, 2011; Janicka et al., 2018; Xue et al., 2019). Exogenous GR24 application increases H+ and malic acid contents (Figures 7A,D). Frontiers in Plant Science Discussion Moreover, the expression levels of three AHA enzyme family genes, MhAHA1, MhAHA3, and MhAHA9, were increased by exogenous GR24 treatment under salinity-alkalinity stress. Therefore, exogenous GR24 alleviates the high-pH stress of apple seedlings by regulating the expression of H+-ATPase genes and inducing the production of organic acid. FIGURE 8 The expression level of the 21 candidate genes which divided into Na+ transporters (MhCHX15, MhSOS1, and MhCAX5) (A), K+ transporters (MhSKOR, MhNHX1, and MhNHX2) (B), H+-ATPase (AHA) enzyme family genes (MhAHA1, MhAHA3, and MhAHA9) (C), antioxidant enzymes (MhGPX6, MhPER65, MhpoxN1, MhSOD, MhPOD and MhCAT) (D), kinase (MhANP2, MhMAPKKK, and MhGK) (E), and transcription factors (MhMYB39, MhERF019, and MhNAC56) (F). The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). Oxidative damage is caused by excessive ROS, which is an important signal molecule that regulates plant metabolism, growth, and stress response (Zheng et al., 2020). The application of GR24 diminishes the H2O2 and MDA contents in Triticum aestivum under drought condition (Sedaghat et al., 2017). In the present experiment, we found that the O2·-, H2O2, and MDA contents were remarkably induced by salinity-alkalinity stress, and exogenous GR24 application can decrease their contents (Figure 3), suggesting that SL may act as ROS scavenger and reduce lipid peroxidation in apple seedlings under salinity-alkalinity stress. Enzymatic antioxidant systems include three main antioxidant enzymes, namely, SOD, POD, and CAT (Abdelaal et al., 2018; Min et al., 2018). Our results indicated that salinity-alkalinity stress diﬀerentially aﬀects the contents of antioxidant enzymes, the SOD, POD and CAT activities were inhibited under salinity-alkalinity stress. When exogenous GR24 was applied, the SOD, POD and CAT activities substantially increased (Figure 3). This ﬁnding was similar with that of exogenous SL treatment under KCl stress (Zheng et al., 2020). Furthermore, six antioxidant enzyme genes (MhGPX6, MhPER65, MhpOXN1, MhSOD, MhPOD and MhCAT) were substantially aﬀected by exogenous GR24, and the tendencies of MhSOD, MhPOD and MhCAT expression levels were correlated with SOD, POD and CAT activities (Figure 8). Thus, exogenous GR24 could alleviate oxidative damage by regulating the expression of antioxidant enzyme genes, enhancing the enzyme activities of SOD, POD, and CAT under salinity-alkalinity stress. FIGURE 8 The expression level of the 21 candidate genes which divided into Na+ transporters (MhCHX15, MhSOS1, and MhCAX5) (A), K+ transporters (MhSKOR, MhNHX1, and MhNHX2) (B), H+-ATPase (AHA) enzyme family genes (MhAHA1, MhAHA3, and MhAHA9) (C), antioxidant enzymes (MhGPX6, MhPER65, MhpoxN1, MhSOD, MhPOD and MhCAT) (D), kinase (MhANP2, MhMAPKKK, and MhGK) (E), and transcription factors (MhMYB39, MhERF019, and MhNAC56) (F). The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). Plants adapt to osmotic stress mainly by regulating the accumulation of osmolytes, such as sugars and amino acids, to reduce cellular osmotic potential and remove excessive ROS (Blumwald, 2003). Our results indicated that electrolyte leakage was remarkably induced by salinity-alkalinity stress but inhibited by exogenous GR24 treatment. This result was consistent with previous ﬁndings, in which SL could protect plants from osmotic stress (Wang et al., 2019). To investigate the function mechanism of GR24 on osmotic stress, we detected the soluble sugar, soluble protein, and proline contents under salinity-alkalinity and GR24 treatment. The results indicated that exogenous GR24 could aﬀect the proline content under salinity-alkalinity stress (Figure 4). Exogenous SL could GR24 treatment in response to oxidative damage caused by salinity-alkalinity stress. High pH can reduce the availability of mineral elements and aﬀect intracellular ion balance (Palmgren and Nissen, 2011). 11 frontiersin.org Ma et al. 10.3389/fpls.2022.901782 FIGURE 9 The expression level of the four genes in SL signal transduction pathway (MhD14-1, MhD14-3, MhMAX2, and MhD53), and four SL biosynthesis genes (MhCYP711, MhCCD7, MhCCD8, and MhD27) under salinity-alkalinity stress and exogenous GR24 treatment for 15 days. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). FIGURE 9 The expression level of the four genes in SL signal transduction pathway (MhD14-1, MhD14-3, MhMAX2, and MhD53), and four SL biosynthesis genes (MhCYP711, MhCCD7, MhCCD8, and MhD27) under salinity-alkalinity stress and exogenous GR24 treatment for 15 days. The data represent the mean ± SD of three biological replicates. Different lowercase letters indicate signiﬁcant differences according to Fisher’s least signiﬁcant difference (P < 0.05). increase proline concentration and alleviate the KCl stress of M. hupehensis. Therefore, exogenous GR24 could protect plants from osmotic stress by aﬀecting the accumulation of proline. stress in rice (Ning et al., 2008). FIGURE 8 The expression levels of MhGK and MhANP2 were induced by salinity-alkalinity stress but inhibited by GR24 treatment (Figure 8). Therefore, these two kinase genes would participate in response to salinity-alkalinity and GR24 treatment. Mitogen-activated protein kinase (MAPK) pathway reportedly participates in the signaling pathway of salt stress in plants, such as peppermint (Mentha piperita) and cucumber (Cucumis sativus) (Xu et al., 2011; Li et al., 2016). In the present study, the transcript level of MhMAPKKK showed almost the opposite trend as that of K+-outward rectiﬁer gene MhSKOR. This ﬁnding indicates that the potential mechanisms of post-translational modiﬁcation play an important role in mediating the signaling pathway of salinity-alkalinity stress. In addition, MYB, NAC and ERF transcription factors serve as connecting links between the upstream signal and the expression of functional genes under salt stress (Blumwald, 2003; Ju et al., 2020). Here, we found that these genes might participate in the GR24 signaling transduction pathway under salinity-alkalinity in apple. Phytohormones mediate various environmental stresses and thus regulate plant growth adaptation (Yu et al., 2020). The involvement of these hormones in plant salinity-alkalinity tolerance and the interactions among them remain to be elucidated. In our study, GR24 was sprayed to the apple leaves. However, the apple roots also exhibited better root activity under saline-alkali stress (Figure 7). Since applying with exogenous GR24 signiﬁcantly improved the IAA content in apple leaves (Figure 6), the good activity phenotype of apple roots might caused by the systemic regulation of IAA. Haim et al. (2021) reported that auxin production occurred in the shoot apical meristem (SAM) and the young leaves before it was transported toward the roots by polar movement through the stem, and auxin could improve the tolerance of plants to abiotic stress. Xu et al. (2013) also covered that the tomato 14-3-3 protein TFT4 modulated basipetal auxin transport, and the PKS5-J3 pathway for maintaining primary root elongation response to alkaline stress. Therefore, we guessed that the better root activity under saline-alkali stress, which was resulted from spraying GR24 on apple leaves, was caused by the increased IAA content in apple leaves and transport to roots response to saline-alkali stress through polar transport Zhan et al., 2018). Moreover, G-protein kinase (GK) is an important kinase in plant response to salt stress (Lian et al., 2018; Shen et al., 2019). Frontiers in Plant Science 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. 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. Conclusion Our study explored that strigolactones could eﬀectively improve the tolerance on salinity-alkalinity stress in apple. Exogenous GR24 could aﬀect ion homeostasis by regulating Na+/K+ transporter genes, eliminate ROS through enhancing the activities of SOD, POD, and CAT, regulate osmotic balance by increasing the proline content, balance root pH through secretion of organic acid, and cooperate with IAA, GA3, ZR, JA responding to saline-alkali stress (Supplementary Table 3). This work will provide theoretical basis for analyzing the mechanism of SL on salinity-alkalinity stress in apple plants. Funding This work was supported by the National Natural Science Foundation of China (32172542 and 32102351), Breeding Plan of Shandong Provincial Qingchuang Research Team (2019), and Funds for Modern Agricultural Industry Technology System in Shandong Province, China (SDAIT-06-06). FIGURE 8 NPK1- related protein kinase (ANP2) plays an important role in abiotic Strigolactone (SL) signaling pathway enzymes include SL receptor D14, transcriptional repressor protein D53/MXL6/7/8, and F-box protein D3/MAX2 (Yao et al., 2016; Shabek et al., 2018). The transcript levels of the four SL signal transduction pathway genes, namely, MhD14-1, MhD14-3, MhMAX2, and MhD53 were substantially induced by exogenous GR24 treatment (Figure 9). Similar results were also observed in the KCl stress of apple seedlings, in which the expression levels of MdD14, MhMAX2, and MhD53 were induced by Frontiers in Plant Science 12 frontiersin.org Ma et al. Ma et al. 10.3389/fpls.2022.901782 Author contributions SL treatment in apple leaves. Furthermore, the decrease of SLs in tomato might be a systemic signal of drought stress (Visentin et al., 2016). Our results showed that the expression of four SL biosynthetic enzyme genes, namely, MhCYP711, MhCCD7, MhCCD8, and MhD27 were decreased by salinity- alkalinity stress but substantially increased by exogenous GR24 treatment (Figure 9). Therefore, the expression levels of these four genes decreased might be an energy-saving strategy for apple to cope with salinity-alkalinity stress. Interestingly, the expression tendency of SL biosynthetic enzyme genes and MhD14-1 were similar with MhCHX15, MhSOS1, and MhAHAs under salinity-alkalinity and GR24 treatment, and those of MhD53 and MhSKOR were opposite, indicating the potential relationship between them. Overall, these ion transporters, kinases, transcription factors, and the SL biosynthesis and signal transduction pathway genes might have complicated regulation and interaction mechanisms. However, the mechanisms of SL signaling pathway under salinity-alkalinity stress require further analysis. XZ and CW planned and designed the research. CB, CM, WL, XX, XL, DG, ZS, and YT performed the experiments, conducted the ﬁeldwork, and analyzed the data. XZ and CM wrote the manuscript. All authors contributed to the article and approved the submitted version. Supplementary material The original contributions presented in this study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fpls.2022.901782/full#supplementary-material Bhoi, A., Yadu, B., Chandra, J., and Keshavkant, S. (2021). 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https://openalex.org/W2937814148	https://discovery.ucl.ac.uk/10072650/1/s41598-019-41758-1.pdf	English	null	Application of ESMACS binding free energy protocols to diverse datasets: Bromodomain-containing protein 4	Scientific reports	2,019	cc-by	13,607	Application of ESMACS binding free energy protocols to diverse datasets: Bromodomain-containing protein 4 David W. Wright 1, Shunzhou Wan 1, Christophe Meyer 2, Herman van Vlijmen 2, Gary Tresadern 2 & Peter V. Coveney 1 Received: 15 November 2018 Accepted: 8 March 2019 Published: xx xx xxxx As the application of computational methods in drug discovery pipelines becomes more widespread it is increasingly important to understand how reproducible their results are and how sensitive they are to choices made in simulation setup and analysis. Here we use ensemble simulation protocols, termed ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent), to investigate the sensitivity of the popular molecular mechanics Poisson-Boltzmann surface area (MMPBSA) methodology. Using the bromodomain-containing protein 4 (BRD4) system bound to a diverse set of ligands as our target, we show that robust rankings can be produced only through combining ensemble sampling with multiple trajectories and enhanced solvation via an explicit ligand hydration shell. The discovery and design of novel drugs is immensely expensive, with one study putting the cost of each new therapeutic molecule that reaches the clinic at US$1.8 billion1. A diversity of computational approaches, specif- ically binding free energy calculations which rely on physics-based molecular dynamics simulations (MD) have been developed2, and blind tests show that many have considerable predictive potential3,4. In this context, recent developments in algorithms and hardware that have reduced the cost and time of these computational approaches have seen an increase in their appeal to the pharmaceutical industry5–9. With commercial approaches that claim accuracy of below 1 kcal mol−1 now on the market10 it is becoming of increasing interest to understand the accu- racy of and uncertainties inherent in different approaches11. These concerns echo wider interest in the scientific community in the lack of reproducible results in the published literature12,13.fi y p p One of the most common computational binding affinity prediction techniques is molecular mechanics Poisson–Boltzmann surface area (MMPBSA)14. This is an approximate post-processing end-state method, which uses continuum solvent models to reduce the computational cost of obtaining results. The speed and ease of setup (compared to rigorous free energy calculations) make MMPBSA an attractive candidate for use throughout the drug discovery pipeline. However, results are often seen to be system dependent and are widely perceived to be less accurate than those obtained from more expensive and theoretically rigorous approaches (such as free energy perturbation, FEP, and thermodynamic integration, TI)2,15. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Centre for Computational Science, Department of Chemistry, University College London, London, WC1H 0AJ, United Kingdom. 2Janssen Research & Development, Turnhoutseweg 30, B-2340, Beerse, Belgium. Correspondence and requests for materials should be addressed to P.V.C. (email: p.v.coveney@ucl.ac.uk) Received: 15 November 2018 Accepted: 8 March 2019 Published: xx xx xxxx Computational Methodsh p The principle behind the ESMACS family of protocols is that many short simulations provide better sampling than single long simulations, facilitating the rapid and reproducible calculation of binding affinities using var- iations of MMPBSA. The ESMACS simulation and analysis workflow has been automated using the Binding Affinity Calculator (BAC)23 which we have recently enhanced using Radical Cybertools24,25 to create HTBAC26. The goal of HTBAC is to provide a programmable interface to create computational pipelines built from selected software tools and services, and execute them on remote resources. It automates much of the complexity of run- ning and marshalling the molecular dynamics simulations, as well as collecting and analyzing data.l Our ESMACS protocols are flexible, allowing for the analysis to be tailored to the target system. Previous targets we have studied include small molecule inhibitors of HIV proteins18,27,28, kinases8,29 and larger more flex- ible ligands such as peptides which bind to MHC17. In all these studies correlation coefficients of better than 0.7 were obtained. MMPBSA is most commonly used to assess binding affinities from a single trajectory of a protein bound to its target ligand but in this work we explore the influence of protein and ligand flexibility using inde- pendent trajectories. Free energy of binding computations. When two reactants combine at constant temperature and pres- sure the binding affinity is characterized by the change in Gibbs free energy, ΔG. MMPBSA is an endpoint free energy calculation; in such methods ΔG is calculated using: ∆ = 〈 〉−〈 〉−〈 〉 G G G G , complex receptor ligand ∆ = 〈 〉−〈 〉−〈 〉 G G G G , (1) complex receptor ligand (1) where 〈 〉 Gcomplex , 〈 〉 Greceptor and 〈 〉 Gligand are the average values of the Gibbs free energy for the complex, receptor protein) and ligand respectively. Sampling of the complex and its two components can be performed independently or conformations of the receptor and ligand extracted from simulation of the complex. The latter approach is more commonly used due to its improved convergence behaviour, a consequence of cancellation between the noisy terms describing the internal energy of the ligand, receptor and complex30. However, recent work has indicated that adaptation ener- gies associated with confining the receptor and ligand in a complex can differ significantly even for closely related complexes9. Here we investigate a range of ESMACS protocols incorporating different component sampling strat- egies. Application of ESMACS binding free energy protocols to diverse datasets: Bromodomain-containing protein 4 David W. Wright 1, Shunzhou Wan 1, Christophe Meyer 2, Herman van Vlijmen 2, Gary Tresadern 2 & Peter V. Coveney 1 Furthermore, the term MMPBSA as used in the lit- erature permits a wide range of variants which incorporate different sampling strategies (for example, all ligand conformers can be drawn from simulation of the complex or from independent runs) and differing solvation and entropy terms. Our previous work has demonstrated that MMPBSA analysis of single simulations is highly unreliable with calculations initiated from the same structures varying by up to 12 kcal mol−1 for small molecules bound to HIV-1 protease and even more for flexible ligands binding to MHC16,17. This served as the inspiration for our ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) proto- cols which use ensemble simulations that have been shown to produce results with reproducible uncertainties of less than 2 kcal mol−1 for a range of systems9,16,18. In this work we seek to assess the performance of the approach in a challenging dataset containing a highly varied set of ligands which interact with water in the protein binding site. We assess the impact on protocol performance of multiple trajectory sampling, ligand parameterization, 1Centre for Computational Science, Department of Chemistry, University College London, London, WC1H 0AJ, United Kingdom. 2Janssen Research & Development, Turnhoutseweg 30, B-2340, Beerse, Belgium. Correspondence and requests for materials should be addressed to P.V.C. (email: p.v.coveney@ucl.ac.uk) Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 1 www.nature.com/scientificreports/ Protocol Contribution to the binding free energy Complex Receptor Ligand 1-traj C C C 1-traj-ar C Constant C 2-traj-fr C R C 2-traj-fl C C L 2-traj-ar C Constant L 3-traj C R L Table 1. Summary of the origin of component contributions in 6 ESMACS protocols indicating whether they come from the ensemble of simulations run for the complex (C) or separate ensembles performed for the receptor (R) and ligands (L). Constant refers to the use of a constant, usually the average value across the studied systems. Table 1. Summary of the origin of component contributions in 6 ESMACS protocols indicating whether they come from the ensemble of simulations run for the complex (C) or separate ensembles performed for the receptor (R) and ligands (L). Constant refers to the use of a constant, usually the average value across the studied systems. inclusion of explicit water molecules and a recently developed approach to calculating the entropic contribution to the binding free energy.h The target of our investigation is the bromodomain-containing protein 4 (BRD4). Application of ESMACS binding free energy protocols to diverse datasets: Bromodomain-containing protein 4 David W. Wright 1, Shunzhou Wan 1, Christophe Meyer 2, Herman van Vlijmen 2, Gary Tresadern 2 & Peter V. Coveney 1 Bromodomains are a major and rapidly evolving focus for the pharmaceutical industry with inhibitors targeting them having shown promising pre-clinical efficacy in pathologies ranging from cancer to inflammation. BRD4, in particular, has recently become something of a benchmark system for free energy calculations15,19–21, including for those based on MMPBSA22. Computational Methodsh When both the receptor and ligand contributions are computed from the complex trajectory we designate this a “1traj protocol”. When all three derive from independent trajectories we refer to this as the “3traj protocol” and when only one or other of the receptor or ligand contributions do so a “2traj protocol”. A suffix (either -fl or -fr, for flexible ligand and receptor respectively) is added to the protocol name to signify which component is derived from the independent simulation. Additional variants involve the use of the average receptor contribution across the complex simulations for all comparable ligands, which is indicated with an –ar (averaged receptor) suf- fix in the protocol name. A summary of all of the protocols, describing from which simulation component data is obtained, is given in Table 1. It should be noticed that the statistical performance of the pair of protocols 1traj-ar and 2traj-fr, and 2traj-ar and 3traj are the same, as the receptor contribution in all cases is constant. Consequently, we do not analyze the 3traj or 2traj-fr protocols explicitly.h y j j p p y The binding free energy change calculated by MMPBSA (ΔGMMPBSA) can be broken down into a number of components: y j j p p y binding free energy change calculated by MMPBSA (ΔGMMPBSA) can be broken down into a number of t ∆ = ∆ + ∆ + ∆ + ∆ + ∆ G G G G G G , MMPBSA ele MM vdW MM MM nonpol sol pol sol int ∆ = ∆ + ∆ + ∆ + ∆ + ∆ G G G G G G , (2) MMPBSA ele MM vdW MM MM nonpol sol pol sol int (2) Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 2 www.nature.com/scientificreports/ Figure 1. Overview of the ESMACS workflow. The 1traj protocol is shown in (a) consisting of an ensemble of 1 to N (25 in this study) simulations of the protein-ligand complex. Each simulation is made up of (min)imization and two (eq)uilibration steps and a single production NAMD run which are each analyzed independently using the MMPBSA.py script. The output of the analysis is then collated and bootstrap statistics produced. The multiple trajectory approaches, shown in (b) follow a similar outline but with independent trajectories also run of the ligand system alone. Figure 1. Overview of the ESMACS workflow. Computational Methodsh The 1traj protocol is shown in (a) consisting of an ensemble of 1 to N (25 in this study) simulations of the protein-ligand complex. Each simulation is made up of (min)imization and two (eq)uilibration steps and a single production NAMD run which are each analyzed independently using the MMPBSA.py script. The output of the analysis is then collated and bootstrap statistics produced. The multiple trajectory approaches, shown in (b) follow a similar outline but with independent trajectories also run of the ligand system alone. where ΔGele MM, ΔGvdW MM and ΔGint MM are the electrostatic, van der Waals and the internal bonded contributions to the molecular mechanics free energy difference, respectively, and ΔGpol sol and ΔGnonpol sol are the polar and non-polar solvation terms, respectively.h The MMPBSA.py31 program, provided as part of the AmberTools 14 package32, was used in the evaluation of all components of the MMPBSA calculation. The electrostatic free energy of solvation, ΔGpol sol, is the part of the calculation described by the Poisson-Boltzmann (PB) calculation. Default values were used for the PB calculation (grid spacing of 0.5 Å, internal and external dielectric constants of 1 and 80, respectively). The non-polar solva- tion free energy calculation is calculated from the solvent accessible surface area using the traditional one com- ponent method (specified using inp = 1 in the input file). In this approach the surface tension, γ, is set to 0.00542 kcal mol−1 Å−2) and the off-set, β, to 0.92 kcal mol−1. The fill ratio parameter was set to 4.0 which does not impact the results but ensures the stability of the calculations. For calculations in which explicit water molecules were incorporated as part of the receptor, the closest N molecules to the ligand were chosen for inclusion. Entropic contribution to binding free energies. A variety of options are available to incorporate entropic contri- butions to ΔG. The most common approach is normal mode analysis33,34 but it can require similar computational effort to the underlying simulations in order to obtain converged results18. Consequently, here we explore the use of another, more computationally efficient, alternative approach proposed by Duan et al.35. In their formulation the “variational entropy” can be derived from the fluctuations of the receptor-ligand interaction energy, Einter. This energy can be calculated using components of the MMPBSA calculation: = + . Computational Methodsh E G G (3) inter ele MM vdW MM (3) The fluctuation in interaction energy is then given by: ∆ = −〈 〉 E E E , (4) inter inter inter (4) where angle braces indicate an ensemble average. This is then used to compute the entropic contribution to binding via: where angle braces indicate an ensemble average. This is then used to compute the entropic contribution to binding via: −∆ = 〈 〉 β∆ T S k T e ln (5) var B Einter −∆ = 〈 〉 β∆ T S k T e ln (5) var B Einter −∆ = 〈 〉 β∆ T S k T e ln var B Einter (5) he Boltzmann constant and β = k T 1/ B . where kB is the Boltzmann constant and β = k T 1/ B . Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 3 www.nature.com/scientificreports/ Figure 2. Chemical structures of ligands from the BRD4 dataset previously studied by Aldeghi et al.15 (1–11) and the tetrahydroquinoline (THQ) scaffold. Full R-group information for the THQ dataset ligands is provided in Fig. 3 and Table 2. Figure 2. Chemical structures of ligands from the BRD4 dataset previously studied by Aldeghi et al.15 (1–11) and the tetrahydroquinoline (THQ) scaffold. Full R-group information for the THQ dataset ligands is provided in Fig. 3 and Table 2. Simulation setup. Ensembles of 25 replica MD simulations were conducted using the package NAMD 2.1136 for each system (complex, receptor or ligand) studied. All simulations were conducted using the protocol incorporated into BAC23. We have previously shown that the use of 25 replica ensembles provides a good balance of computational cost and calculation uncertainty for a number of varied systems8,17,18. Simulation setup. Ensembles of 25 replica MD simulations were conducted using the package NAMD 2.1136 for each system (complex, receptor or ligand) studied. All simulations were conducted using the protocol incorporated into BAC23. We have previously shown that the use of 25 replica ensembles provides a good balance of computational cost and calculation uncertainty for a number of varied systems8,17,18. p y y Each system was minimized with all heavy protein atoms restrained at their initial positions (with a restrain- ing force constant of 4 kcal mol−1 Å−2). Initial velocities were then generated independently for each replica from a Maxwell–Boltzmann distribution at 50 K. Computational Methodsh Each system was virtually heated to 300 K over 60 ps and subsequently maintained at this temperature using a thermostat (employing a coupling coefficient of 1 ps−1) during which time the restraints applied during minimization were retained. Once the system reached the correct temperature the pressure was maintained at 1 bar using a Berendesen barostat (with a pressure coupling constant of 0.1 ps). Subsequent to the heating, a series of equilibration runs, totaling 2 ns, were conducted, during which the restraints on heavy atoms were gradually reduced. The restraint reduction occurs in ten 100 ps steps, after each one the force constant was halved. Finally, 4 ns production simulations were executed with snapshots output for analysis every 100 ps. A 2 fs time step was used for all MD simulation steps. The workflow of the ESMACS protocols is shown in Fig. 1. For each system run through the 1traj protocol an ensemble of independent NAMD simulations is exe- cuted, consisting of four steps. The first minimization (min), which is followed by two equilibration steps (labelled eq1 and eq2 respectively). In Eq. 1 the system is heated while restraints are applied to heavy atoms. In Eq. 2 restraints are gradually reduced before free simulation is undertaken. After 2 ns of aggregate equilibration the 4 ns production phase is initiated. It is the production trajectory which is analysed by MMPBSA.py. A script is then run to aggregate these results from the ensemble of simulations and values of ΔGMMPBSA computed along with bootstrap statistics. In multiple trajectory approaches a second ensemble of ligand-only simulations is conducted and fed into the aggregation and bootstrapping script. Full simulation details are provided in the main text. Experimental Datasets. This study investigates a combination of BRD4 ligand binding datasets which have been the subjects of earlier studies. The first, previously studied by Aldeghi et al.15 using a combination of FEP based abso- lute binding free energy and MMPBSA techniques, contains a diverse set of 11 ligands which will be referred to as the diverse (DIV) dataset. The second was recently studied by our group in collaboration with GlaxoSmithKline9 (using a combination of ESMACS and ensemble thermodynamic integration approaches) and contains 16 ligands, all based on a single tetrahydroquinoline (THQ) template (consequently we identify this as the THQ dataset). The compounds were selected to represent a range of chemical functionality and binding affinities, despite their shared scaffold. www.nature.com/scientificreports/ 3(a–h), with the common THQ scaffold in Fig. 3(i). Compounds THQ1–9 are neutral, 10–12 and 16 are positively charged (+1), and 13–15 negatively charged (−1). All compounds are the 2-(S) 4-(R) isomers except compound 16 which is 2-(R) 4-(S). Table 2. Composition of the ligands of the THQ dataset. The groups found at R4 are shown in Fig. 3(a–h), with the common THQ scaffold in Fig. 3(i). Compounds THQ1–9 are neutral, 10–12 and 16 are positively charged (+1), and 13–15 negatively charged (−1). All compounds are the 2-(S) 4-(R) isomers except compound 16 which is 2-(R) 4-(S). Figure 3. (a–h) Structures of the groups of the side groups which are added to the tetrahydroquinoline (THQ) scaffold shown in (i) to create the ligands in the THQ dataset. Full composition information for the ligands is provided in Table 2. Figure 3. (a–h) Structures of the groups of the side groups which are added to the tetrahydroquinoline (THQ) scaffold shown in (i) to create the ligands in the THQ dataset. Full composition information for the ligands is provided in Table 2. Figure 3. (a–h) Structures of the groups of the side groups which are added to the tetrahydroquinoline (THQ) scaffold shown in (i) to create the ligands in the THQ dataset. Full composition information for the ligands is provided in Table 2. of rigorously derived uncertainty estimates in the experimental data, must be borne in mind when assessing protocol performance. In Table 3 we provide the full experimental binding affinities for both the diverse (DIV) and tetrahydro- quinoline scaffold (THQ) datasets. of rigorously derived uncertainty estimates in the experimental data, must be borne in mind when assessing protocol performance. In Table 3 we provide the full experimental binding affinities for both the diverse (DIV) and tetrahydro- quinoline scaffold (THQ) datasets. Structural models. The ligands from both datasets were simulated bound to the two BRD4 structural models based on PDBs 2OSS and 4BJX respectively (these are the initial structures used in Aldeghi et al.15 and Wan et al.9). The former represents the apo BRD4 and the latter the protein bound to a THQ based ligand. The secondary structure of both models is very similar (see Fig. 4a) and the RMSD between the two structures is 0.44 Å. All crystallographic water molecules were retained, including four which are conserved in the binding site of both models. Computational Methodsh The first 11 compounds are labeled 1 to 9 according to the scheme used by Aldeghi et al.15, the THQ based ligands are labeled THQ1 to THQ16 (the numbers correspond to those used in Wan et al.9). The chemical structure of the first 11 compounds and the THQ scaffold are shown in Fig. 2. Details of the groups found at positions R1 to R4 in the THQ based ligands are detailed in Fig. 3 and Table 2. Ligand 4 was parameterized with a charge of +1. Compounds THQ10 to THQ12 and THQ16 are positively charged (+1), and compounds THQ13 to THQ15 are negatively charged (−1). p y g p g y g Experimental binding free energies (ΔGexpt) for the first dataset were obtained from a combination of SPR, Alphascreen and Isothermal Titration Calorimetry (ITC) experiments15, whereas those for the THQ dataset are derived from IC50 values from FRET9. These techniques are very different from one another and will necessarily introduce var- ying levels of uncertainty into the data they provide. The divergence in the origin of the measurements is representative of the sources of experimental data to which free energy calculations are typically compared. This, alongside the lack Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 4 Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Ligand ID R1 R2 R3 R4 THQ1 H Me Me (a) THQ2 H Me Me H THQ3 H Me Me (f) THQ4 H Me Me (b) THQ5 H Me Me (c) THQ6 H Me Me (d) THQ7 H Me Me (e) THQ8 H Me Et (f) THQ9 H Me i-Pr (f) THQ10 H Me Me (g) THQ11 H Et Me (g) THQ12 H Pr Me (g) THQ13 H Pr Me (h) THQ14 H Et Me (h) THQ15 Cl Me Me (h) THQ16 H Me Me (g) Table 2. Composition of the ligands of the THQ dataset. The groups found at R4 are shown in Fig. 3(a–h), with the common THQ scaffold in Fig. 3(i). Compounds THQ1–9 are neutral, 10–12 and 16 are positively charged (+1), and 13–15 negatively charged (−1). All compounds are the 2-(S) 4-(R) isomers except compound 16 which is 2-(R) 4-(S). Ligand ID R1 R2 R3 R4 THQ1 H Me Me (a) THQ2 H Me Me H THQ3 H Me Me (f) THQ4 H Me Me (b) THQ5 H Me Me (c) THQ6 H Me Me (d) THQ7 H Me Me (e) THQ8 H Me Et (f) THQ9 H Me i-Pr (f) THQ10 H Me Me (g) THQ11 H Et Me (g) THQ12 H Pr Me (g) THQ13 H Pr Me (h) THQ14 H Et Me (h) THQ15 Cl Me Me (h) THQ16 H Me Me (g) Table 2. Composition of the ligands of the THQ dataset. The groups found at R4 are shown in Fig. 3(a–h), with the common THQ scaffold in Fig. 3(i). Compounds THQ1–9 are neutral, 10–12 and 16 are positively charged (+1), and 13–15 negatively charged (−1). All compounds are the 2-(S) 4-(R) isomers except compound 16 which is 2-(R) 4-(S). Ligand ID R1 R2 R3 R4 THQ1 H Me Me (a) THQ2 H Me Me H THQ3 H Me Me (f) THQ4 H Me Me (b) THQ5 H Me Me (c) THQ6 H Me Me (d) THQ7 H Me Me (e) THQ8 H Me Et (f) THQ9 H Me i-Pr (f) THQ10 H Me Me (g) THQ11 H Et Me (g) THQ12 H Pr Me (g) THQ13 H Pr Me (h) THQ14 H Et Me (h) THQ15 Cl Me Me (h) THQ16 H Me Me (g) Table 2. Composition of the ligands of the THQ dataset. The groups found at R4 are shown in Fig. www.nature.com/scientificreports/ The poses of the lig- ands in the DIV dataset were extracted from crystal structures (PDBs: 3U5J, 3U5L, 4OGI, 4OGJ, 3MXF, 4MR3, 4MR4, 3SVG, 4J0R and 4HBV), except for one ligand, labelled 10, which was modeled (based on PDB 3SVG) and docked into 2OSS as two conformers. These are the same two conformers used in Aldeghi et al.15, differing by a 180° flip of the trifluorophenyl moiety. The modelled poses were aligned and copied into the 4BJX based models. Poses of the THQ ligands were based on that of I-BET726 as found in the 4BJX structure. g System setup, including the creation of a water box and addition of neutralizing ions, was performed using AmberTools 1737,38. The majority of simulations were conducted using protein parameters taken from the stand- ard Amber force field for bioorganic systems (ff14SB)39. Reproducibility studies of the THQ ligands were con- ducted using an earlier version of the forcefield, ff99SBildn40.ih if Drug parameters were produced using the general Amber force field (GAFF)41. The majority of the simula- tions presented here employ ligands prepared using the Gaussian/RESP protocol. In this approach, Gaussian 9842 was used to perform geometric optimization of the inhibitor with 6–31G** basis functions, and the restrained electrostatic potential (RESP) procedure was used to calculate the partial atomic charges. Reproducibility studies Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 5 www.nature.com/scientificreports/ Figure 4. BRD4 structure in cartoon representation with conserved binding site waters shown as van der Waals spheres. (a) A comparison of the structural models created as derived from the 2OSS (green) and 4BJX (purple) PDBs. (b) The initial binding mode of ligand 1 (shown in chemical representation) in the 2OSS derived BRD4 structure. Figure 4. BRD4 structure in cartoon representation with conserved binding site waters shown as van der Waals spheres. (a) A comparison of the structural models created as derived from the 2OSS (green) and 4BJX (purple) PDBs. (b) The initial binding mode of ligand 1 (shown in chemical representation) in the 2OSS derived BRD4 structure. of the DIV dataset were conducted using AM1-BCC43 derived charges. All charge assignment and input file gen- eration was performed in the Antechamber component of AmberTools. Statistics and uncertainties. All statistics presented use their standard definitions with the exception of the mean unsigned error (MUE). www.nature.com/scientificreports/ It is well known that MMPBSA results have a significant offset from experimental values (typically of the order of 15 to 25 kcal mol−1) due to a range of factors, in particularly the neglect of entropic contribu- tions33,34. Consequently we present values corrected for the systematic (mean signed) error and designate them cMUE. tions33,34. Consequently we present values corrected for the systematic (mean signed) error and designate them cMUE. We compute uncertainties for all metrics through bootstrapping analysis. This method involves resampling with replacement the N input data points (in this case, the replica averages of ΔGMMPBSA) to provide a new bootstrap sample also containing N data points. This process is repeated many times (in our case 5000 times) and the statistic of interest of each bootstrap population calculated. The standard deviation of these values provides an estimate of the uncertainty associated with an average derived from a given sample; this is what is quoted as the bootstrap error measure of our statistics. For correlation coefficients samples are drawn from the overall averages for each ligand paired with the relevant experimental value. In addition to this metric, when making a direct comparison of specific correlation coefficients we will also quote 95% confidence intervals. These intervals are calculated by sorting the bootstrap sample distribution of correlation coefficients and taking the values falling at the 2.5 and 97.5 percentiles. Results All THQ values were derived from IC50 values from FRET experiments. Ligand ID pIC50 ΔGexpt (kcal mol−1) Diverse (DIV) 1 — −9.8 (0.1) 2 — −9.6 (0.1) 3 — −9.0 (0.1) 4 — −8.9 (0.1) 5 — −8.8 (0.1) 6 — −8.2 (0.1) 7 — −7.8 (0.1) 8 — −7.4 (0.1) 9 — −7.3 (0.1) 10 — −6.3 (0.1) 11 — −5.6 (0.1) Tetrahydroquinoline scaffold (THQ) THQ1 7.0 −9.6 (0.1) THQ2 5.6 −7.7 (0.1) THQ3 6.8 −9.3 (0.1) THQ4 6.8 −9.3 (0.1) THQ5 7.9 −10.8 (0.1) THQ6 5.6 −7.7 (0.1) THQ7 5.8 −8.0 (0.1) THQ8 6.5 −8.9 (0.1) THQ9 <4.3 >−5.9 (0.1) THQ10 7.6 −10.4 (0.4) THQ11 6.8 −9.3 (0.1) THQ12 5.5 −7.5 (0.1) THQ13 5.4 −7.4 (0.1) THQ14 6.7 −9.2 (0.3) THQ15 7.8 −10.7 (0.1) THQ16 5.4 −7.4 (0.4) Table 3. Experimental binding affinities for both the dive d t t Th l d h t k f Ald hi t Table 3. Experimental binding affinities for both the diverse (DIV) and tetrahydroquinoline scaffold (THQ) datasets. The values used here were taken from Aldeghi et al.15 for the DIV and Wan et al.9 for the THQ datasets respectively. Values for ligands 1–4 and 6–8 were derived from ITC experiments, 5 from SPR and 9–11 from Alphascreen. All THQ values were derived from IC50 values from FRET experiments. Table 3. Experimental binding affinities for both the diverse (DIV) and tetrahydroquinoline scaffold (THQ) datasets. The values used here were taken from Aldeghi et al.15 for the DIV and Wan et al.9 for the THQ datasets respectively. Values for ligands 1–4 and 6–8 were derived from ITC experiments, 5 from SPR and 9–11 from Alphascreen. All THQ values were derived from IC50 values from FRET experiments. pronouncedly THQ16. This is in contrast to nearly all other ligands where the values obtained from simulations with either model are well within the error margin, many sitting on top of one another in Fig. 5. It can also be seen in Table 4 that the impact of the incorporation of receptor ‘strain’ in the 1traj-ar and 2traj-ar protocols is different in the DIV and THQ subsets. In the 4BJX simulations the DIV rankings are notably less good than that in the 1traj, whilst they are fairly similar in the 2OSS case. Whereas for THQ, we find that account- ing for the receptor and ligand flexibility is necessary to obtain a good ranking in both cases. Results Here we evaluate the performance of a range of ESMACS protocols in reproducing the experimental rankings across the full diverse ligand dataset, the robustness of this ranking to choices in system setup and the influence of non-standard MMPBSA components. Standard ESMACS Performance and Robustness to Initial Structure Variation. Comparison of the results of all ESMACS protocols across the full DIV + THQ dataset shows a distinct trend in which inclusion of the receptor average energy considerably improves the predictions obtained for both initial protein models. In both cases 1traj results have a Spearman rank coefficient, rs, of 0.46 [CI: 0.16–0.84 for both] which improves to 0.66 [CI: 0.50–0.94]/0.60 [CI: 0.40–0.91] (2OSS/4BJX) when both ligand and receptor flexibility are accounted for in the 2traj-ar protocol. In the DIV dataset better ranking can be obtained using receptor flexibility alone, but in order to obtain good rankings for THQ both additional contributions are required. This is the same behaviour observed in the simulation results for the THQ dataset in Wan et al.9; however the overall ranking is worse (the original study obtained an rs of 0.78 [CI: 0.53–0.92]), primarily due to the stronger predicted binding affinity for the experimentally least potent drug, THQ16, in the present study.h p g Q p y The improvement between 1traj and 2traj-ar is illustrated in Fig. 5, which shows that outliers are moved closer to the overall trend line (particularly apparent for the DIV ligands 3, 4 and 5 which were also outliers in Aldeghi et al.15). These three ligands have similar experimental binding energies but a difference of 15 kcal mol−1 in 1traj and 10 kcal mol−1 in 2traj-ar is seen in ΔGMMPBSA The ranking improvement is larger for the THQ ligands than the DIV dataset, with the 1traj results exhibiting little if any correlation with experiment. The main THQ outliers in the 1traj results are THQ12, THQ13 and THQ9. The first two are moved closer to the trend in the 2traj-ar results but THQ9 remains more negative than might be expected. Results Another feature of the 2traj-ar data here is that greater separation is seen between the results obtained from the two BRD4 structures for TH12, THQ13 and most Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 6 www.nature.com/scientificreports/ Ligand ID pIC50 ΔGexpt (kcal mol−1) Diverse (DIV) 1 — −9.8 (0.1) 2 — −9.6 (0.1) 3 — −9.0 (0.1) 4 — −8.9 (0.1) 5 — −8.8 (0.1) 6 — −8.2 (0.1) 7 — −7.8 (0.1) 8 — −7.4 (0.1) 9 — −7.3 (0.1) 10 — −6.3 (0.1) 11 — −5.6 (0.1) Tetrahydroquinoline scaffold (THQ) THQ1 7.0 −9.6 (0.1) THQ2 5.6 −7.7 (0.1) THQ3 6.8 −9.3 (0.1) THQ4 6.8 −9.3 (0.1) THQ5 7.9 −10.8 (0.1) THQ6 5.6 −7.7 (0.1) THQ7 5.8 −8.0 (0.1) THQ8 6.5 −8.9 (0.1) THQ9 <4.3 >−5.9 (0.1) THQ10 7.6 −10.4 (0.4) THQ11 6.8 −9.3 (0.1) THQ12 5.5 −7.5 (0.1) THQ13 5.4 −7.4 (0.1) THQ14 6.7 −9.2 (0.3) THQ15 7.8 −10.7 (0.1) THQ16 5.4 −7.4 (0.4) Table 3. Experimental binding affinities for both the diverse (DIV) and tetrahydroquinoline scaffold (THQ) datasets. The values used here were taken from Aldeghi et al.15 for the DIV and Wan et al.9 for the THQ datasets respectively. Values for ligands 1–4 and 6–8 were derived from ITC experiments, 5 from SPR and 9–11 from Alphascreen. All THQ values were derived from IC50 values from FRET experiments. Ligand ID pIC50 ΔGexpt (kcal mol−1) Diverse (DIV) 1 — −9.8 (0.1) 2 — −9.6 (0.1) 3 — −9.0 (0.1) 4 — −8.9 (0.1) 5 — −8.8 (0.1) 6 — −8.2 (0.1) 7 — −7.8 (0.1) 8 — −7.4 (0.1) 9 — −7.3 (0.1) 10 — −6.3 (0.1) 11 — −5.6 (0.1) Tetrahydroquinoline scaffold (THQ) THQ1 7.0 −9.6 (0.1) THQ2 5.6 −7.7 (0.1) THQ3 6.8 −9.3 (0.1) THQ4 6.8 −9.3 (0.1) THQ5 7.9 −10.8 (0.1) THQ6 5.6 −7.7 (0.1) THQ7 5.8 −8.0 (0.1) THQ8 6.5 −8.9 (0.1) THQ9 <4.3 >−5.9 (0.1) THQ10 7.6 −10.4 (0.4) THQ11 6.8 −9.3 (0.1) THQ12 5.5 −7.5 (0.1) THQ13 5.4 −7.4 (0.1) THQ14 6.7 −9.2 (0.3) THQ15 7.8 −10.7 (0.1) THQ16 5.4 −7.4 (0.4) Table 3. Experimental binding affinities for both the diverse (DIV) and tetrahydroquinoline scaffold (THQ) datasets. The values used here were taken from Aldeghi et al.15 for the DIV and Wan et al.9 for the THQ datasets respectively. Values for ligands 1–4 and 6–8 were derived from ITC experiments, 5 from SPR and 9–11 from Alphascreen. Results Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 7 www.nature.com/scientificreports/ Protocol Dataset cMUE* PI r rs 2OSS Structure 1traj DIV + THQ 3.25 (0.51) 0.48 0.51 (0.16) 0.46 (0.18) DIV 3.38 (0.68) 0.74 0.64 (0.19) 0.69 (0.23) THQ 2.01 (0.37) 0.11 0.19 (0.22) 0.09 (0.27) 1traj-ar DIV + THQ 3.30 (0.49) 0.62 0.61 (0.14) 0.60 (0.14) DIV 3.98 (0.68) 0.75 0.67 (0.18) 0.72 (0.19) THQ 2.05 (0.30) 0.46 0.47 (0.17) 0.42 (0.22) 2traj-ar DIV + THQ 3.57 (0.54) 0.65 0.62 (0.12) 0.66 (0.11) DIV 4.09 (0.78) 0.66 0.62 (0.18) 0.64 (0.21) THQ 2.41 (0.67) 0.67 0.54 (0.13) 0.65 (0.17) 2traj-fl DIV + THQ 3.33 (0.59) 0.59 0.55 (0.14) 0.57 (0.14) DIV 3.41 (0.88) 0.75 0.59 (0.19) 0.72 (0.20) THQ 2.09 (0.55) 0.41 0.40 (0.17) 0.39 (0.23) 4BJX Structure 1traj DIV + THQ 3.32 (0.53) 0.48 0.50 (0.17) 0.46 (0.18) DIV 3.54 (0.80) 0.79 0.65 (0.18) 0.74 (0.20) THQ 2.14 (0.43) 0.07 0.11 (0.24) 0.04 (0.27) 1traj-ar DIV + THQ 4.05 (0.48) 0.52 0.56 (0.14) 0.49 (0.15) DIV 3.51 (0.78) 0.60 0.68 (0.18) 0.55 (0.27) THQ 2.55 (0.46) 0.24 0.30 (0.18) 0.16 (0.25) 2traj-ar DIV + THQ 3.97 (0.44) 0.61 0.63 (0.12) 0.60 (0.13) DIV 3.42 (0.82) 0.57 0.67 (0.17) 0.55 (0.24) THQ 2.70 (0.48) 0.51 0.51 (0.15) 0.46 (0.22) 2traj-fl DIV + THQ 3.46 (0.51) 0.54 0.56 (0.14) 0.52 (0.16) DIV 3.52 (0.86) 0.75 0.63 (0.18) 0.73 (0.20) THQ 2.20 (0.56) 0.27 0.36 (0.19) 0.22 (0.26) Table 4. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies, measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error provided in brackets where appropriate. Results are provided for the diverse (DIV) and tetrahydroquinoline (THQ) datasets and both combined (DIV + THQ). MUE corrected for mean signed error in kcal mol−1. Results Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the AM1-BCC method to parameterize ligands. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error shown in brackets where appropriate. Results are provided for the diverse (DIV) dataset alone bound to protein models based on PDB 2OSS. MUE corrected for mean signed error in kcal mol−1. Protocol cMUE* PI r rs 1traj 3.57 (0.72) 0.67 0.65 (0.20) 0.61 (0.26) 1traj-ar 5.16 (2.18) 0.62 0.42 (0.22) 0.56 (0.26) 2traj-ar 5.16 (2.22) 0.62 0.39 (0.21) 0.56 (0.27) 2traj-fl 3.49 (0.78) 0.68 0.61 (0.19) 0.62 (0.26) Table 5. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the AM1-BCC method to parameterize ligands. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error shown in brackets where appropriate. Results are provided for the diverse (DIV) dataset alone bound to protein models based on PDB 2OSS. MUE corrected for mean signed error in kcal mol−1. Protocol cMUE PI r rs 1traj 3.57 (0.72) 0.67 0.65 (0.20) 0.61 (0.26) 1traj-ar 5.16 (2.18) 0.62 0.42 (0.22) 0.56 (0.26) 2traj-ar 5.16 (2.22) 0.62 0.39 (0.21) 0.56 (0.27) 2traj-fl 3.49 (0.78) 0.68 0.61 (0.19) 0.62 (0.26) Table 5. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the AM1-BCC method to parameterize ligands. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error shown in brackets where appropriate. Results are provided for the diverse (DIV) dataset alone bound to protein models based on PDB 2OSS. MUE corrected for mean signed error in kcal mol−1. Table 5. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the AM1-BCC method to parameterize ligands. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error shown in brackets where appropriate. Results are provided for the diverse (DIV) dataset alone bound to protein models based on PDB 2OSS. MUE corrected for mean signed error in kcal mol−1. Inclusion of Explicit Water. Results Overall the results from the 2OSS structure are better than those from 4BJX. However, it should be noted that the ΔGMMPBSA values for all drugs using the 1traj protocol agree within error (see Fig. 5a). Robustness of Ranking to Parameterization. Two of the key decisions in ligand binding free energy calculations are the choices of the forcefield and how small molecules are parameterized. For simulations using Amber forcefields the choice of procedures for ligand preparation is usually whether to use AM1-BCC or Gaussian/RESP based protocols to determine atom charges in combination with the GAFF general purpose forcefield parameters. Following the choice in Wan et al.9 we used Gaussian/RESP for the majority of simula- tions in this work, but to evaluate the influence of this we re-ran the DIV dataset in the 2OSS model using the AM1-BCC methodology. Figure 6a shows that the ΔGMMPBSA values for the large majority of the ligands are highly correlated between the two schemes (within 1–2 kcal mol−1). This and the similar correlation with experi- ment (shown in Table 5) indicates that our results are robust with respect to this choice.hfi p The Wan et al.9 study employed the Amber ff99ildn forcefield for the protein, whilst in this study we have used ff14. In general the results obtained for all ligands are consistent but two ligands at either end of the rankings, THQ9 and THQ15, differ significantly as shown in Fig. 6b. The ranking performance with ff99ildn is described in Table 6. Comparing to those for ff14 (the THQ subset values in Table 4) shows ff99ildn provides better results, especially those for 2traj-ar in the 4BJX model (rs of 0.80 compared to 0.46). There are many factors which may cause this difference but one we identified was the possibility that the balance between direct and water mediated interactions might be altered by modifications to the amino acid side chain parameters. This in part motivated our investigation of the impact of including explicit water molecules in the receptor component of our calculations (see the following section). Results Protocol Dataset cMUE* PI r rs 2OSS Structure 1traj DIV + THQ 3.25 (0.51) 0.48 0.51 (0.16) 0.46 (0.18) DIV 3.38 (0.68) 0.74 0.64 (0.19) 0.69 (0.23) THQ 2.01 (0.37) 0.11 0.19 (0.22) 0.09 (0.27) 1traj-ar DIV + THQ 3.30 (0.49) 0.62 0.61 (0.14) 0.60 (0.14) DIV 3.98 (0.68) 0.75 0.67 (0.18) 0.72 (0.19) THQ 2.05 (0.30) 0.46 0.47 (0.17) 0.42 (0.22) 2traj-ar DIV + THQ 3.57 (0.54) 0.65 0.62 (0.12) 0.66 (0.11) DIV 4.09 (0.78) 0.66 0.62 (0.18) 0.64 (0.21) THQ 2.41 (0.67) 0.67 0.54 (0.13) 0.65 (0.17) 2traj-fl DIV + THQ 3.33 (0.59) 0.59 0.55 (0.14) 0.57 (0.14) DIV 3.41 (0.88) 0.75 0.59 (0.19) 0.72 (0.20) THQ 2.09 (0.55) 0.41 0.40 (0.17) 0.39 (0.23) 4BJX Structure 1traj DIV + THQ 3.32 (0.53) 0.48 0.50 (0.17) 0.46 (0.18) DIV 3.54 (0.80) 0.79 0.65 (0.18) 0.74 (0.20) THQ 2.14 (0.43) 0.07 0.11 (0.24) 0.04 (0.27) 1traj-ar DIV + THQ 4.05 (0.48) 0.52 0.56 (0.14) 0.49 (0.15) DIV 3.51 (0.78) 0.60 0.68 (0.18) 0.55 (0.27) THQ 2.55 (0.46) 0.24 0.30 (0.18) 0.16 (0.25) 2traj-ar DIV + THQ 3.97 (0.44) 0.61 0.63 (0.12) 0.60 (0.13) DIV 3.42 (0.82) 0.57 0.67 (0.17) 0.55 (0.24) THQ 2.70 (0.48) 0.51 0.51 (0.15) 0.46 (0.22) 2traj-fl DIV + THQ 3.46 (0.51) 0.54 0.56 (0.14) 0.52 (0.16) DIV 3.52 (0.86) 0.75 0.63 (0.18) 0.73 (0.20) THQ 2.20 (0.56) 0.27 0.36 (0.19) 0.22 (0.26) Table 4. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies, measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error provided in brackets where appropriate. Results are provided for the diverse (DIV) and tetrahydroquinoline (THQ) datasets and both combined (DIV + THQ). MUE corrected for mean signed error in kcal mol−1. Table 4. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies, measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error provided in brackets where appropriate. Results are provided for the diverse (DIV) and tetrahydroquinoline (THQ) datasets and both combined (DIV + THQ). MUE corrected for mean signed error in kcal mol−1. Protocol cMUE* PI r rs 1traj 3.57 (0.72) 0.67 0.65 (0.20) 0.61 (0.26) 1traj-ar 5.16 (2.18) 0.62 0.42 (0.22) 0.56 (0.26) 2traj-ar 5.16 (2.22) 0.62 0.39 (0.21) 0.56 (0.27) 2traj-fl 3.49 (0.78) 0.68 0.61 (0.19) 0.62 (0.26) Table 5. Results Results are provided for the THQ dataset alone bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Protocol cMUE PI r rs 2OSS Structure 1traj 2.16 (0.39) 0.35 0.31 (0.21) 0.28 (0.25) 1traj-ar 2.62 (0.51) 0.28 0.37 (0.18) 0.25 (0.24) 2traj-ar 2.45 (0.48) 0.60 0.54 (0.13) 0.58 (0.17) 2traj-fl 2.21 (0.36) 0.53 0.50 (0.17) 0.45 (0.23) 4BJX Structure 1traj 2.00 (0.38) 0.33 0.31 (0.21) 0.26 (0.24) 1traj-ar 1.73 (0.33) 0.69 0.62 (0.11) 0.58 (0.18) 2traj-ar 1.79 (0.41) 0.84 0.77 (0.07) 0.80 (0.10) 2traj-fl 2.09 (0.37) 0.55 0.51 (0.16) 0.47 (0.22) Table 6. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the ff99ildn protein forcefield. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error provided in brackets where appropriate. Results are provided for the THQ dataset alone bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Table 6. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the ff99ildn protein forcefield. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error provided in brackets where appropriate. Results are provided for the THQ dataset alone bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Table 6. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the ff99ildn protein forcefield. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error provided in brackets where appropriate. Results are provided for the THQ dataset alone bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Figure 5. Comparison of experimental and computed binding affinities for the combined DIV (circle) and THQ (triangle) datasets. Computational results were obtained using (a) 1traj and (b) 2traj-ar MMPBSA based ESMACS protocols. Results are shown for simulations initiated from models based on PDBs 2OSS (green) and 4BJX (purple). Solid lines represent lines of best fit, dashed ones optimal correlations. Figure 5. Results Aldeghi et al.22 found that the inclusion of explicit water molecules as part of the receptor in MMPBSA calculations improved the correlation with experiment in the DIV dataset. Here we explore whether this finding is reproducible using ensemble simulations and is robust to the addition of THQ ligands to the dataset under investigation. We use the same strategy in selecting water molecules for inclusion as the previous work, namely using the closest N to the ligand in each frame of the simulation trajectory.f We found a large difference in the impact of explicit water molecules between the combined DIV + THQ and DIV alone datasets. The correlations within the THQ dataset do not benefit from the inclusion of the additional water molecules in any protocol. For the combined dataset we find that up to around 5 explicit water molecules improves the rankings for all protocols (see Fig. 7a). After 50 water molecules are included 1traj performance drops to show no significant correlation with experiment and is only slightly improved as more molecules are added. A similar pattern is observed for the 1traj-ar and 2traj-ar results although, after the initial improvements, performance is more stable until 100 water molecules are included when an even sharper fall off is observed.h p pf For the DIV dataset as shown in Fig. 7b the improvements are yet more marked. The biggest improvement is seen in the 1traj results. Furthermore, the MUE for these rankings does not increase with adding more water near peak performance 3.38/3.54 for 0 and 3.08/3.08 for 5 water molecules (2OSS/4BJX). In line with the results of Aldeghi et al.22 Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 8 www.nature.com/scientificreports/ Protocol cMUE PI r rs 2OSS Structure 1traj 2.16 (0.39) 0.35 0.31 (0.21) 0.28 (0.25) 1traj-ar 2.62 (0.51) 0.28 0.37 (0.18) 0.25 (0.24) 2traj-ar 2.45 (0.48) 0.60 0.54 (0.13) 0.58 (0.17) 2traj-fl 2.21 (0.36) 0.53 0.50 (0.17) 0.45 (0.23) 4BJX Structure 1traj 2.00 (0.38) 0.33 0.31 (0.21) 0.26 (0.24) 1traj-ar 1.73 (0.33) 0.69 0.62 (0.11) 0.58 (0.18) 2traj-ar 1.79 (0.41) 0.84 0.77 (0.07) 0.80 (0.10) 2traj-fl 2.09 (0.37) 0.55 0.51 (0.16) 0.47 (0.22) Table 6. Performance of different MMPBSA based ESMACS protocols in reproducing experimental binding free energies using the ff99ildn protein forcefield. Performance is measured by mean unsigned error (MUE), Pearson’s predictivity index (PI), correlation coefficient (r) and Spearman’s rank coefficient (rs). Bootstrapped error provided in brackets where appropriate. Results Water 2OSS 4BJX cMUE* rs cMUE* rs 1traj 0 3.25 (0.51) 0.46 (0.18) 3.32 (0.53) 0.46 (0.18) 1 3.59 (0.45) 0.52 (0.16) 3.62 (0.46) 0.46 (0.17) 2 3.76 (0.47) 0.54 (0.16) 3.78 (0.48) 0.48 (0.17) 3 3.91 (0.48) 0.51 (0.16) 3.88 (0.49) 0.48 (0.16) 4 4.01 (0.50) 0.52 (0.16) 3.96 (0.50) 0.48 (0.16) 5 4.11 (0.52) 0.52 (0.15) 4.03 (0.50) 0.47 (0.17) 2traj-ar 0 3.57 (0.54) 0.66 (0.11) 3.97 (0.44) 0.60 (0.13) 1 3.70 (0.43) 0.65 (0.11) 4.14 (0.41) 0.64 (0.12) 2 3.96 (0.45) 0.70 (0.11) 4.62 (0.47) 0.67 (0.12) 3 4.28 (0.49) 0.68 (0.12) 5.04 (0.52) 0.67 (0.12) 4 4.57 (0.51) 0.68 (0.12) 5.35 (0.56) 0.66 (0.12) 5 4.79 (0.54) 0.68 (0.12) 5.59 (0.58) 0.66 (0.12) Table 7. Performance of 1traj and 2traj-ar MMPBSA based ESMACS protocols in reproducing experimental binding free energies incorporating different numbers of explicit water molecules. Performance is measured by mean unsigned error (MUE) and Spearman’s rank coefficient (rs) (bootstrapped error provided in brackets). Results are provided for the combined diverse (DIV) and THQ datasets bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Table 7. Performance of 1traj and 2traj-ar MMPBSA based ESMACS protocols in reproducing experimental binding free energies incorporating different numbers of explicit water molecules. Performance is measured by mean unsigned error (MUE) and Spearman’s rank coefficient (rs) (bootstrapped error provided in brackets). Results are provided for the combined diverse (DIV) and THQ datasets bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Table 7. Performance of 1traj and 2traj-ar MMPBSA based ESMACS protocols in reproducing experimental binding free energies incorporating different numbers of explicit water molecules. Performance is measured by mean unsigned error (MUE) and Spearman’s rank coefficient (rs) (bootstrapped error provided in brackets). Results are provided for the combined diverse (DIV) and THQ datasets bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Figure 6. Comparison of 1traj ESMACS results using different forcefield choices. In (a) the ranking of the DIV dataset is shown with the same protein forcefield (ff14) but different ligand parameterization methods (Gaussian-RESP in dark green, AM1-BCC in lighter green). Results Comparison of experimental and computed binding affinities for the combined DIV (circle) and THQ (triangle) datasets. Computational results were obtained using (a) 1traj and (b) 2traj-ar MMPBSA based ESMACS protocols. Results are shown for simulations initiated from models based on PDBs 2OSS (green) and 4BJX (purple). Solid lines represent lines of best fit, dashed ones optimal correlations. the peak performance has > . r 0 9 s ; however, unlike in the previous work here we see this at 2 water molecules included with a decline after 5 (as opposed to a peak at 20 and consistent performance thereafter). A number of factors could impact this including our use of ensembles of 4 ns trajectories (compared to single 16 ns runs) and Gaussian/RESP charges (as opposed to AM1-BCC). Overall though, it is important to retain at least four of the conserved water mole- cules in the binding site for the ESMACS calculations in order to obtain consistently good rankings across datasets. Moreover, the impact of adding water molecules differs between runs initiated with different starting structures, as shown in Table 7. the peak performance has > . r 0 9 s ; however, unlike in the previous work here we see this at 2 water molecules included with a decline after 5 (as opposed to a peak at 20 and consistent performance thereafter). A number of factors could impact this including our use of ensembles of 4 ns trajectories (compared to single 16 ns runs) and Gaussian/RESP charges (as opposed to AM1-BCC). Overall though, it is important to retain at least four of the conserved water mole- cules in the binding site for the ESMACS calculations in order to obtain consistently good rankings across datasets. Moreover, the impact of adding water molecules differs between runs initiated with different starting structures, as shown in Table 7. Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 9 www.nature.com/scientificreports/ Protocol No. Results Results are provided for the combined diverse (DIV) and THQ datasets bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Table 8. Performance of 1traj and 2traj-ar MMPBSA based ESMACS protocols in reproducing experimental binding free energies incorporating both variational entropy and different numbers of explicit water molecules. Performance is measured by mean unsigned error (MUE) and Spearman’s rank coefficient (rs) (bootstrapped error provided in brackets). Results are provided for the combined diverse (DIV) and THQ datasets bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Table 8. Performance of 1traj and 2traj-ar MMPBSA based ESMACS protocols in reproducing experimental binding free energies incorporating both variational entropy and different numbers of explicit water molecules. Performance is measured by mean unsigned error (MUE) and Spearman’s rank coefficient (rs) (bootstrapped error provided in brackets). Results are provided for the combined diverse (DIV) and THQ datasets bound to protein models based on both PDBs 2OSS and 4BJX. MUE corrected for mean signed error in kcal mol−1. Figure 7. Impact of the inclusion of explicit water molecules as part of the receptor in ESMACS calculations on the Spearman rank coefficient (rs), exhibited for both (a) combined DIV and THQ and (b) DIV alone datasets. Results are shown for simulations initiated from models based on PDBs 2OSS (green) and 4BJX (purple) and three protocols; 1traj (circles), 1traj-ar (triangles) and 2traj-ar (squares). Main figures show detailed view of the inclusion of up to 25 water molecules, inset shows how performance falls off as 50 or more water molecules are accounted for. Figure 7. Impact of the inclusion of explicit water molecules as part of the receptor in ESMACS calculations on the Spearman rank coefficient (rs), exhibited for both (a) combined DIV and THQ and (b) DIV alone datasets. Results are shown for simulations initiated from models based on PDBs 2OSS (green) and 4BJX (purple) and three protocols; 1traj (circles), 1traj-ar (triangles) and 2traj-ar (squares). Main figures show detailed view of the inclusion of up to 25 water molecules, inset shows how performance falls off as 50 or more water molecules are accounted for. from a lower baseline rapidly whilst those from 2OSS remain consistent until 5 water molecules are added, at which point the results from both structures give an rs of around 0.6. Results In (b) results for the THQ dataset are compared using the ff14 (dark green is based on PDB 2OSS, purple on 4BJX) and ff99ildn (cyan based on PDB 2OSS, orange on 4BJX) forcefields. Solid lines represent lines of best fit, dashed ones optimal correlations. Figure 6. Comparison of 1traj ESMACS results using different forcefield choices. In (a) the ranking of the DIV dataset is shown with the same protein forcefield (ff14) but different ligand parameterization methods (Gaussian-RESP in dark green, AM1-BCC in lighter green). In (b) results for the THQ dataset are compared using the ff14 (dark green is based on PDB 2OSS, purple on 4BJX) and ff99ildn (cyan based on PDB 2OSS, orange on 4BJX) forcefields. Solid lines represent lines of best fit, dashed ones optimal correlations. The combined DIV + THQ 4BJX 1traj ranking shows only a consistent result, with no improvement, as the first 5 water molecules were incorporated, whereas in 2OSS the ranking improves from an rs of 0.46 [CI: 0.16– 0.84] to 0.54 [CI: 0.16–0.84] after the first two water molecules are included. In 1traj-ar the 4BJX results improve Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 10 www.nature.com/scientificreports/ Protocol No. Water 2OSS 4BJX cMUE* rs cMUE* rs 1traj 0 2.86 (0.34) 0.34 (0.20) 2.87 (0.41) 0.35 (0.20) 2 4.66 (1.34) 0.19 (0.22) 4.37 (1.14) 0.24 (0.22) 5 5.07 (1.35) 0.14 (0.21) 4.80 (1.39) 0.18 (0.21) 2traj-ar 0 3.35 (0.51) 0.43 (0.16) 3.39 (0.41) 0.46 (0.16) 2 5.57 (1.37) 0.30 (0.19) 4.70 (0.99) 0.40 (0.19) 5 5.57 (1.48) 0.35 (0.20) 5.35 (1.11) 0.46 (0.18) Table 8. Performance of 1traj and 2traj-ar MMPBSA based ESMACS protocols in reproducing experimental b d f b h l d d ff b f l l l Protocol No. Water 2OSS 4BJX cMUE* rs cMUE* rs 1traj 0 2.86 (0.34) 0.34 (0.20) 2.87 (0.41) 0.35 (0.20) 2 4.66 (1.34) 0.19 (0.22) 4.37 (1.14) 0.24 (0.22) 5 5.07 (1.35) 0.14 (0.21) 4.80 (1.39) 0.18 (0.21) 2traj-ar 0 3.35 (0.51) 0.43 (0.16) 3.39 (0.41) 0.46 (0.16) 2 5.57 (1.37) 0.30 (0.19) 4.70 (0.99) 0.40 (0.19) 5 5.57 (1.48) 0.35 (0.20) 5.35 (1.11) 0.46 (0.18) Table 8. Performance of 1traj and 2traj-ar MMPBSA based ESMACS protocols in reproducing experimental binding free energies incorporating both variational entropy and different numbers of explicit water molecules. Performance is measured by mean unsigned error (MUE) and Spearman’s rank coefficient (rs) (bootstrapped error provided in brackets). Results A similar pattern is seen in 2traj-ar, but with the peak performance at 2 water molecules of 0.70 [CI: 0.54–0.97]/0.67 [CI: 0.49–0.96] (2OSS/4BJX) as shown in Table 7. The increase in MUE which accompanies the improvement in correlation indicates that the effects are not uniform across all ligands. Marginal gains in correlation coefficient should not be over emphasized (as can be seen in Table 7, improvements are often within error); we rather wish to draw attention to the trend that inclusion of water molecules likely to be involved in mediating stable ligand-protein interactions improves (or at least does not degrade) calculation performance. The most important observation is that the addition of explicit water mol- ecules improves the reproducibility of the ranking when using different starting models. from a lower baseline rapidly whilst those from 2OSS remain consistent until 5 water molecules are added, at which point the results from both structures give an rs of around 0.6. A similar pattern is seen in 2traj-ar, but with the peak performance at 2 water molecules of 0.70 [CI: 0.54–0.97]/0.67 [CI: 0.49–0.96] (2OSS/4BJX) as shown in Table 7. The increase in MUE which accompanies the improvement in correlation indicates that the effects are not uniform across all ligands. Marginal gains in correlation coefficient should not be over emphasized (as can be seen in Table 7, improvements are often within error); we rather wish to draw attention to the trend that inclusion of water molecules likely to be involved in mediating stable ligand-protein interactions improves (or at least does not degrade) calculation performance. The most important observation is that the addition of explicit water mol- ecules improves the reproducibility of the ranking when using different starting models. Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 11 www.nature.com/scientificreports/ Figure 8. Comparison of experimental and computed binding affinities incorporating variational entropy for the combined DIV (circle) and THQ (triangle) dataset. Computational results obtained using (a) 1traj and (b) 2traj-ar MMPBSA based ESMACS protocols. Results are shown for simulations initiated from models based on PDBs 2OSS (green) and 4BJX (purple). Solid lines represent lines of best fit, dashed ones optimal correlations. Figure 8. Comparison of experimental and computed binding affinities incorporating variational entropy for the combined DIV (circle) and THQ (triangle) dataset. Computational results obtained using (a) 1traj and (b) 2traj-ar MMPBSA based ESMACS protocols. Results Results are shown for simulations initiated from models based on PDBs 2OSS (green) and 4BJX (purple). Solid lines represent lines of best fit, dashed ones optimal correlations. Variational Entropy. Accounting correctly, and computationally efficiently, for the entropic component of binding free energies remains a challenge for MMPBSA based computations. Here we investigated the use of the variational entropy technique on the ranking of different ESMACS protocols. In all cases the variational entropy was computed using the fluctuations from the 1traj simulations. As shown in Table 8 the inclusion of this term results in a reduction in the performance of all protocols in simulations based on both initial models. Furthermore, the incorporation of explicit water molecules into the receptor reduces this to an even greater extent. Looking in more detail we see that some compounds suffer a deterioration in prediction whilst others manifest an improvement. For instance, Fig. 8 shows that the three DIV outliers 3, 4 and 5 are closer to the trend line than in Fig. 5, whereas THQ12 and THQ13 are more poorly predicted. The entropic term is based on the variation in interaction energy during the complex simulation. As it compares versus the average it captures properties of the interaction energy surface. For molecules such as 6, 8, 9 and 10 that have few degrees of freedom, the interaction energy surface is likely to be steep, with small changes in conformation or translations leading to a rapid loss of interaction energy. Meanwhile, larger more flexible compounds such as 4 and 5 (which has a flexi- ble benzhydryl core) can adapt to conformational changes of the receptor and maintain a favourable interaction energy, leading to a flatter potential surface. The results suggest that this entropic term is suited to the latter but not the former examples. Correctly capturing entropic contributions is key to obtaining truly reliable rankings in diverse datasets and further work in this area is required. Also, components of the MMPBSA calculation (par- ticularly the surface area term) incorporate some entropic contributions and such double counting may account at least in part for the poor performance of variational entropy here. www.nature.com/scientificreports/ significant despite the relatively modest size of the dataset (which contains a total of 27 ligands). It should be noted that increase in computational cost is minimal here as the only additional simulations required are of the ligand (which are much smaller than either complex or receptor) with the receptor energy replaced by a con- stant. Hence, for prospective day to day applications, we recommend accounting for both ligand and receptor strain through independent ligand simulations and either further simulation of the apo receptor (as in the 3 traj ESMACS protocol) or the use of an average value for the receptor energies (2traj-ar). p g p g j A key consideration in the use of binding free energy calculations in real world (industrial or clinical) settings is the reproducibility of the results. Other considerations include computational cost and calculation stability. ESMACS protocols offer advantages in both these regards as they make use of relatively simple and fast classical MD simulations compared to many parallel simulations of intermediate states as required in alchemical calcu- lations of absolute binding free energies44. We have shown that the results obtained in this study are robust to changing the ligand charge generation protocol (to use AM1-BCC instead of Gaussian/RESP) and the forcefield used to parameterize the protein (from Amber ff14SB to ff99SBildn). The use of ensemble simulations is the key to obtaining this reproducibility as individual replicas in ensembles varied by as much as 15 kcal mol−1 (which is in line with our own and other groups previous results9,16,18,45). Despite this, performance differences were found for all initial protocols when simulations were initiated from different crystal structures.h pf y This observation, along with the fact that some ligands which have very similar experimental binding ener- gies were widely separated even using protocols which accounted for receptor flexibility (1traj-ar and 2traj-ar), prompted us to investigate potential enhancements of the pure MMPBSA protocol. Specifically, we looked at the inclusion of an explicit ligand hydration shell in the receptor and variational entropy which had previously been investigated for single replica simulations by Aldeghi et al.22 (though they also only investigated what we would term “1traj” calculations). These additional components capture chemical and physical features of the system neglected by MMPBSA but at minimal computational cost, a key consideration for practical binding affinity cal- culation applications. www.nature.com/scientificreports/ The entropy term reduced extreme outliers but at the expense of decreased overall ranking performance. This observation replicates that obtained by Aldeghi et al.22 for the DIV dataset bound to BRD4, although they found the term improved results for sensitivity based datasets including multiple proteins. When less than five water molecules were incorporated into the receptor our rankings were improved with the best ranking across the full dataset obtained using this in combination with the 2traj-ar protocol. The most important observation of our work, however, is that the inclusion of these bound water molecules considerably reduced the performance difference between simulations initiated from models based on different crystal structures. A criticism of continuum based methods is that they are incapable of capturing the effect of crucial water molecules, possible activity cliffs, etc, that are now a well understood feature of structure-activity relationship (SAR) land- scapes and medicinal chemistry lead optimization. Here it is shown again how this challenge can be met, with the simple inclusion of explicit water molecules. Future work should address how to consider this in prospective application scenarios and in a wider range of protein targets.h pp g p g The reason for the improved performance observed for the diverse datasets in this study is presumably due to the capture of interactions between the ligand and the closest of the four conserved water molecules also found in the binding site. This observation is in line with other work in which system dependent numbers of water mole- cules were found to improve rankings46–49 and the broader phenomenon of the impact of crucial water molecules on SAR landscapes. Incorporation of the water molecules was highly effective in differentiating the ligands with diverse binding modes but less effective in the set of related THQ-scaffold based compounds. The fact that our observations fit a general pattern, and that the level of explicit water hydration which improves results is similar to the number of conserved water molecules suggests that the approach can be applied more generally. gg pp pp g y Overall we have shown that, for a diverse set of ligands, in order to deliver reproducible results from ESMACS (MMPBSA) calculations it is necessary to account for receptor and ligand strain and account explicitly for water molecules bound alongside ligands. Essential to obtaining these results is the use of ensemble simulations to gen- erate meaningfully quantified uncertainties. Data Availability Simulation input topologies and coordinates (alongside ligand parameters) for all protein-ligand systems and collated MMPBSA results are made available via Zenodo, 10.5281/zenodo.1484050. Trajectories are available from the corresponding author on reasonable request. Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 Discussion In summary, we have investigated the influence of different analysis choices on the results of ensemble MMPBSA based free energy calculations. The basis of our tests are two datasets which cover common computational chem- istry challenges - one which is based on a set of related ligands and the other a highly diverse set of ligands with differing binding modes. In order to obtain successful rankings across the two datasets we found it necessary to incorporate receptor and ligand strains. Using the 2traj-ar ESMACS protocol we obtained Spearman correlations of between 0.60 [CI: 0.46–0.91] and 0.66 [CI: 0.50–0.94] for two different starting structures despite differences in charge and scaffold in the ligands. The lower confidence bounds of both these estimates are comparable to the average correlation coefficient from the 1traj protocol 0.46 [CI: 0.16–0.84], suggesting the result is statistically Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758-1 12 www.nature.com/scientificreports/ www.nature.com/scientificreports/ W., Jha, S. & Coveney, P. V. Quantized water access to the HIV-1 protease active site as a proposed mechanism for cooperative mutations in drug affinity. Biochemistry (Mosc.) 51, 6487–6489 (2012).fi , , g , , J , y, Q p p p for cooperative mutations in drug affinity. Biochemistry (Mosc.) 51, 6487–6489 (2012).fi fi veney, P. V. Rapid and accurate ranking of binding affinities of e 29. Wan, S. & Coveney, P. V. Rapid and accurate ranking of binding affinities of epidermal growth factor receptor sequences with selected lung cancer drugs. J. R. Soc. Interface 8, 1114–1127, https://doi.org/10.1098/rsif.2010.0609 (2011).h 29. Wan, S. & Coveney, P. V. 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Journal of Chemical Theory and Computation 5, 422–429, https://doi.org/10.1021/ct8003707, PMID: 19461869 (200 Scientific Reports \| (2019) 9:6017 \| https://doi.org/10.1038/s41598-019-41758- 14 www.nature.com/scientificreports/ Acknowledgements g The authors thank the EU H2020 projects ComPat (http://www.compat-project.eu/, Grant No. 671564), CompBioMed (http://www.compbiomed.eu/, Grant No. 675451) and VECMA (http://www.vecma.eu/, Grant No. 800925), NSF Award (https://www.nsf.gov/pubs/2017/nsf17542/nsf17542.htm, Award No. NSF 1713749), the MRC Medical Bioinformatics project (MR/L016311/1), and funding from the UCL Provost. We made use of the BlueWaters supercomputer at the National Center for Supercomputing Applications of the University of Illinois at Urbana–Champaign https://bluewaters.ncsa.illinois.edu), access to which was made available through the aforementioned NSF award. We acknowledge the Leibniz Supercomputing Centre for providing access to SuperMUC (https://www.lrz.de/services/compute/) and the very able assistance of its scientific support staff. Additional calculation were conducted using an award of computer time on the Titan machine provided by the US Department of Energy’s Innovative and Novel Computational Impact on Theory and Experiment (INCITE) program (through the INSPIRE project). This research used resources of the Oak Ridge Leadership Computing Facility at the Oak Ridge National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC05-00OR22725. Author Contributions D.W.W. performed and analyzed simulations and wrote the main manuscript text. S.W. performed additional simulations and analysis. The study was designed by C.M., H.v.V., G.T., D.W.W. and P.V.C. All authors contributed to and reviewed the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-41758-1 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, 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. 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https://openalex.org/W1953541549	https://europepmc.org/articles/pmc3050219?pdf=render	English	null	4-Methyl-5-phenyl-1<i>H</i>-pyrazol-3(2<i>H</i>)-one	Acta crystallographica. Section E	2,010	cc-by	3,970	Table 1 Hydrogen bond geometry (A, ). D—H A D—H H A D A D—H A N1B—H1NB O1Ai 0.913 (17) 1.796 (17) 2.7001 (11) 170.0 (16) N1A—H1NA O1B 0.935 (19) 1.78 (2) 2.6987 (14) 165.9 (16) N2A—H2NA O1Aii 0.93 (2) 1.768 (19) 2.6917 (12) 173.9 (17) N2B—H2NB O1Biii 0.934 (18) 1.752 (18) 2.6850 (13) 177.0 (16) Symmetry codes: (i) x þ 1 2; y þ 1 2; z þ 1 2; (ii) x þ 1 2; y þ 1 2; z; (iii) x þ 1; y; z þ 1 2. ‡ Thomson Reuters ResearcherID: C-7581-2009. § Thomson Reuters ResearcherID: A-3561-2009. organic compounds organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Experimental Crystal data C10H10N2O Mr = 174.20 Monoclinic, C2=c a = 25.9337 (4) A˚ b = 10.8100 (1) A˚ c = 14.1426 (2) A˚ = 118.961 (1) V = 3468.98 (8) A˚ 3 Z = 16 Mo K radiation = 0.09 mm1 T = 100 K 0.45 0.39 0.25 mm Data collection Bruker SMART APEXII CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2009) Tmin = 0.961, Tmax = 0.978 36992 measured reﬂections 5087 independent reﬂections 4389 reﬂections with I > 2(I) Rint = 0.036 Reﬁnement R[F 2 > 2(F 2)] = 0.044 wR(F 2) = 0.119 S = 1.03 5087 reﬂections 253 parameters H atoms treated by a mixture of independent and constrained reﬁnement max = 0.45 e A˚ 3 min = 0.22 e A˚ 3 Wan-Sin Loh,a‡ Hoong-Kun Fun,a§ R. Venkat Ragavan,b V. Vijayakumarb and S. Sarveswarib aX-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia, and bOrganic Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, India Correspondence e-mail: hkfun@usm.my Experimental Crystal data C10H10N2O Mr = 174.20 Monoclinic, C2=c a = 25.9337 (4) A˚ b = 10.8100 (1) A˚ c = 14.1426 (2) A˚ = 118.961 (1) V = 3468.98 (8) A˚ 3 Z = 16 Mo K radiation = 0.09 mm1 T = 100 K 0.45 0.39 0.25 mm Data collection Bruker SMART APEXII CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2009) Tmin = 0.961, Tmax = 0.978 36992 measured reﬂections 5087 independent reﬂections 4389 reﬂections with I > 2(I) Rint = 0.036 Reﬁnement R[F 2 > 2(F 2)] = 0.044 wR(F 2) = 0.119 S = 1.03 5087 reﬂections 253 parameters H atoms treated by a mixture of independent and constrained reﬁnement max = 0.45 e A˚ 3 min = 0.22 e A˚ 3 Received 8 December 2010; accepted 12 December 2010 Received 8 December 2010; accepted 12 December 2010 Key indicators: single-crystal X-ray study; T = 100 K; mean (C–C) = 0.002 A˚; R factor = 0.044; wR factor = 0.119; data-to-parameter ratio = 20.1. Key indicators: single-crystal X-ray study; T = 100 K; mean (C–C) = 0.002 A˚; R factor = 0.044; wR factor = 0.119; data-to-parameter ratio = 20.1. The asymmetric unit of the title compound, C10H10N2O, contains two crystallographically independent molecules with similar geometries, which exist in the keto form. The C O bond lengths are 1.2878 (12) A˚ in molecule A and 1.2890 (12) A˚ in molecule B, indicating that the compound undergoes enol-to-keto tautomerism during the crystallization process. In molecule A, the pyrazole ring is approximately planar [maximum deviation = 0.007 (1) A˚ ] and forms a dihedral angle of 36.67 (6) with the attached phenyl ring. In molecule B, the dihedral angle formed between the pyrazole ring [maximum deviation = 0.017 (1) A˚ ] and the phenyl ring is 41.19 (6). In the crystal, intermolecular N—H O hydrogen bonds link neighbouring molecules into dimers generating R2 2(8) ring motifs. These dimers are linked into ribbons along [101] via intermolecular N—H O hydrogen bonds, forming R4 2(10) ring motifs. Table 1 Hydrogen-bond geometry (A˚ , ). Acta Cryst. (2011). E67, o151–o152 Related literature For background to pyrazole derivatives and their anti- microbial activity, see: Ragavan et al. (2009, 2010). For bond- length data, see: Allen et al. (1987). For the structure of the enol form of this molecule, see: Shahani et al. (2010). For other related structures, see: Loh et al. (2010a,b,c). For hydrogen- bond motifs, see: Bernstein et al. (1995). For the stability of the temperature controller used in the data collection, see: Cosier & Glazer (1986). Data collection: APEX2 (Bruker, 2009); cell reﬁnement: SAINT (Bruker, 2009); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL and PLATON (Spek, 2009). HKF and WSL thank Universiti Sains Malaysia (USM) for the Research University Grant (1001/PFIZIK/811160). WSL also thanks the Malaysian government and USM for the award of a Research Fellowship. VV is grateful to the DST–India for funding through the Young Scientist Scheme (Fast Track Proposal). Loh et al. o151 o151 Acta Cryst. (2011). E67, o151–o152 Acta Cryst. (2011). E67, o151–o152 Loh et al. doi:10.1107/S160053681005213X organic compounds Loh, W.-S., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Sarveswari, S. (2010a). Acta Cryst. E66, o2925. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: SJ5074). Loh, W.-S., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Venkatesh, M. (2010b). Acta Cryst. E66, o2563–o2564. Loh, W.-S., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Venkatesh, M. (2010c). Acta Cryst. E66, o3050–o3051. Acta Cryst. (2011). E67, o151–o152 Loh, W.-S., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Sarveswari, S. (2010a). Acta Cryst. E66, o2925. Loh, W.-S., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Venkatesh, M. (2010b). Acta Cryst. E66, o2563–o2564. Loh, W.-S., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Venkatesh, M. (2010c). Acta Cryst. E66, o3050–o3051. Ragavan, R. V., Vijayakumar, V. & Sucheta Kumari, N. (2009). Eur. J. Med. Chem. 44, 3852–3857. Ragavan, R. V., Vijayakumar, V. & Sucheta Kumari, N. (2010). Eur. J. Med. Chem. 45, 1173–1180. Shahani, T., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Sarveswari, S. (2010). Acta Cryst. E66, o1697–o1698. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Loh, W.-S., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Sarveswari, S. (2010a). Acta Cryst. E66, o2925. Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. Bruker (2009). APEXII, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Cosier, J. & Glazer, A. M. (1986). J. Appl. Cryst. 19, 105–107. References Ragavan, R. V., Vijayakumar, V. & Sucheta Kumari, N. (2009). Eur. J. Med. Chem. 44, 3852–3857. Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. Bruker (2009). APEXII, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Cosier, J. & Glazer, A. M. (1986). J. Appl. Cryst. 19, 105–107. Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19. Ragavan, R. V., Vijayakumar, V. & Sucheta Kumari, N. (2010). Eur. J. Med. Chem. 45, 1173–1180. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Sarveswari Shahani, T., Fun, H.-K., Ragavan, R. V., Vijayakumar, V. & Sarveswari, S. (2010). Acta Cryst. E66, o1697–o1698. g Bruker (2009). APEXII, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Cosier, J. & Glazer, A. M. (1986). J. Appl. Cryst. 19, 105–107. o152 Loh et al. C10H10N2O Acta Cryst. (2011). E67, o151–o152 Comment Antibacterial and antifungal activities of the azoles are most widely studied and some of them are in clinical practice as anti-microbial agents. However, the azole-resistant strains have led to the development of new anti-microbial compounds. In particular, pyrazole derivatives are extensively studied and used as anti-microbial agents. Pyrazoles represent an important class of heterocyclic compounds and many pyrazole derivatives are reported to have a broad spectrum of biological prop- erties such as anti-inflammatory, antifungal, herbicidal, anti-tumour, cytotoxic, molecular modelling and antiviral activities. Pyrazole derivatives also act as anti-angiogenic agents, A3 adenosine receptor antagonists, neuropeptide YY5 receptor ant- agonists as well as kinase inhibitors for the treatment of type 2 diabetes, hyperlipidemia, obesity and thrombopiotinmimetics. Recently urea derivatives of pyrazoles have been reported as potent inhibitors of p38 kinase. Since the high electronegativity of halogens (particularly chlorine and fluorine) in the aromatic part of the drug molecules plays an important role in enhan- cing their biological activity, we are interested to have 4-fluoro or 4-chloro substitution in the aryls of 1,5-diaryl pyrazoles. These properties and applications are discussed in our previous reports on the synthesis of novel pyrazole derivatives and their microbial activities (Ragavan et al., 2009, 2010). The enol-form of this compound has been already reported in the literature (Shahani et al., 2010). The title compound (Fig. 1), consists of two crystallographically independent molecules, with similar geometries and exists in the keto-form. This indicates that the compound undergoes an enol-to-keto tautomerism during the crystallization process with the bond length of C═O being 1.2878 (12) Å in molecule A and 1.2890 (12) Å in molecule B. In molecule A, the pyrazole ring (N1A/N2A/C7A–C9A) is approximately planar (maximum deviation of 0.007 (1) Å at N1A) and forms a dihedral angle of 36.67 (6)° with the attached phenyl ring (C1A–C6A). In molecule B, the dihedral angle formed between the pyrazole ring (N1B/N2B/C7B–C9B) [maximum deviation of 0.017 (1) Å at C9B] and the phenyl ring (C1B–C6B) is 41.19 (6)°. Bond lengths (Allen et al., 1987) and angles are within the normal ranges and are comparable to the related structures (Loh et al., 2010a,b,c). In the crystal packing (Fig. 2), intermolecular N2A—H2NA···O1A and N2B—H2NB···O1B hydrogen bonds (Table 1) link the neighbouring molecules to form dimers, generating R22(8) ring motifs (Bernstein et al., 1995). Comment These set of dimers are linked into ribbons along the [101], via intermolecular N1A—H1NA···O1B and N1B—H1NB···O1A hydrogen bonds (Table 1), forming R42(10) ring motifs (Bernstein et al., 1995). 4-Methyl-5-phenyl-1H-pyrazol-3(2H)-one W.-S. Loh, H.-K. Fun, R. V. Ragavan, V. Vijayakumar and S. Sarveswari The compound was synthesized using a literature method (Ragavan et al., 2009, 2010) and recrystallized from ethanol-chlo- roform; 1:1. M. p.: 493–494 K, yield: 72%. Acta Cryst. (2011). E67, o151-o152 [ doi:10.1107/S160053681005213X ] Acta Cryst. (2011). E67, o151-o152 [ doi:10.1107/S160053681005213X ] Acta Cryst. (2011). E67, o151-o152 [ doi:10.1107/S160053681005213X ] supplementary materials supplementary materials Experimental The compound was synthesized using a literature method (Ragavan et al., 2009, 2010) and recrystallized from ethanol-chlo- roform; 1:1. M. p.: 493–494 K, yield: 72%. sup-1 supplementary materials Refinement N– bound H atoms were located from a difference Fourier map and refined freely [N–H = 0.913 (17) to 0.935 (16) Å]. The remaining H atoms were positioned geometrically with bond lengths C–H = 0.93 to 0.96 Å and were refined using a riding model, with Uiso(H) = 1.2 or 1.5 Ueq(C). A rotating group model was applied to the methyl groups. Figures Fig. 1. The molecular structure of the title compound, showing 50% probability displacemen ellipsoids and the atom-numbering scheme. Fig. 2. The crystal packing of the title compound, viewed along the b axis. H atoms not in- volved in the intermolecular interactions (dashed lines) have been omitted for clarity. 4-Methyl-5-phenyl-1H-pyrazol-3(2H)-one Crystal data C10H10N2O F(000) = 1472 Mr = 174.20 Dx = 1.334 Mg m−3 Monoclinic, C2/c Mo Kα radiation, λ = 0.71073 Å Hall symbol: -C 2yc Cell parameters from 9946 reflections a = 25.9337 (4) Å θ = 2.4–30.1° b = 10.8100 (1) Å µ = 0.09 mm−1 c = 14.1426 (2) Å T = 100 K β = 118.961 (1)° Block, colourless V = 3468.98 (8) Å3 0.45 × 0.39 × 0.25 mm Z = 16 Data collection Bruker SMART APEXII CCD area-detector diffractometer 5087 independent reflections Radiation source: fine-focus sealed tube 4389 reflections with I > 2σ(I) graphite Rint = 0.036 Figures Fig. 1. The molecular structure of the title compound, showing 50% probability displaceme ellipsoids and the atom-numbering scheme. Fig. 2. The crystal packing of the title compound, viewed along the b axis. H atoms not in- volved in the intermolecular interactions (dashed lines) have been omitted for clarity. 4-Methyl-5-phenyl-1H-pyrazol-3(2H)-one Fig. 1. The molecular structure of the title compound, showing 50% probability displacement ellipsoids and the atom-numbering scheme. Fig. 2. The crystal packing of the title compound, viewed along the b axis. H atoms not in- volved in the intermolecular interactions (dashed lines) have been omitted for clarity. supplementary materials (SADABS; Bruker, 2009) Tmin = 0.961, Tmax = 0.978 36992 measured reflections k = −15→15 l = −19→18 Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.044 wR(F2) = 0.119 S = 1.03 5087 reflections 253 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo 2) + (0.0684P)2 + 2.050P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.45 e Å−3 Δρmin = −0.22 e Å−3 Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo 2) + (0.0684P)2 + 2.050P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.45 e Å−3 Δρmin = −0.22 e Å−3 Refinement 4-Methyl-5-phenyl-1H-pyrazol-3(2H)-one Crystal data C10H10N2O F(000) = 1472 Mr = 174.20 Dx = 1.334 Mg m−3 Monoclinic, C2/c Mo Kα radiation, λ = 0.71073 Å Hall symbol: -C 2yc Cell parameters from 9946 reflections a = 25.9337 (4) Å θ = 2.4–30.1° b = 10.8100 (1) Å µ = 0.09 mm−1 c = 14.1426 (2) Å T = 100 K β = 118.961 (1)° Block, colourless V = 3468.98 (8) Å3 0.45 × 0.39 × 0.25 mm Z = 16 Data collection Bruker SMART APEXII CCD area-detector diffractometer 5087 independent reflections Radiation source: fine-focus sealed tube 4389 reflections with I > 2σ(I) graphite Rint = 0.036 φ and ω scans θmax = 30.1°, θmin = 1.8° Absorption correction: multi-scan h = −36→36 F(000) = 1472 Dx = 1.334 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 9946 reflections θ = 2.4–30.1° µ = 0.09 mm−1 T = 100 K Block, colourless 0.45 × 0.39 × 0.25 mm sup-2 supplementary materials supplementary materials sup-4 H5AA 0.2794 0.0391 0.4211 0.023 C6A 0.34665 (4) 0.06155 (9) 0.38784 (8) 0.01417 (19) C7A 0.31075 (4) 0.10488 (9) 0.27595 (8) 0.01305 (19) C8A 0.25384 (4) 0.07360 (9) 0.19815 (8) 0.01399 (19) C9A 0.24055 (4) 0.14984 (9) 0.10712 (8) 0.01324 (19) C10A 0.21385 (4) −0.02449 (10) 0.20107 (9) 0.0171 (2) H10A 0.2367 −0.0958 0.2385 0.026* H10B 0.1855 −0.0472 0.1286 0.026* H10C 0.1937 0.0066 0.2380 0.026* O1B 0.43937 (3) 0.28341 (7) 0.28820 (6) 0.01641 (16) N1B 0.58492 (4) 0.29961 (8) 0.48713 (7) 0.01537 (18) N2B 0.54157 (4) 0.29083 (8) 0.38216 (7) 0.01525 (18) C1B 0.64581 (5) 0.25037 (10) 0.72121 (9) 0.0177 (2) H1BA 0.6549 0.1929 0.6825 0.021* C2B 0.68154 (5) 0.26106 (11) 0.83201 (9) 0.0225 (2) H2BA 0.7144 0.2102 0.8676 0.027* C3B 0.66848 (5) 0.34762 (12) 0.89026 (10) 0.0233 (2) H3BA 0.6926 0.3549 0.9645 0.028* C4B 0.61925 (5) 0.42316 (11) 0.83712 (9) 0.0208 (2) H4BA 0.6107 0.4816 0.8758 0.025* C5B 0.58278 (5) 0.41160 (10) 0.72656 (9) 0.0168 (2) H5BA 0.5494 0.4610 0.6916 0.020* C6B 0.59605 (4) 0.32598 (9) 0.66741 (8) 0.01363 (19) C7B 0.55927 (4) 0.31664 (9) 0.54975 (8) 0.01354 (19) C8B 0.49830 (4) 0.31808 (9) 0.48467 (8) 0.01383 (19) C9B 0.48786 (4) 0.29746 (9) 0.37751 (8) 0.01359 (19) C10B 0.45201 (4) 0.34052 (10) 0.51676 (9) 0.0184 (2) H10D 0.4627 0.2991 0.5838 0.028* H10E 0.4149 0.3091 0.4617 0.028* H10F 0.4487 0.4277 0.5254 0.028* H1NB 0.6234 (7) 0.3100 (16) 0.5038 (13) 0.034 (4)* H1NA 0.3696 (7) 0.2271 (15) 0.2649 (13) 0.030 (4)* H2NA 0.2954 (8) 0.2691 (17) 0.0857 (14) 0.042 (5)* H2NB 0.5490 (7) 0.2858 (15) 0.3239 (13) 0.032 (4)* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1A 0.0111 (3) 0.0236 (4) 0.0122 (4) −0.0017 (3) 0.0041 (3) 0.0009 (3) N1A 0.0108 (4) 0.0179 (4) 0.0116 (4) −0.0012 (3) 0.0034 (3) 0.0017 (3) N2A 0.0099 (4) 0.0182 (4) 0.0109 (4) −0.0012 (3) 0.0031 (3) 0.0018 (3) C1A 0.0151 (5) 0.0252 (5) 0.0193 (6) 0.0041 (4) 0.0084 (4) 0.0052 (4) C2A 0.0156 (5) 0.0334 (6) 0.0210 (6) 0.0065 (4) 0.0051 (4) 0.0075 (5) C3A 0.0239 (5) 0.0223 (5) 0.0155 (5) 0.0037 (4) 0.0057 (4) 0.0052 (4) C4A 0.0249 (5) 0.0220 (5) 0.0180 (5) 0.0000 (4) 0.0122 (5) 0.0030 (4) C5A 0.0163 (5) 0.0224 (5) 0.0179 (5) −0.0006 (4) 0.0083 (4) 0.0020 (4) C6A 0.0138 (4) 0.0135 (4) 0.0135 (5) 0.0002 (3) 0.0053 (4) 0.0003 (3) C7A 0.0117 (4) 0.0148 (4) 0.0128 (5) 0.0005 (3) 0.0061 (4) 0.0005 (3) Special details Experimental. The crystal was placed in the cold stream of an Oxford Cryosystems Cobra open-flow nitrogen cryostat (Cosier & Glazer, 1986) operating at 100.0 (1) K. Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq O1A 0.19371 (3) 0.15583 (7) 0.01430 (6) 0.01628 (16) N1A 0.33170 (4) 0.19356 (8) 0.23566 (7) 0.01429 (17) N2A 0.28884 (4) 0.22212 (8) 0.13372 (7) 0.01378 (17) C1A 0.40804 (5) 0.05241 (11) 0.43463 (9) 0.0198 (2) H1AA 0.4262 0.0681 0.3931 0.024 C2A 0.44188 (5) 0.02010 (12) 0.54257 (10) 0.0249 (2) H2AA 0.4826 0.0147 0.5730 0.030* C3A 0.41532 (5) −0.00418 (11) 0.60540 (10) 0.0222 (2) H3AA 0.4382 −0.0251 0.6779 0.027* C4A 0.35439 (5) 0.00296 (10) 0.55947 (9) 0.0209 (2) H4AA 0.3364 −0.0139 0.6012 0.025* C5A 0.32019 (5) 0.03512 (10) 0.45141 (9) 0.0188 (2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 supplementary materials supplementary materials Atomic displacement parameters (Å2) sup-4 supplementary materials C8A 0.0121 (4) 0.0157 (4) 0.0143 (5) −0.0006 (3) 0.0065 (4) −0.0001 (3) C9A 0.0102 (4) 0.0165 (4) 0.0128 (5) −0.0005 (3) 0.0054 (4) −0.0010 (3) C10A 0.0145 (4) 0.0183 (5) 0.0176 (5) −0.0039 (3) 0.0071 (4) 0.0001 (4) O1B 0.0108 (3) 0.0237 (4) 0.0127 (4) −0.0020 (3) 0.0041 (3) −0.0001 (3) N1B 0.0097 (4) 0.0232 (4) 0.0108 (4) −0.0011 (3) 0.0030 (3) −0.0021 (3) N2B 0.0110 (4) 0.0226 (4) 0.0110 (4) −0.0009 (3) 0.0044 (3) −0.0014 (3) C1B 0.0163 (4) 0.0181 (5) 0.0158 (5) 0.0025 (4) 0.0056 (4) 0.0000 (4) C2B 0.0198 (5) 0.0258 (5) 0.0163 (6) 0.0041 (4) 0.0042 (4) 0.0042 (4) C3B 0.0227 (5) 0.0324 (6) 0.0121 (5) −0.0028 (4) 0.0062 (4) −0.0006 (4) C4B 0.0222 (5) 0.0254 (5) 0.0177 (5) −0.0039 (4) 0.0119 (4) −0.0056 (4) C5B 0.0151 (4) 0.0192 (5) 0.0160 (5) 0.0003 (3) 0.0073 (4) −0.0017 (4) C6B 0.0121 (4) 0.0155 (4) 0.0118 (5) −0.0014 (3) 0.0046 (4) −0.0004 (3) C7B 0.0127 (4) 0.0142 (4) 0.0130 (5) 0.0005 (3) 0.0056 (4) −0.0004 (3) C8B 0.0123 (4) 0.0157 (4) 0.0131 (5) 0.0001 (3) 0.0058 (4) 0.0002 (3) C9B 0.0116 (4) 0.0141 (4) 0.0146 (5) −0.0004 (3) 0.0060 (4) 0.0006 (3) C10B 0.0141 (4) 0.0243 (5) 0.0182 (5) −0.0004 (4) 0.0089 (4) −0.0020 (4) Geometric parameters (Å, °) O1A—C9A 1.2878 (12) O1B—C9B 1.2890 (12) N1A—C7A 1.3560 (13) N1B—C7B 1.3533 (13) N1A—N2A 1.3628 (12) N1B—N2B 1.3640 (12) N1A—H1NA 0.935 (16) N1B—H1NB 0.913 (17) N2A—C9A 1.3655 (12) N2B—C9B 1.3641 (12) N2A—H2NA 0.928 (19) N2B—H2NB 0.933 (17) C1A—C2A 1.3875 (16) C1B—C2B 1.3861 (16) C1A—C6A 1.4006 (14) C1B—C6B 1.4004 (14) C1A—H1AA 0.9300 C1B—H1BA 0.9300 C2A—C3A 1.3878 (17) C2B—C3B 1.3926 (17) C2A—H2AA 0.9300 C2B—H2BA 0.9300 C3A—C4A 1.3896 (16) C3B—C4B 1.3901 (17) C3A—H3AA 0.9300 C3B—H3BA 0.9300 C4A—C5A 1.3893 (16) C4B—C5B 1.3866 (16) C4A—H4AA 0.9300 C4B—H4BA 0.9300 C5A—C6A 1.3994 (14) C5B—C6B 1.3979 (14) C5A—H5AA 0.9300 C5B—H5BA 0.9300 C6A—C7A 1.4708 (14) C6B—C7B 1.4668 (14) C7A—C8A 1.3895 (13) C7B—C8B 1.3920 (13) C8A—C9A 1.4233 (14) C8B—C9B 1.4221 (14) C8A—C10A 1.4978 (13) C8B—C10B 1.4946 (14) C10A—H10A 0.9600 C10B—H10D 0.9600 C10A—H10B 0.9600 C10B—H10E 0.9600 C10A—H10C 0.9600 C10B—H10F 0.9600 C7A—N1A—N2A 108.49 (8) C7B—N1B—N2B 108.33 (8) C7A—N1A—H1NA 129.6 (10) C7B—N1B—H1NB 129.5 (11) N2A—N1A—H1NA 121.6 (10) N2B—N1B—H1NB 120.7 (11) N1A—N2A—C9A 109.34 (8) N1B—N2B—C9B 109.45 (9) N1A—N2A—H2NA 123.7 (11) N1B—N2B—H2NB 123.4 (10) C9A—N2A—H2NA 125.5 (11) C9B—N2B—H2NB 127.0 (10) sup-5 supplementary materials supplementary materials sup-6 C2A—C1A—C6A 120.42 (10) C2B—C1B—C6B 120.09 (10) C2A—C1A—H1AA 119.8 C2B—C1B—H1BA 120.0 C6A—C1A—H1AA 119.8 C6B—C1B—H1BA 120.0 C1A—C2A—C3A 120.42 (10) C1B—C2B—C3B 120.25 (10) C1A—C2A—H2AA 119.8 C1B—C2B—H2BA 119.9 C3A—C2A—H2AA 119.8 C3B—C2B—H2BA 119.9 C2A—C3A—C4A 119.66 (11) C4B—C3B—C2B 119.85 (11) C2A—C3A—H3AA 120.2 C4B—C3B—H3BA 120.1 C4A—C3A—H3AA 120.2 C2B—C3B—H3BA 120.1 C5A—C4A—C3A 120.25 (10) C5B—C4B—C3B 120.20 (10) C5A—C4A—H4AA 119.9 C5B—C4B—H4BA 119.9 C3A—C4A—H4AA 119.9 C3B—C4B—H4BA 119.9 C4A—C5A—C6A 120.49 (10) C4B—C5B—C6B 120.22 (10) C4A—C5A—H5AA 119.8 C4B—C5B—H5BA 119.9 C6A—C5A—H5AA 119.8 C6B—C5B—H5BA 119.9 C5A—C6A—C1A 118.74 (10) C5B—C6B—C1B 119.37 (10) C5A—C6A—C7A 120.29 (9) C5B—C6B—C7B 120.73 (9) C1A—C6A—C7A 120.89 (9) C1B—C6B—C7B 119.89 (9) N1A—C7A—C8A 109.11 (9) N1B—C7B—C8B 109.25 (9) N1A—C7A—C6A 120.23 (9) N1B—C7B—C6B 119.73 (9) C8A—C7A—C6A 130.60 (9) C8B—C7B—C6B 130.99 (9) C7A—C8A—C9A 105.93 (8) C7B—C8B—C9B 105.79 (8) C7A—C8A—C10A 129.40 (9) C7B—C8B—C10B 128.55 (10) C9A—C8A—C10A 124.56 (9) C9B—C8B—C10B 125.63 (9) O1A—C9A—N2A 122.59 (9) O1B—C9B—N2B 121.99 (9) O1A—C9A—C8A 130.31 (9) O1B—C9B—C8B 130.90 (9) N2A—C9A—C8A 107.10 (9) N2B—C9B—C8B 107.09 (9) C8A—C10A—H10A 109.5 C8B—C10B—H10D 109.5 C8A—C10A—H10B 109.5 C8B—C10B—H10E 109.5 H10A—C10A—H10B 109.5 H10D—C10B—H10E 109.5 C8A—C10A—H10C 109.5 C8B—C10B—H10F 109.5 H10A—C10A—H10C 109.5 H10D—C10B—H10F 109.5 H10B—C10A—H10C 109.5 H10E—C10B—H10F 109.5 C7A—N1A—N2A—C9A −1.33 (11) C7B—N1B—N2B—C9B −2.31 (11) C6A—C1A—C2A—C3A 0.33 (18) C6B—C1B—C2B—C3B −0.54 (17) C1A—C2A—C3A—C4A 0.55 (19) C1B—C2B—C3B—C4B 0.32 (18) C2A—C3A—C4A—C5A −0.48 (18) C2B—C3B—C4B—C5B 0.73 (17) C3A—C4A—C5A—C6A −0.47 (17) C3B—C4B—C5B—C6B −1.54 (16) C4A—C5A—C6A—C1A 1.33 (16) C4B—C5B—C6B—C1B 1.30 (15) C4A—C5A—C6A—C7A −175.25 (10) C4B—C5B—C6B—C7B −177.31 (9) C2A—C1A—C6A—C5A −1.26 (16) C2B—C1B—C6B—C5B −0.27 (15) C2A—C1A—C6A—C7A 175.29 (10) C2B—C1B—C6B—C7B 178.36 (10) N2A—N1A—C7A—C8A 1.22 (11) N2B—N1B—C7B—C8B 0.43 (11) N2A—N1A—C7A—C6A −176.28 (8) N2B—N1B—C7B—C6B 178.45 (8) C5A—C6A—C7A—N1A 141.18 (10) C5B—C6B—C7B—N1B 139.78 (10) C1A—C6A—C7A—N1A −35.32 (14) C1B—C6B—C7B—N1B −38.83 (14) C5A—C6A—C7A—C8A −35.71 (16) C5B—C6B—C7B—C8B −42.70 (16) C1A—C6A—C7A—C8A 147.80 (11) C1B—C6B—C7B—C8B 138.69 (11) N1A—C7A—C8A—C9A −0.64 (11) N1B—C7B—C8B—C9B 1.50 (11) sup-6 supplementary materials supplementary materials C6A—C7A—C8A—C9A 176.51 (10) C6B—C7B—C8B—C9B −176.23 (10) N1A—C7A—C8A—C10A 175.59 (10) N1B—C7B—C8B—C10B −176.60 (10) C6A—C7A—C8A—C10A −7.25 (18) C6B—C7B—C8B—C10B 5.67 (18) N1A—N2A—C9A—O1A −178.72 (9) N1B—N2B—C9B—O1B −175.40 (9) N1A—N2A—C9A—C8A 0.91 (11) N1B—N2B—C9B—C8B 3.21 (11) C7A—C8A—C9A—O1A 179.42 (10) C7B—C8B—C9B—O1B 175.58 (10) C10A—C8A—C9A—O1A 2.96 (17) C10B—C8B—C9B—O1B −6.24 (17) C7A—C8A—C9A—N2A −0.17 (11) C7B—C8B—C9B—N2B −2.85 (11) C10A—C8A—C9A—N2A −176.63 (9) C10B—C8B—C9B—N2B 175.32 (10) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N1B—H1NB···O1Ai 0.913 (17) 1.796 (17) 2.7001 (11) 170.0 (16) N1A—H1NA···O1B 0.935 (19) 1.78 (2) 2.6987 (14) 165.9 (16) N2A—H2NA···O1Aii 0.93 (2) 1.768 (19) 2.6917 (12) 173.9 (17) N2B—H2NB···O1Biii 0.934 (18) 1.752 (18) 2.6850 (13) 177.0 (16) Symmetry codes: (i) x+1/2, −y+1/2, z+1/2; (ii) −x+1/2, −y+1/2, −z; (iii) −x+1, y, −z+1/2. Hydrogen-bond geometry (Å, °) sup-7 supplementary materials Fig. 1 Fig. 1 Fig. 1 sup-8 supplementary materials Fig. 2 sup-9
https://openalex.org/W2134149037	https://www.scielo.br/j/mr/a/ydBtXpLwZ8b9pkDmvHqgm8w/?lang=en&format=pdf	English	null	Characterization of the physical and mechanical properties of femoral bone defects filled with polyanionic collagen scaffolds in ovariectomized rats	Materials research	2,010	cc-by	5,056	Received: January 9, 2010; Revised: May 13, 2010 The aim of this study was to evaluate the effect of scaffolds native or polyanionic collagen matrix (submitted to alkaline treatment for 48 or 96 hours, PCM48 or PCM96, respectively) on the repair of osteoporosis bone fractures resulting from the gonadal hormone alterations caused by ovariectomy in rats undergoing hormone replacement therapy. The physical and mechanical characteristics of bone were analyzed. Macroscopic analysis revealed the absence of pathological alterations in the implanted areas. The percent mineral matter and bone mineral density of the femurs were lower in ovariectomized rats. The mechanical strength of newly formed bone was greater in the area receiving the PCM96 scaffolds compared to the area implanted with the native scaffolds. The PCM96 scaffold is the best choice for bone repair in animals with hormone deficiency since it promotes faster bone growth and good mechanical strength. Keywords: bone healing, hormone deficiency, osteoporosis, ovariectomy, polyanionic collagen *e-mail: arnaldo.santos@ufabc.edu.br © 2010 © 2010 Materials Research. 2010; 13(2): 239-244 Characterization of the Physical and Mechanical Properties of Femoral Bone Defects Filled with Polyanionic Collagen Scaffolds in Ovariectomized Rats Characterization of the Physical and Mechanical Properties of Femoral Bone Defects Filled with Polyanionic Collagen Scaffolds in Ovariectomized Rats aDepartamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil bDepartamento de Morfologia e Patologia, Faculdade de Medicina de Jundiaí, Jundiaí, SP, Brasil cCentro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP, Brasil dBiotech Biomédica Produtos Médicos e Odontológico Ltda ME, São Carlos, SP, Brasil eDepartamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP, Brasil fCentro Estadual de Educação Tecnológica Paula Souza, Faculdade de Tecnologia de Bauru, Bauru, SP, Brasil aDepartamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil bDepartamento de Morfologia e Patologia, Faculdade de Medicina de Jundiaí, Jundiaí, SP, Brasil cCentro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP, Brasil dBiotech Biomédica Produtos Médicos e Odontológico Ltda ME, São Carlos, SP, Brasil eDepartamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP, Brasil fCentro Estadual de Educação Tecnológica Paula Souza, Faculdade de Tecnologia de Bauru, Bauru, SP, Brasil 2.7. Analysis of the physical properties of the femur The femurs were kept in a desiccator for 24 hours, and the immersed weight (IW, weight of bone immersed in distilled water (Archimedes Principle)) and wet weight (WW, wet weight of bone after 24 hours in a desiccator) were then calculated. After this procedure, the bones were dehydrated in an oven at 100 °C for 24 hours and the dry weight (DW, weight of dehydrated bone after 24 hours at 100 °C) was calculated. For the determination of mineral composition, the bones were placed in a muffle furnace at 800 °C for 24 hours and the mineral weight (MW) was determined. Bone and mineral densities were obtained by calculation of the bone volume according to the Archimedes Principle. The bone percentages of water and mineral matter were also calculated using the following formulas12: bone volume = WW – IW (cm3)/water density; bone density = WW/bone volume (g.cm–3); bone mineral density = MW/bone volume (g.cm–3), and bone percentage of mineral matter = MW/WW (%). On the basis of the results of the biomechanical compression test and bone mineral density, bone quality was calculated using the following formula: S = F max / BMD, where S = maximum normalized force (bone quality) [N/(mg.mm–3)]; F max = maximum load applied to the recipient area until the occurrence of 2 mm displacement of the support [N], and BMD = bone mineral density (mg.mm–3). 2.4. Surgical procedure for the implantation of the native and polyanionic collagen matrices The scaffolds were implanted 4 months after ovariectomy. The animals were anesthetized as described above and an incision was made in the skin on the medial side of the thigh, exposing the femoral quadriceps muscle which was sectioned longitudinally in its distal third and separated anterolaterally. With the distal end of the diaphysis of the left femur exposed and the periosteum separated, a bone defect was created with a 5 mm surgical bur coupled to the pen of a mini‑motor. Next, the defect was filled with the artificial matrices. The periosteum was repositioned and closed with No. 6.0 suture. The musculature and skin were closed with No. 4.0 suture. 2.5. Morphometric analysis The raw material used for the preparation of the three-dimensional collagen scaffolds was bovine pericardium provided by Braile Biomédica S/A (São José do Rio Preto, SP, Brazil). The samples were prepared and provided by the Institute of Chemistry of São Carlos, University of São Paulo. The following samples were obtained: untreated native three-dimensional collagen matrices (NCM), and polyanionic three-dimensional collagen matrices obtained by alkaline treatment of the collagen matrix for 48 and 96 hours (PCM48 and PCM96, respectively). Using a square grid with 100 points coupled to the eyepiece of a light microscope, the volume density of newly formed bone at the site of the collagen implant was calculated for each recipient area. Newly formed bone was quantified by stereology according to the Delesse principle cited by Mandarin de Lacerda10 using the following formula: VV = PP/PT (%), where VV = volume density or relative volume, PP = number of points over newly formed bone, and PT = total number of points of the system. 2.3. Ovariectomy and hormone replacement therapy The animals were weighed and anesthetized by intramuscular administration of a dose of 0.10 mg/100 g body weight ketamine (Francotar, Sespo Ind, Jacareí, SP, Brazil) and xylazine hydrochloride (2% Virbaxyl, Virbac Brasil, São Paulo, SP, Brazil) at a proportion of 1:1. A 2-cm incision was made in the skin with a scalpel lateral to the spine to completely remove both ovaries from the pelvic cavity. During the postoperative period the animals received dipyrone in water for 2 weeks. Estradiol hexahydrobenzoate (Benzogynestryl, Hoechst Marion Roussel, French) was used. The drug was diluted in peanut oil (All Chemistry, São Paulo, SP, Brazil) and the animals were injected subcutaneously with 20 µg at an average interval of 48 hours from the time of scaffold implantation to the day of sacrifice after 8 weeks. 1. Introduction presented good biocompatibility, and may represent a suitable alternative for bone implants5,6. However, studies have shown that the health status of bone is essential for its interaction with biomaterials7 and may alter the expected results. Pan et al.8 investigated the influence of estrogen deficiency on bone remodeling adjacent to hydroxyapatite implants in the tibia of ovariectomized rats and noted a decrease in both bone volume and implant-bone contact in the recipient area. Thus, osteoporosis might compromise the bone-implant interaction. The ovary is an endocrine gland responsible for the production of estrogen and progesterone. A decrease in estrogen secretion is observed in cases of early menopause, late menarche1,2 and ovariectomy3. Deficiency in this hormone results in uncontrolled bone remodeling characterized by a decrease in osteoblastic activity and in bone matrix and reduced deposition of calcium and phosphorus in bone. These alterations can damage bone microarchitecture, predisposing to the occurrence of osteoporosis1,2. Shen et al.3 demonstrated that a reduction of estrogen concentration in ovariectomized rats provoked a decrease in bone mineral density and biomechanical changes in the femur, two factors that are important for the prevention of pathological femoral fractures, the most feared event in osteoporosis. Pathological fractures are generally treated surgically by placement of a total prosthesis or by fixation with pins/screws depending on the type and severity of the fractures4. One approach to prevent the installation or even to treat osteoporosis is hormone replacement therapy. Although widely used, there are no data in the literature showing the possible effects of osteoporosis or hormone replacement therapy on bone-implant interactions9. In view of this fact and of the advantages offered by artificial collagen matrices, the objective of the present study was to evaluate the volume density of newly formed bone at the implant site and the mechanical strength and physical properties of femoral defects filled with polyanionic collagen scaffolds in rats with ovariectomy- induced osteoporosis. Polyanionic collagen matrices implanted into experimental animals were found to cause no inflammatory response and Cunha et al. 240 Materials Research Materials Research 2.6. Analysis of the biomechanical properties of the femur Ninety-six adult female albino Wistar rats (Rattus norvegicus, 12 weeks old), provided by the Multi-Institutional Animal House of the State University of Campinas, were used. The animals were divided into the following groups: group 1, non-ovariectomized animals (NO); group 2, unilaterally ovariectomized animals (UO); group 3, bilaterally ovariectomized animals not submitted to hormone replacement therapy (BOWHRT); group 4, bilaterally ovariectomized animals submitted to hormone replacement therapy (BOHRT). Each group was divided into three subgroups (n = 8 per subgroup) which received the NCM, PCM48 and PCM96 scaffold implants, respectively. The experimental procedure was approved by the Ethics Committee of the institution (CEPex, process 042/07). The biomechanical parameters were obtained by compression testing in an Instron 4444 Universal testing machine. A load cell with a maximum capacity of 1 kN was used. Before the test, the bones were thawed at room temperature and kept in saline until the time of the test. The bone was fixed to a support and force was applied perpendicularly to the longitudinal axis of the recipient area in the anteroposterior direction using a cylindric rod with a 3 mm roller fixed to its end. A pre-load of 5 N was initially used for accommodation. Next, the load was applied over the recipient area at a constant velocity of 2 mm/min until a 2 mm displacement of the support was observed, and the data were recorded. On the basis of these data, force-deformation curves were constructed for the different tests and the biomechanical parameters were determined using the Matlad software. Stress-strain curves were obtained for the different assays and the mechanical strength of bone can be calculated from these curves11. 2. Materials and Methods Karnovsky’s fixative, dehydrated in an increasing ethanol series, cleared in xylene, and embedded in Paraplast. Semi-serial 5 µm cross‑sections were obtained and stained with hematoxylin and eosin. Karnovsky’s fixative, dehydrated in an increasing ethanol series, cleared in xylene, and embedded in Paraplast. Semi-serial 5 µm cross‑sections were obtained and stained with hematoxylin and eosin. 2.8. Statistical analysis Linear models were fitted to each of the response variables, with the response being the variable analyzed and the factors the group to which the animals belonged (NO, UO, BOWHRT and BOHRT) and the type of implant (NCM, PCM48 and PCM96). The model was also fitted considering the interaction between group and type of implant. Significance tests were applied to determine the effect of each factor on the response variable. The levels of significance (p < 0.05) were adjusted using the Tukey-Kramer test. The animals were sacrificed 8 weeks after implantation of the scaffold into the femur by intramuscular administration of an overdose of the anesthetic (ketamine/xylazine hydrochloride), followed by pneumothorax induced by sectioning of the diaphragm through the abdominal cavity. The femurs with the recipient area were removed, photographed and submitted to mechanical testing and subsequent analysis of their physical properties. Next, the samples were fixed in Characterization of the Physical and Mechanical Properties of Femoral Bone Defects Filled with Polyanionic Collagen Scaffolds in Ovariectomized Rats Characterization of the Physical and Mechanical Properties of Femoral Bone Defects Filled with Polyanionic Collagen Scaffolds in Ovariectomized Rats 2010; 13(2) 241 3.1. Macroscopic and quantitative analysis 3.1. Macroscopic and quantitative analysis In NO animals, the NCM and PCM48 implants presented a lower bone interaction in the recipient area than the PCM96 implant (Figure 1a1-a3), as demonstrated macroscopically by the fact that the bone lesion created was still open and the implant was exposed (Figure 1a1-a2, arrows). In the case of the PCM96 implant, the lesion was partially closed and the implant was covered with periosteum (Figure 1a3, arrow). Similar results 3. Results were observed for UO and BOWHRT animals (Figures 1b1-b3 and 1c1-c3, respectively). Macroscopic analysis of BOHRT animals showed that, for the three implants studied, the lesions created continued to be partially open and were not covered with periosteum (Figure 1d1-d3, arrows). With respect to the quantity of newly formed bone in the implant area, lower bone formation was observed in the BOWHRT group, irrespective of the type of scaffold used. No significant difference in the morphometric data was observed between the NO, UO and BOHRT groups. In addition, the volume of newly formed bone was lower in areas receiving the NCM than in those treated with the polyanionic matrices (Figure 2). 3.2. Biomechanical properties Figure 1. Macroscopic analysis of the implant areas. NCM: native collag matrix; PCM48: polyanionic collagen matrix submitted to alkaline treatme for 48 hours; PCM96: polyanionic collagen matrix submitted to alkali treatment for 96 hours. Figure 3 shows the mean maximum strength according to the type of implant (NCM, PCM48, and PCM96) and group (NO, UO, BOHRT, and BOWHRT). Analysis of this variable showed a highly Figure 2. Mean quantity of newly formed bone determined by morphometric analysis. NCM: native collagen matrix; PCM48: polyanionic collagen matrix submitted to alkaline treatment for 48 hours; PCM96: polyanionic collagen matrix submitted to alkaline treatment for 96 hours; NO: non‑ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. Figure 2. Mean quantity of newly formed bone determined by morphometric analysis. NCM: native collagen matrix; PCM48: polyanionic collagen matrix submitted to alkaline treatment for 48 hours; PCM96: polyanionic collagen matrix submitted to alkaline treatment for 96 hours; NO: non‑ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. Figure 3. Mean mechanical strength of newly formed bone. NCM: native collagen matrix; PCM48: polyanionic collagen matrix submitted to alkaline treatment for 48 hours; PCM96: polyanionic collagen matrix submitted to alkaline treatment for 96 hours; NO: non-ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. Figure 3. Mean mechanical strength of newly formed bone. NCM: native collagen matrix; PCM48: polyanionic collagen matrix submitted to alkaline treatment for 48 hours; PCM96: polyanionic collagen matrix submitted to alkaline treatment for 96 hours; NO: non-ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. Figure 1. Macroscopic analysis of the implant areas. NCM: native collagen matrix; PCM48: polyanionic collagen matrix submitted to alkaline treatment for 48 hours; PCM96: polyanionic collagen matrix submitted to alkaline treatment for 96 hours. 242 Cunha et al. Materials Research 4 Di i Figure 4. a) Mean percent mineral matter; and b) mineral density of bone in the different experimental groups. 3.2. Biomechanical properties NO: non-ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. Figure 5. a) Mean bone quality and; b) mean bone density in the different experimental groups. NO: non-ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. et al. Materials Research Materials Research 242 Figure 5. a) Mean bone quality and; b) mean bone density in the different experimental groups. NO: non-ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. Figure 4. a) Mean percent mineral matter; and b) mineral density of bone in the different experimental groups. NO: non-ovariectomized animals; UO: unilaterally ovariectomized animals; BOWHRT: bilaterally ovariectomized animals not submitted to hormone replacement therapy; BOHRT: bilaterally ovariectomized animals submitted to hormone replacement therapy. p < 0.05 indicates statistical significance. 4. Discussion Previous studies from our group have shown that osteoblastic cells cultured on polyanionic collagen matrices are able to induce the biomineralization process in vitro13 and in vivo5,6. In addition, when implanted into nude animals, these polyanionic collagen matrices containing osteoblastic cells induce new bone formation in calvarial bone defects14. In view of the highly promising initial results of this material, we decided to study its behavior in a situation in which the recipient bone is of poor quality. In the present experimental model, osteoporotic bone was evaluated, which presents structural and mechanical characteristics distinct from those of healthy bone. significant group/implant interaction (p < 0.05). With respect to the NCM, a significant difference was observed between groups, except between the NO and UO groups and between the BOWRHT and BOHRT groups. No significant differences between groups were found for the PCM48 or PCM96. Comparison of the three matrices in each group showed no difference between the native and polyanionic scaffolds in the NO or UO group. In the BOWHRT and BOHRT groups, mechanical strength was greater in the area receiving the PCM96 scaffold compared to the area implanted with the NCM. Functionally, the most important mechanical properties of bone are its strength and rigidity. These and other biometric features are best evaluated by analysis of their behavior under loads, i.e., under the influence of externally applied forces9,11. This fact becomes important when evaluating the quality of bone and of the synthetic graft material to be used for the treatment of pathological fractures, the most common complications of hormone deficiency-induced osteoporosis. Several studies have reported the deleterious effects of ovariectomy on bone properties, including reduced resistance to external mechanical forces11,15,16,17. 3.3. Physical properties Percent mineral matter was lower in the BOWHRT group compared to the other groups (Figure 4a). No significant differences in mineral density were observed between groups (Figure 4b). For the bone quality, similar results occurred between NO and OU and between BOHRT and BOWHRT; however significant differences were found between NO/OU and BOHRT/BOWHRT (Figure 5a). Mean bone density was lower in the BOWHRT group compared to the other groups (Figure 5b). Characterization of the Physical and Mechanical Properties of Femoral Bone Defects Filled with Polyanionic Collagen Scaffolds in Ovariectomized Rats 2010; 13(2) 243 Katsumata et al.23 and Shen et al.3 observed a decrease of bone mineral content and density in the femur of ovariectomized rats compared to the intact group, but there was no difference between groups in terms of fracture resistance or rigidity. Wang et al.25 evaluated the influence of osteoporosis on the middle and late periods of tibia fracture healing in ovariectomized rats, analyzing histomorphological changes, bone mineral density and biomechanical properties. In that study, bone mineral density and callus failure stress were lower in the ovariectomized group compared to the sham group. In the present study, a decrease in bone mineral density and percent mineral matter was observed in ovariectomized rats not submitted to hormone replacement therapy, but these animals did not differ in terms of mechanical strength of the area receiving the polyanionic scaffolds. Hypoestrogenemia increases bone resorption and reduces trabecular bone mass, thus affecting the biomechanical strength of bone tissue18. This strength depends on bone structure, including the global geometry of bone and the microscopic pattern of the trabecular network19. Compared to normal bone, osteoporotic bone is characterized by a reduced number of trabeculae, trabecular thinning, and loss of trabecular connectivity. The overall result is a deterioration of bone strength and an increased fracture susceptibility20, however, these authors did not use any type of implant. Ozawa et al.17 used titanium implants to treat bone defects created in the femur of ovariectomized rats. Biomechanical tests performed 2 weeks later showed that the mechanical strength of the recipient area in ovariectomized rats was only half the value found in non-ovariectomized animals. In a recent study, Wang et al.21 induced an osteoporotic state for the evaluation of CaSO4 resorption and observed that ovariectomized animals exhibited pathological bone changes consistent with the osteoporosis phenotype. 3.3. Physical properties The authors suggested that these derangements in bone microarchitecture and physiology accelerated cement resorption and altered the bone response to CaSO4 21. Ovariectomized animals are frequently used as models in the study of postmenopausal osteoporosis; however, some discrepancies exist in the literature regarding bone-implant interaction when biomaterials such as titanium and ceramics are used. For example, some studies reported significant bone loss around these biomaterials, whereas others found no difference in bone growth in the recipient areas of ovariectomized animals17. Differences in the results of human studies have also been reported. In a study involving 30 postmenopausal women ranging in age from 46 to 62 years with a diagnosis of osteoporosis and alveolar bone defects, a biphasic calcium phosphate/poly-DL-lactide-co-glycolide (BCP/PLGA) composite was implanted into the osteoporotic alveolar bone. Six weeks after implantation, bone density was lower in the region of osteoporotic bone receiving the implant than in untreated control bone. On the other hand, 24 weeks after implantation bone density was significantly higher when compared to the untreated region28. In a previous study, we have shown that the quantity of newly formed bone was significantly lower in animals receiving the NCM compared to PCM48 and PCM96, but no data were obtained regarding the characteristics or quality of the bone formed22. In the present study, no difference in mechanical strength was observed between the groups receiving the polyanionic scaffolds despite a higher concentration of newly formed bone in non-ovariectomized animals. The rapid loss of metaphyseal trabecular bone as a result of the imbalance between bone resorption and formation caused by ovariectomy may had a negative influence on the mechanical quality of the recipient area in animals receiving the polyanionic scaffolds. Analysis of some physical properties of the femurs revealed a significant difference between intact animals and ovariectomized rats not submitted to hormone replacement therapy. Similar results have been reported by Hietala29 and Nordsletten et al.16 who observed a reduction in femoral bone density and volume in ovariectomized rats. However, in the present study bone volume was higher in ovariectomized animals not submitted to hormone replacement therapy, although wet weight, a variable used to calculate bone volume and density, was similar in the groups. Controversial data regarding the lack of change in wet weight have also been reported in other studies11,16. 3.3. Physical properties The mechanical strength of the areas implanted with the native scaffold was higher in intact animals compared to ovariectomized rats not submitted to hormone replacement therapy. However, an increase in bone strength and rigidity does not necessarily indicate improvement in the biomechanical properties of bone tissue23. We therefore used a three-point bending biomechanical test. According to Keller and Spengler24, in this test bone deformation is due to changes in angular length or shape. This deformation does not exceed 3% considering the elastic amplitude of the stress-strain curve because bone restores its original shape or length after the load is removed. If load application is continued, the bone tissue reaches its point of deformation followed by rupture, a phase called plastic or non-elastic phase. In the present study, the bone sample contained an implant and we therefore chose to perform a compression test at the implant site. In the three-point bending test, fracture generally occurs in the proximal part, whereas in the present case the implant was located in the distal part. The load for accommodation of the sample should not exceed 10% of the maximal load; therefore, 5 N was used which was sufficient to accommodate the sample. In the present experiment, macroscopic and quantitative analyses showed that the sites receiving the polyanionic collagen matrices (PCM48 and PCM96) presented the best interaction and capacity of bone neoformation when compared to areas implanted with the native collagen scaffold. In addition, in ovariectomized rats bone strength was greater in areas implanted with the PCM96 scaffold. The present findings are also compatible with previous results from our group showing a significant advantage of PCM96 compared to other scaffolds when implanted into animals with experimentally induced osteoporosis22. However, it is not sufficient that these implants induce bone formation. It is also necessary that this newly formed bone is of good quality, i.e., its strength is compatible with that of normal bone. In the present study, bone tissue of satisfactory quality was obtained with the PCM96 matrix in all situations studied. The PCM48 matrix also promoted fairly good mechanical strength, except for animals with experimental osteoporosis. Trabecular architecture is an important factor in osteoporosis and has been quantified by mean of many parameters. However, variations within specimens have been largely ignored. Furthermore, age, disease or drug treatment increases variations in trabecular thickness, which might interfere with the mechanical properties of bone25. References 16. Nordsletten L, Kaastad TS, Madsen JE, Reikeras O, Ovstebo R, Stromme JH et al. The development of femoral osteopenic in ovariectomized rats in no reduced by high intensity treadmill training: a mechanical and densitometric study. Calcified Tissue International. 1994; 55(6):436‑443. 1. Rehman HU and Masson EA. Neuroendocrinology of female aging. Gender Medicine. 2005; 2(1):41-56. 2. Rasgon N, Shelton S and Halbreich U. Perimenopausal mental disorders: epidemiology and phenomenology. CNS Spectrums. 2005; 10(10):471‑478. 17. Ozawa S, Ogawa T, Iida K, Sukotjo C, Hasegawa H, Nishimura RD et al. Ovariectomy hinders the early stage of bone-implant integration: histomorphometric, biomechanical, and molecular analyses. Bone. 2002; 30(1):137-143. 3. Shen V, Birchman R, Wu DD and Lindsay R. Skeletal effects of parathyroid hormone infusion in ovariectomized rats with or without estrogen repletion. Journal of Bone and Mineral Research. 2000; 15(4):740-746. 18. Ikeda S, Tsurukami H, Ito M, Sakai A., Sakata T, Nishida S et al. Effects of trabecular bone contour on ultimate strength of lumbar vertebra after bilateral ovariectomy in rats. Bone. 2001; 28(6):625-633. 4. Kanis JA. Consequences of osteoporosis. In Kanis JA, (editor). Textbook of Osteoporosis. London: Blackwell; 1996. p. 200-225. 19. Benhamou CL, Lespessailles E and Royant V. Bone structure and mechanical resistance of the bone tissue. Presse Medicale. 1996; 25(6):249-254. 5. Rosa FP, Lia RCC, Souza KOF, Goissis G and Marcantonio Jr. E. Tissue response to polyanionic collagen:elastin matrices implanted in rat calvaria, Biomaterials. 2003; 24(2):207-212. 20. Hawker GA. The epidemiology of osteoporosis. Journal of Rheumatology. 1996; 45(9):2-5. 6. Rocha LB, Adam RL, Leite NJ and Metze K and Rossi MA. Biomineralization of polyanionic collagen-elastin matrices during calvarial bone repair. Journal of Biomedical Material Research. 2006; 79A(2):237-245. 21. Wang ML, Massie J, Perry A, Steven R and Kim CW. A rat osteoporotic spine model for the evaluation of bioresorbable bone cements. Spine Journal. 2007; 7(4):466-474. 7. Albrektsson T, Branemark PI, Hansson HA and Lindstrom J. Osseointegrated titanium implants. Acta Orthopaedica Scandinavica. 1981; 52(2):155-170. 22. Cunha MR, Santos Jr. AR, Goissis G and Genari SC. Implants of polyanionic collagen matrix in bone defects of ovariectomized rats. Journal of Materials Science: Materials in Medicine. 2008; 19(3):1341‑1348. 8. Pan J, Shirota T, Ohno K and Michi K. Effect of ovariectomy on bone remodeling adjacent to hydroxyapatite-coated implants in the tibia of mature rats. Journal of Oral and Maxillofacial Surgery. 2000; 58(8):877‑882. 23. Katsumata T, Nakamura T, Ohnishi H and Sakurama T. 3.3. Physical properties Comparison of the scaffolds within each group showed no difference in the NO or UO group, whereas a difference was observed in the BOHRT and BOWHRT groups, with the area receiving the PCM96 scaffold presenting greater strength than the area implanted with the native scaffold. The present results suggest that polyanionic scaffolds are a promising biomaterial for the regeneration of bone defects due to their qualities. In cases of osteoporosis due to hormone deficiency, the PCM96 scaffold is the best choice for bone repair since it promotes faster bone growth and good mechanical strength. According to Lepola et al.26, bone mineral content is one of the determinants of biomechanical bone strength and ovariectomy causes a loss of bone mineral content, especially in trabecular areas27. Cunha et al. 244 Materials Research Materials Research References Intermittent cyclical etidronate treatment maintains the mass, structure and the mechanical property of bone in ovariectomized rats. Journal of Bone and Mineral Research. 1995; 10(6):921-931. 9. Fini M, Giavaresi G, Torricelli P, Borsari V, Giardino R, Nicolini A et al. Osteoporosis and biomaterial osteointegration. Biomedicine and Pharmacotherapy. 2004; 58(9):487-493. 24. Keller TS and Spengler DM. Regulation of bone stress and strain in the immature and mature rat femur. Journal of Biomechanics. 1989; 22(11‑12):1115-1127. 10. Mandarim de Lacerda CA. What is the interest of normal and pathological morphological research to be quantitative? The example of the stereology. Brazilian Journal of Morphological Science. 1999; 16:131-139. 25. Wang JW, Li W, Xu SW, Yang DS, Wang Y, Lin M et al. Osteoporosis influences the middle and late periods of trabeculae healing in a rat osteoporotic model. Chinese Journal Traumatology. 2005; 8(2):111‑116. 11. Peng Z, Tuukkanen J, Zhang H, Jamsa T and Vaananen HK. The mechanical strength of bone in different rat models of experimental osteoporosis. Bone. 1994; 15(5):523-532. 26. Lepola VT, Hannuniemi R, Kippo K, Lauren L, Jalovaara P and Vaananen H.K. Long term effects of clodronate on growing rat bone. Bone. 1996; 18(2):191-196. 12. Martin RB. Effects of simulated weightlessness on bone properties in rats. Journal of Bone and Mineral Metabolism. 1990; 21:91-97. 27. Westerlind KC, Wronski TJ, Ritman EL, Luo ZP, An KN, Bell NH et al. Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain. Physiology. 1999; 94:4199-4104. 13. Moreira PL, An YH, Santos Jr AR and Genari SC. In vitro analysis of anionic collagen scaffolds for bone repair. Journal of Biomedical Material Research. 2004; 71B(2):229-237. 14. Cunha MR, Santos Jr AR and Genari SC. Cultura de osteoblastos sobre membranas de colágeno polianiônico: avaliação preliminar do potencial de indução da formação de tecido ósseo visando reparação tecidual. Boletim de Medicina Veterinária. 2005; 1(1):73-85. 28. Zorica A, Nenad I, Dragan P and Dragan U. Substitution of osteoporotic alveolar bone by biphasic calcium phosphate/poly-DL-lactide- co‑glycolide biomaterials. Journal of Biomaterials Applications. 2007; 21(3):317-328. 15. Fonseca D and Ward WE. Daidzein together with high calcium preserve bone mass and biomechanical strength at multiple sites in ovariectomized mice. Bone. 2004; 35(2):489-497. 29. Hietala EL. The effect of ovariectomy on periosteal bone formation and bone resorption in adult rats. Bone and Mineral. 1993; 20(1):57-65.
https://openalex.org/W4388684091	https://ukinstitute.org/journals/ib/article/download/5123/pdf	Malay	null	Pelatihan Deteksi Dini Penyakit Tidak Menular Pada Kader Posbindu Di Wilayah Kerja Puskesmas Kapoiala	Indonesia Berdaya	2,023	cc-by-sa	2,914	ABSTRACT Secara global, WHO (World Health Organization) memperkirakan penyakit tidak menular menyebabkan sekitar 71% dari jumlah kematian diseluruh dunia. Data tahun 2018 di Indonesia menunjukkan kenaikan prevalensi penyakit tidak menular seperti kanker, stroke, penyakit ginjal kronik, diabetes melitus dan hipertensi dibanding lima tahun sebelumnya. Saat ini Kementerian Kesehatan RI sudah melaksanakan program deteksi dini dan pencegahan penyakit tidak menular dimasyarakat dengan melakukan kegiatan promotif, preventif, kuratif dan rehabilitatif bagi individu dan masyarakat melalui Posbindu PTM di sekolah, tempat kerja, lapas/rutan, terminal, kampung nelayan. Pelatihan dilakukan oleh tim FK Universitas Halu Oleo sebagai kegiatan pengabdian kepada masyarakat, dengan peserta berjumlah 15 kader Puskemas Kapoiala, 5 Petugas Puskesmas dan 6 orang dari FK Universitas Halu Oleo. Tujuan pelatihan yaitu peserta mampu melakukan pengelolaan dan pelaksanaan Posbindu Penyakit Tidak Menular (PTM). Materi yang diberikan dengan metoda ceramah, tanya jawab, diskusi, praktek keterampilan yaitu memberikan pengetahuan tentang PTM, Posbindu PTM, memberikan kemampuan & keterampilan dalam memantau faktor risiko PTM serta konseling. Terdapat peningkatan nilai posttest yang signifikan dibanding pretest, yang berarti ada peningkatan pengetahuan dan keterampilan deteksi dini faktor risiko PTM. Kegiatan ini menginiasiasi terbentuknya Posbindu PTM di Kelurahan Kapoiala Kabupaten Konawe. Keywords: Posbindu, Pelatihan, Kader, Penyakit Tidak Menular PENDAHULUAN Pada pemeriksaan asam urat, didapatkan kadar asam urat normal 66,7%, sedangkan kadar asam urat tinggi 33,3%. Hasil pemeriksaan gula darah sewaktu, didapatkan didapatkan kadar gula darah normal 92,3%, sedangkan kadar gula darah tinggi 7,7% (Purnamasari et al., 2020). Masih tingginya kejadian hipertensi di Kabupaten Konawe khususnya di wilayah kerja Puskesmas Kapoiala, memerlukan suatu penanganan serius dengan cara meningkatkan peran Puskesmas melalui Pos Pembinaan Terpadu Penyakit Tidak Menular (Posbindu PTM) khususnya meningkatkan peran kader Posbindu PTM dalam rangka pencegahan dan pengendalian penyakit hipertensi. Posbindu PTM merupakan wujud peran serta masyarakat dalam kegiatan deteksi dini, pemantauan dan tindak lanjut dini faktor risiko Penyakit Tidak Menular secara mandiri, rutin, terpadu, dan berkesinambungan (Widarti et al., 2018). Makin meningkatnya risiko PTM, maka perlu adanya edukasi dan pendampingan kepada masyarakat pesisir untuk melaksanakan deteksi dini atau skrining factor risiko PTM, termasuk sosialisasi Gerakan Masyarakat Sehat (GERMAS), melalui pelayanan posbindu PTM, terutama kepada kelompok sasaran yang berisiko tinggi (Rahmawati et al., 2018). Pengendalian PTM lebih dititik beratkan pada promotif dan preventif untuk mencegah faktor risiko, sehingga tidak akan berdampak pada sosial ekonomi. Salah satu strategi pengendalian PTM khususnya penyakit hipertensi dilakukan dengan melibatkan peran serta masyarakat. Sedangkan bentuk pemberdayaan masyarakat dapat dilakukan melalui kegiatan posbindu PTM. Pada pelaksanaannya, peran utama kegiatan posbindu PTM dilakukan oleh kader posbindu (Wahyuni et al., 2019)). Peran kader posbindu adalah sebagai pelaksana pengendalian faktor risiko PTM bagi masyarakat di sekitarnya melalui Posbindu PTM. Adapun fungsi kader dalam Posbindu adalah Koordinator penyelenggaraan Posbindu PTM, Penggerak masyarakat untuk mengikuti Posbindu PTM, Pemantau pengukuran faktor risiko PTM, Konselor peserta Posbindu PTM, Pencatat hasil kegiatan Posbindu PTM (Susanti et al., 2020). Hasil penelitian sebelumnya menyatakan bahwa pemberdayaan kader dalam kegiatan program kesehatan akan memberikan keuntungan antara lain adalah kemudahan koordinasi dan penekanan biaya program kesehatan. Kader posbindu merupakan relawan yang dipandang memiliki kemampuan lebih dibanding masyarakat lainnya (12). Hasil wawancara dengan mitra, khususnya kepala puskesmas, menunjukkan bahwa di wilayah kerja Puskesmas Kapoiala, semua desa sudah memiliki Pos Pembinaan Terpadu Penyakit Tidak Menular (Posbindu PTM), akan tetapi sekitar 75% kader posbindu, belum pernah mengikuti pelatihan, sehingga kemampuan dan ketrampilan dalam pelaksanakan Skrining Faktor Risiko PTM masih kurang. Kapasitas kader belum berani untuk secara mandiri memberikan edukasi kepada masyarakat. Sedangkan frekuensi kunjungan sasaran masyarakat juga ke psobindu yang masih kurang dari 25%, dimana pencatatan dan pelaporan kasus PTM yang masih belum tertib. PENDAHULUAN Kejadian hipertensi sebagai bagian dari Penyakit Tidak Menular (PTM) menjadi penyebab utama mortalitas dan morbiditas penyakit kardiovaskular di seluruh dunia (Akbar et al., 2021; Angelina et al., 2020). Upaya penurunan tekanan darah tinggi akan dapat mengurangi risiko komplikasi penyakit kardiovaskular (Arsyati & Chandra, 2020). Kondisi dinyatakan hipertensi atau tekanan darah tinggi, jika pada saat pemeriksaan tekanan darah menunjukkan tekanan darah sistolik lebih dari atau sama dengan 140 mmHg dan tekanan darah diastolik lebih dari atau sama dengan 90 mmHg(Hayati & Fatimaningrum, 2015). Secara global, diperkirakan 26% populasi dunia (972 juta orang) menderita hipertensi. Berdasarkan data Riset Kesehatan Dasar, bahwa prevalensi kasus hipertensi sebanyak berdasarkan hasil pengukuran tekanan darah penduduk usia ≥ 18 tahun sebesar 25,8 per 100.000 penduduk meningkat menjadi 34,1 per 100.000 penduduk (Megawati & Wiramihardja, 2019). Jumlah estimasi penderita hipertensi berusia ≥15 tahun pada tahun 2018 di Provinsi Sulawesi Tenggara sebesar 372.159 kasus (Mulyono & Khasanah, 2020). Sedangkan untuk Kabupaten Konawe sejak tahun 2015, Secara global, diperkirakan 26% populasi dunia (972 juta orang) menderita hipertensi. Berdasarkan data Riset Kesehatan Dasar, bahwa prevalensi kasus hipertensi sebanyak berdasarkan hasil pengukuran tekanan darah penduduk usia ≥ 18 tahun sebesar 25,8 per 100.000 penduduk meningkat menjadi 34,1 per 100.000 penduduk (Megawati & Wiramihardja, 2019). Jumlah estimasi penderita hipertensi berusia ≥15 tahun pada tahun 2018 di Provinsi Sulawesi Tenggara sebesar 372.159 kasus (Mulyono & Khasanah, 2020). Sedangkan untuk Kabupaten Konawe sejak tahun 2015, jumlah kasus hipertensi mengalami peningkatan kasus setiap tahunnya, dimana diharapkan adanya pula peningkatan penanganan kejadian hipertensi, melalui deteksi dini, penyuluhan pola hidup sehat di posbindu, pada setiap wilayah kerja puskesmas (Noya et al., 2021). ) corresponding authorLa Ode AlifarikiEmail: penderita hipertensi berusia ≥15 tahun pada tahun 2018 di Provinsi Sulawesi Tenggara sebesar 372.159 kasus (Mulyono & Khasanah, 2020). Sedangkan untuk Kabupaten Konawe sejak tahun 2015, jumlah kasus hipertensi mengalami peningkatan kasus setiap tahunnya, dimana diharapkan adanya pula peningkatan penanganan kejadian hipertensi, melalui deteksi dini, penyuluhan pola hidup sehat di posbindu, pada setiap wilayah kerja puskesmas (Noya et al., 2021). ) corresponding authorLa Ode AlifarikiEmail: Hasil skrining faktor risiko penyakit tidak menular (SFR-PTM) terhadap 96 responden, yang terdiri dari 28 laki-laki, dan 68 perempuan pada masyarakat pesisir Kecamatan Kapoiala, menunjukkan, hasil pemeriksaan tekanan darah, didapatkan tekanan darah normal 12%, sedangkan tekanan darah tinggi 88%. Hasil pemeriksaan kadar kolesterol, kadar normal 50,0%, sedangkan kadar kolesterol tinggi 50,0%. PENDAHULUAN Realita ini sangat relevan dengan distribusi kejadian hipertensi yang masih tinggi di wilayah kerja Puskesmas Kapoiala, Kabupaten Konawe. Hasil pengamatan di lapangan ditemukan bahwa masyarakat di wilayah kerja Puskesmas Kapoiala masih memiliki kebiasaan berisiko terhadap munculnya kejadian hipertensi, seperti kebiasaan merokok yang masih tinggi, kebiasaan kurang gerak, kebiasaan konsumsi makanan gorengan dan tinggi garam. Kondisi ini relevan dengan hasil penelitian La Ode Alifariki (2015) yang menyatakan bahwa faktor risiko kejadian penyakit hipertensi antara lain stres, pola makan rendah serat, makanan tinggi garam atau natrium, aktivitas fisik rendah, dan kebiasaan merokok (La Ode Alifariki et al., 2021). Melalui pemberdayaan kapasitas peranan kader dalam kegiatan Posbindu PTM sangat diharapkan agar masalah kesehatan yang dialami masyarakat, terutama penyakit hipertensi di wilayah kerja Puskesmas Kapoiala dapat ditekan. Kegiatannya meliputi deteksi dini atau skrining faktor risiko penyakit tidak menular melalui wawancara, pemeriksaan fisik berupa pemeriksaan tekanan darah, indek massa tubuh, pemeriksaan kadar gula darah, pemberian edukasi dan rujukan. Jenis kegiatan dalam Posbindu PTM ini sangat sinkron dengan program promosi esehatan Kementerian Kesehatan melalui program CERDIK (Cek kesehatan secara berkala, Enyahkan asap rokok, Rajin beraktivitas fisik, Diet seimbang, Istirahat cukup dan Kelola stres) (Darmiyanti & Adiputri, 2020) dan PATUH (Periksa kesehatan secara rutin dan ikuti anjuran dokter, Atasi penyakit dengan pengobatan yang tepat dan teratur, tetap diet sehat dengan gizi seimbang, Upayakan beraktivitas fisik dengan aman dan Hindari rokok, alkohol serta zat karsinogenik lainnya) (Akbar et al., 2021). Kegiatan pengabdian kepada masyarakat akan dilaksanakan oleh tim pengabdian dosen Fakultas Kedokteran Universitas Halu Oleo dengan melibatkan peran serta mahasiswa sebagai salah satu komitmen mewujudkan “Merdeka Belajar Kampus Merdeka”. Melalui kegiatan pengabdian kepada masyarakat ini mahasiswa diberi kesempatan luas bersama tim pengabdian mencari solusi nyata masalah kejadian kasus PTM di masyarakat. BAHAN DAN METODE Kegiatan ini dilaksanakan pada bulan Agustus-Oktober 2023. Kegiatan pelayanan kesehatan yang akan dilaksanakan di daerah pesisir, wilayah kerja dari Puskesmas Kapoiala, Kecamatan Kapoiala, Kabupaten Konawe Khalayak Sasaran Sasaran dalam kegiatan ini adalah warga masyarakat tidak mampu yang berdomisili di wilayah pesisir Kecamatan Kapoiala, Kabupaten Konawe. Teknik pelaksanaan kegiatan ini adalah terlebih dahulu mengumpulkan peserta khalayak sasaran di balai pertemuan. Kemudian dilanjutkan dengan penyuluhan kesehatan, pembinaan kader kesehatan keluarga (posbindu) pada masyarakat pesisir, yang dilakukan oleh petugas kesehatan, dan pelaksana kegiatan pengabdian masyarakat dari tim dosen FK Universitas Halu Oleo. HASIL DAN PEMBAHASAN Unit Pelaksana Teknis Dinas (UPTD) Puskesmas Kapoiala Kecamatan Kapoiala, terletak ±85 km dari Kabupaten Konawe Provinsi Sulawesi Tenggara, yang terletak dibagian selatan khatulistiwa antara 48′45′′-55′00′′ lintang Selatan dan antara 26′15′- 31′15′′ Bujur Timur. Berada dipinggiran sungai Konaweeha dan sebagian wilayah pesisir pantai yang menjadi perlintasan daerah. Disebelah utara terdapat obyek wisata pantai Batu Gong, memungkinkan Puskesmas Kapoiala sebagai tempat untuk mendapatkan pelayanan kesehatan apabila terjadi cedera namun, Puskesmas Kapoiala adalah puskesmas rawat jalan sehingga belum berjalan secara optimal, karena pantai tersebut sering dikunjungi pada saat hari libur saja. Saat ini, sebagian kader Posbindu di wilayah Puskesmas Kapoiala masih belum dilatih untuk melakukan deteksi dini PTM. Sebagai bentuk peran serta Fakultas Kedokteran Universitas Halu Oleo (FK UHO) dilakukan kegiatan pengabdian masyarakat untuk melatih kader kesehatan sehingga diharapkan kader-kader dapat memberikan pengetahuan dan dapat mengendalikan PTM kepada masyarakat yang tinggal di wilayah kerja Puskesmas Kapoiala. Dilakukan pelatihan juga bagi kader FK UHO agar dapat mendeteksi faktor resiko PTM. Dengan dilatihnya kader di wilayah Puskesmas Kapoiala diharapkan meningkatnya peran kader serta masyarakat setempat dalam pencegahan dan penemuan dini faktor risiko PTM sehingga akan menurunkan angka kesakitan yang dilaksanakan secara periodik. Kegiatan pengabdian masyarakat ini dilaksanakan pada hari kamis 05 Oktober 2023 di Puskesmas Kapoiala. Kegiatan ini diikuti oleh 13 orang kader, 2 orang mahasiswa, dan 3 orang dosen serta beberapa petugas puskesmas Kapoiala. Kegiatan ini dimulai dengan menilai pengetahuan awal peserta pelatihan kader melalui pretest. Peserta harus menjawab 30 butir pertanyaan tentang pengetahuan Posbindu PTM yang dibuat oleh tim berdasarkan pada buku Petunjuk Teknis Posbindu Bagi Kader. Didapatkan nilai pretest terkecil 33,3 dan terbesar 73,3, dengan nilai rata-rata 62,023. Hasil pretest dengan nilai cukup sebanyak 3 peserta (24%) dan nilai baik sebanyak 10 peserta (77%), dan tidak ada nilai sangat baik. Kegiatan dilanjutkan dengan pemberian materi tentang “Tugas Kader Posbindu”, pengisian KMS PTM, pemberian materi “Edukasi Pencegahan dan Pengendalian Faktor Risiko PTM”. Setelah pemberian teori selesai, dilanjutkan dengan praktik ketrampilan sebagai kader Posbindu PTM. Pada kegiatan praktik ketrampilan ini disiapkan 5 meja sebagai tahapan pemeriksaan oleh kader. a. Meja I: Pengisian Nomor Induk Kependudukan (NIK) dan data lain Pencatatan hasil wawancara faktor risiko PTM. b. Meja II: Pencatatan hasil wawancara faktor risiko PTM. c. Meja III: Pencatatan hasil pengukuran tinggi badan, penimbangan berat badan, dan menghitung Indeks Massa Tubuh. c. Meja III: Pencatatan hasil pengukuran tinggi badan, penimbangan berat badan, dan menghitung Indeks Massa Tubuh. d. HASIL DAN PEMBAHASAN Meja IV: Pencatatan hasil pengukuran tekanan darah (tensimeter digital), pengukuran gula darah dan kolesterol. e. Meja V: Pencatatan hasil identifikasi faktor risiko PTM, edukasi faktor risiko PTM, tindak lanjut dini faktor risiko PTM, dan pengisian hasil layanan. e. Meja V: Pencatatan hasil identifikasi faktor risiko PTM, edukasi faktor risiko PTM, tindak lanjut dini faktor risiko PTM, dan pengisian hasil layanan. Untuk menilai hasil pelatihan kader Posbindu dilakukan evaluasi setelah pelatihan (posttest) dengan nilai terendah 66,6 dan nilai tertinggi 80 dengan nilai rata-rata 70,74. Hasil posttest dengan nilai sangat baik sebanyak 1 peserta (8%) dan nilai baik sebanyak 12 (92%) dan nilai cukup sudah tidak ada. Dengan demikian ada peningkatan dari pretest pada nilai cukup dari 24% menjadi 0%, nilai baik dari 77% naik 20% menjadi 92%, sedangkan nilai sangat baik dari 0% menjadi 8%. Setelah kegiatan ini dilaksanakan, didapatkan peningkatan pengetahuan tentang deteksi dini faktor risiko PTM, kader Posbindu PTM terampil melakukan pengukuran BB, TB dan lingkar pinggang, jugal melakukan pemeriksaan gula darah dan kolesterol, Kegiatan ini menginisiasi terbentuknya Posbindu PTM pada Puskesmas Kapoiala. Dokumentasi kegiatan pengabdian kepada masyarakat ini dapat ditampilkan sebagai berikut: Gambar 1. Dokumentasi Kegaiatan PKM Gambar 1. Dokumentasi Kegaiatan PKM DISKUSI Kegiatan identifikasi faktor risiko PTM, edukasi dan tindak lanjut dini merupakan tahapan layanan terakhir setelah teridentifikasi faktor risiko yang ada. Pengendalian faktor risiko PTM, tidak selalu harus dilakukan dengan obat-obatan. Pada tahap dini, kondisi faktor risiko PTM dapat dicegah dengan mengendalikan faktor risiko dan berperilaku hidup yang sehat seperti berhenti merokok, diet seimbang, rajin beraktifitas fisik, pengelolaan stres dan lain-lain. Edukasi dilakukan oleh kader Posbindu untuk meningkatkan pengetahuan dan kemampuan masyarakat dalam mengendalikan faktor risiko PTM. Bila ada klien memerlukan pengobatan, kader akan merujuk ke Puskesmas Adanya Posbindu PTM di wilayah kerja Puskesmas Kapoiala diharapkan dapat meningkatkan cakupan deteksi dini PTM. Faktor keluarga dan tokoh masyarakat memberikan pengaruh terhadap keaktifan masyarakat untuk datang ke Posbindu. Kegiatan pengabdian selanjutnya dapat bermitra dengan tokoh masyarakat untuk memperluas cakupan pelayanan. Kegiatan pertama dalam pelatihan ini diawali dengan sosialisasi dan pemaparan materi terlebih dahulu dengan tujuan peserta pengabdian masyarakat mengetahui dan memahami terlebih dahulu konsep mengenai pengelolaan posyandu, materi yang disampaikan yaitu posyandu, peran dan fungsi kader, tumbuh kembang anak, pengisian KMS (kartu menuju sehat). Selama proses kegiatan berlangsung peserta menyimak materi dengan antusias dan peserta mencatat hal-hal yang penting dan yang di akan ditanyakan. Kegiatan kedua dalam kegiatan pengabdian masyarakat yaitu kegiatan demonstrasi kegiatan posyandu melalui 5 meja yaitu mulai dari tahap pendaftaran atau pendataan anak, penilaian antropometri yaitu penimbangan berat badan dan tinggi badan, pengisian KMS (Kartu Menuju Sehat), Pemeriksaan kesehatan pada anak oleh tenaga kesehatan, Penyuluhan atau pemberian penkes, pemberian makanan tambahan untuk anak. Dalam kegiatan demonstrasi pengelolaan kader melalui 5 meja, kader di bagi kelompok, masing masing kelompok berjumlah 3 orang dan masing masing anggota kelompok mempunyai peran yang berbeda, kelompok menganalisis kasus yang diberikan oleh fasilitator, kemudian peserta diberi kesempatan untuk mendemontrasikan 5 meja dalam posyandu. Kegiatan demonstrasi diberikan waktu selama 20 menit bagi setiap kelompok. Selama kegiatan demontrasi pengelolaan 5 meja dalam posyandu, peserta tampak menyimak dengan seksama dan setiap peserta wajib untuk memberikan masukan terhadap kelompok yang sudah mendemontrasikan. Kegiatan ketiga yaitu melakukan evaluasi terhadap kegiatan pelatihan kader yang sudah dilakukan. Kegiatan Evaluasi yang dilakukan pada kader yang dilakukan yaitu dengan cara memberikan posttest, memberikan umpan balik kepada kader terhadap materi yang sudah disampaikan baik di hari pertama dan kedua, hasil observasi dari fasilitator adanya keaktifan peserta dalam kegiatan pelatihan. Kegiatan evaluasi ini dilakukan selama 30 menit. Hasil evaluasi dari pelaksanaan kegiatan pelatihan kader adalah bahwa kegiatan ini mencapai tujuan yang telah ditetapkan. Ucapan Terimakasih Kami ucapkan terima kasih banyak kepada Ketua LPPM Universitas Halu Oleo yang telah memberikan izin pelaksanaan kegiatan pengabdian ini, dan Kepada seluruh masyarakat yang telah berpartisipasi menyukseskan kegiatan ini. DISKUSI Pertama adalah adanya keaktifan peserta dalam mengikuti kegiatan pelatihan hal ini terbukti dengan daftar hadir peserta yang selama 1 hari mengikuti pelatihan yaitu sebanyak 100%, komitmen bersedia menjalankan tugas sebagai kader yaitu sebanyak 100% dapat dilihat pada. Kedua yaitu antusias peserta dalam menerima materi pelatihan, peserta memberikan umpan balik yang benar terhadap pertanyaan yang diberikan oleh pemateri melalui wawancara dan penyebaran kuesioner sebelum dan sesudah sosialisasi. Adanya peningkatan pengetahuan mengenak pengelolaan posyandu sebelum dilakukan sosialisasi dan setelah dilakukan sosialisasi. Ketiga yaitu kader mampu mendemontrasikan pengelolan 5 meja posyandu dengan benar. Adanya peningkatan kemampuan kader sebelum dilakukan pelatihan dan sesudah dilakukan pelatihan. Berdasarkan hasil evaluasi tersebut memperkuat fakta bahwa pelatihan ini berhasil mencapai keberhasilan seperti yang diharapkan. Selama pelatihan respon yang didapatkan sangatlah positif baik dari pemateri, fasilitator dan peserta selama pelatihan berlangsung. Conflict of Interests The authors declared that no potential conflicts of interest with respect to the authorship and publication of this article. KESIMPULAN DAN SARAN Pelaksanaan pengabdian kepada masyarakat ini telah menghasilkan beberapa hal yakni terbentuknya Posbindu penyakit tidak menular di Kelurahan Kapoiala Kabupaten Konawe. REFERENCES Akbar, F., Darmiati, D., Arfan, F., & Putri, A. A. Z. (2021). 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https://openalex.org/W3198387470	https://bmcmedresmethodol.biomedcentral.com/track/pdf/10.1186/s12874-021-01359-x	English	null	Investigator initiated trials versus industry sponsored trials - translation of randomized controlled trials into clinical practice (IMPACT)	BMC Medical research methodology	2,021	cc-by	14,133	Blümle et al. BMC Medical Research Methodology (2021) 21:182 https://doi.org/10.1186/s12874-021-01359-x Blümle et al. BMC Medical Research Methodology (2021) 21:182 https://doi.org/10.1186/s12874-021-01359-x Open Access Investigator initiated trials versus industry sponsored trials - translation of randomized controlled trials into clinical practice (IMPACT) Anette Blümle1,2* , Katharina Wollmann1, Karin Bischoff1, Philipp Kapp1, Szimonetta Lohner3, Edris Nury1, Kai Nitschke1, Jasmin Zähringer1, Gerta Rücker4† and Martin Schumacher4† * Correspondence: anette.bluemle@uniklinik-freiburg.de * Correspondence: anette.bluemle@uniklinik-freiburg.de Gerta Rücker and Martin Schumacher shared last authorship. 1Institute for Evidence in Medicine (for Cochrane Germany Foundation), Faculty of Medicine and Medical Center, University of Freiburg, Breisacher Str. 86, 79110 Freiburg, Germany 2 y 2Clinical Trials Unit, Faculty of Medicine and Medical Center, University of Freiburg, Elsässer Straße 2, 79110 Freiburg, Germany Full list of author information is available at the end of the article Abstract Background: Healthcare decisions are ideally based on clinical trial results, published in study registries, as journal articles or summarized in secondary research articles. In this research project, we investigated the impact of academically and commercially sponsored clinical trials on medical practice by measuring the proportion of trials published and cited by systematic reviews and clinical guidelines. Methods: We examined 691 multicenter, randomized controlled trials that started in 2005 or later and were completed by the end of 2016. To determine whether sponsorship/funding and place of conduct influence a trial’s impact, we created four sub-cohorts of investigator initiated trials (IITs) and industry sponsored trials (ISTs): 120 IITs and 171 ISTs with German contribution compared to 200 IITs and 200 ISTs without German contribution. We balanced the groups for study phase and place of conduct. German IITs were funded by the German Research Foundation (DFG), the Federal Ministry of Education and Research (BMBF), or by another non-commercial research organization. All other trials were drawn from the German Clinical Trials Register or ClinicalTrials.gov. We investigated, to what extent study characteristics were associated with publication and impact using multivariable logistic regressions. Results: For 80% of the 691 trials, results were published as result articles in a medical journal and/or study registry, 52% were cited by a systematic review, and 26% reached impact in a clinical guideline. Drug trials and larger trials were associated with a higher probability to be published and to have an impact than non-drug trials and smaller trials. Results of IITs were more often published as a journal article while results of ISTs were more often published in study registries. International ISTs less often gained impact by inclusion in systematic reviews or guidelines than IITs. * Correspondence: anette.bluemle@uniklinik-freiburg.de Gerta Rücker and Martin Schumacher shared last authorship. 1Institute for Evidence in Medicine (for Cochrane Germany Foundation), Faculty of Medicine and Medical Center, University of Freiburg, Breisacher Str. 86, 79110 Freiburg, Germany 2Clinical Trials Unit, Faculty of Medicine and Medical Center, University of Freiburg, Elsässer Straße 2, 79110 Freiburg, Germany Full list of author information is available at the end of the article Background prospective study registration, there are still trials that are not included in a study registry [13]. Thus, unpub- lished studies and their results are difficult to identify. In recent years, several authorities and research orga- nizations became aware of the problems arising from withholding study results. The World Health Organization (WHO), the World Medical Association (WMA) and the All Trials initiative [14], have alerted that it is unethical to conduct human research without subsequently publishing the results. They also pointed out that vast financial resources spent on clinical re- search are wasted when research results are not pub- lished. Hence, these research organizations took various steps to prevent incomplete, biased or non-reporting of research results [15]. prospective study registration, there are still trials that are not included in a study registry [13]. Thus, unpub- lished studies and their results are difficult to identify. Decisions in healthcare are ideally built on three pillars, the experience of the clinician, the wishes and values of the patient, and the best available external evidence, i.e. results from clinical research [1]. Available, findable and accessible clinical research results are mandatory for a successful transfer of this knowledge into evidence- based practice and further research [2]. Beside research results, also information about detailed study methods is important, since only they allow to appraise the validity, reliability and applicability of clinical evidence to clinical practice [3]. In recent years, several authorities and research orga- nizations became aware of the problems arising from withholding study results. The World Health Organization (WHO), the World Medical Association (WMA) and the All Trials initiative [14], have alerted that it is unethical to conduct human research without subsequently publishing the results. They also pointed out that vast financial resources spent on clinical re- search are wasted when research results are not pub- lished. Hence, these research organizations took various steps to prevent incomplete, biased or non-reporting of research results [15]. It has long been known that only a part of the clinical studies conducted ultimately reach the stage of full pub- lication in peer-reviewed journals [4]. For example, more than half of the study results presented as an abstract at scientific meetings fail to be published as a full-text art- icle [5]. Thus, important study information cannot be considered for health care decisions and further research planning, which in turn could expose patients and future study participants to unnecessary risks [6]. Background Systematic reviews and meta-analyses can come to an erroneous overall effect estimate and conclusion when unpublished data cannot be considered [7]. If experiences and results obtained from trials are not disseminated, they are not only lost for health care, but also for further research. Moreover, personnel resources and scarce research funds are badly invested or even wasted. To the best of our knowledge, it is still unclear under what conditions expenses are invested to support clinical trials pay off in a way that the findings have an impact on healthcare decisions. As an order of magnitude, in 2018 the German Research Foundation (DFG) alone spent 22 Million euros for the conduction of 47 trials within their clinical trials program [16]. Trial discontinu- ation could be identified as one factor for non- publication of clinical trials [17]. Another major step for- ward would be to identify trial specific risk factors for non-publication or for having no impact on medical practice. The aim of this project was to examine the transfer process of clinical trial information into medical prac- tice. First, we determined the proportion of the trials that were published, the type (methods and/or results) and place (as journal article, register entry) of published information and the proportion of trials cited by second- ary research articles (reviews and/or clinical guidelines). We then analyzed whether there is an association of pre-defined study characteristics (sponsoring/funding, study phase, drug/non-drug intervention, number of participants, number of primary outcome, medical field) with publication or use by secondary research articles. An important step for increasing both, the transpar- ency in research and the visibility of unpublished studies was the implementation of study registries as well as the call for prospective study registration by several research organizations [8–10]. In Germany, funding organizations such as the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) and the Federal Ministry of Education and Research (Bundesministerium für Bil- dung und Forschung, BMBF) require the registration of the trial in a public registry and publication of the trial protocol following grant approval [11, 12]. Prospective study registration is a major step forward, but it is equally important to make the results of a trial publicly available, which is possible through study registries. However, even several years after these urgent calls for a © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 2 of 20 Conclusion: An encouraging high proportion of the clinical trials were published, and a considerable proportion gained impact on clinical practice. However, there is still room for improvement. For publishing study results, study registries have become an alternative or complement to journal articles, especially for ISTs. IITs funded by governmental bodies in Germany reached an impact that is comparable to international IITs and ISTs. Keywords: Randomized controlled trials as topic, Registries, Access to information, Evidence-based medicine, Publishing, Systematic reviews as topic, Practice guidelines as topic, Knowledge translation, Health impact assessment, Clinical decision-making Study cohort information: method article only (solely the study methods are described in detail), result article only (study results are described and usually the methods very briefly), and both. This classification allowed us to determine what kind of study information was used in secondary research articles and clinical guidelines. y In brief, we set up a MS Access database consisting of 691 trials (hereafter referred to as study cohort). Eligible for inclusion were clinical trials that were conducted at multiple study sites, were randomized controlled (RCTs), investigated drugs or non-drugs, started in 2005 or later and were completed by the end of 2016. To find out whether sponsorship/funding or place of conduct influ- ence a trial’s impact, we created and compared sub- cohorts of investigator initiated trials (IITs) and industry sponsor trials (ISTs) with and without German contribu- tion (Table 1). For the IIT-sub-cohort “Public Germany” we included trials funded by the DFG and BMBF (Public Germany gov), which we retrieved from the funder’s da- tabases “German Project Information System” (GEPRIS) of the DFG and the website of the BMBF [19, 20]. These IITs served as basis for the determination of the eligibil- ity criteria for the trials to be included in the comparison sub-cohorts. The largest trial of the reference sub-cohort included 4005 participants so that we only considered trials up to this sample size for inclusion in the other sub-cohorts. To achieve a reasonable number of German IITs, we complemented the reference sub-cohort by an equal number of IITs funded by other German non- commercial organizations (Public Germany other), which we randomly drew from the trials registries Clini- calTrials.gov and German Clinical Trials Register (DRKS) (Table 1). Trials included in the sub-cohort Commercial Germany were also drawn from these two registries, whereas trials included in the international sub-cohorts (Public International and Commercial Inter- national) were solely drawn from ClinicalTrials.gov. Methods The rationale and design of this project is described in detail in a previous publication [18]. Page 3 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 Trial information in study registries Beside study registration, publishing trial results in study registries is required since several years [14, 22, 23]. In the DRKS, study related documents can be attached or linked to the trial record. In ClinicalTrials.gov, results can be entered directly into the trial record as a separate register tab or are automatically searched and attached by the study registries themselves [24]. To minimize possibly biasing study characteristics, we aimed to generate comparable sub-cohorts by balancing for effects of the study phase and the location of partici- pating study sites (proportion of German study sites). According to the distribution given in the sub-cohort Public Germany, we balanced the three comparison sub- cohorts Public International, Commercial Germany and Commercial International for the study phase, of both drug trials and non-drug trials, and the sub-cohort Com- mercial Germany additionally for the proportion of Ger- man study sites on all study sites (Table 1). In addition to the publication as a journal article, we determined whether or not study information was avail- able in study registries. Beside their registration in Clini- cialTrials.gov and DRKS, 189 (27%) of the trials were additionally registered in EudraCT and 35 (5%) in the ISRCTN registry. Trials with results available in study registries are hereafter referred to as “results in regis- tries” [18]. Search strategy: sources where journal articles were identified First, we searched for publications in different biomed- ical databases and other sources using an incremental search strategy (Additional file 2). As search terms, we combined various study information such as the registry identification number, study title, acronym, PICO- aspects, and/or name of applicant or principal investiga- tor. Searches for primary study reports were conducted between 6 February 2018 and 30 August 2018. We then downloaded the references of all identified published ar- ticles into an Endnote database. We considered full arti- cles reporting a trial’s methods and/or results. We also downloaded all the study protocols we came across dur- ing our literature search. Definition We independently double-extracted the pre-defined study characteristics such as sample size, study phase, number of pre-defined primary outcomes, and medical fields [21] from the study registries, as we were inter- ested in whether they were associated with research im- pact. For further details concerning the project methods please refer to the methods paper [18]. Hereinafter, we use the following definitions: for publi- cations in journal articles we use the expression “pub- lished articles”. We distinguish between articles solely concerning a trial’s methods, called “method articles”, and articles also reporting study results (“result articles”). Beside publication as journal article, results can be pub- lished in study registries; in this case, we use the expres- sion “results in registries”. For published trial results, i.e. as result article or as results in registries, we use the gen- eral term “published results”. Identification of corresponding publications Identification of corresponding publications For each trial, we identified related publications and classified them according to the published trial Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 4 of 20 Table 1 Characteristics of included trials Characteristics IIT Public Germany gov No. of trials (%) IIT Public Germany other No. of trials (%) IIT Public Germany (total) No. of trials (%) IIT Public International No. of trials (%) IST Commercial Germany No. of trials (%) IST Commercial International No. of trials (%) Total No. Identification of corresponding publications of trials (%) Total 60 60 120 (100) 200 (100) 171 (100) 200 (100) 691 (100) Registered ina ClinicalTrials.gov 32 (53) 16 (27) 48 (40) 200 (100) 158 (92) 200 (100) 606 (88) DRKSb 14 (23) 48 (80) 62 (52) – 19 (11) – 81 (12) ISRCTNc 27 (45) 5 (8) 32 (27) 3 (1) – – 35 (5) EudraCTd 40 (67) 10 (17) 50 (42) 18 (9) 88 (52) 33 (17) 189 (27) Study status Completed 43 (72) 59 (98) 102 (85) 200 (100) 170 (100) 200 (100) 672 (97) Prematurely ended 12 (20) 1 (2) 13 (11) 1 (< 1) 14 (2) Still ongoinge 5 (8) – 5 (4) 5 (< 1) Collaboration International 19 (32) 7 (12) 26 (22) 44 (22) 71 (42) 69 (35) 210 (30) National 40 (66) 53 (88) 93 (78) 156 (78) 100 (58) 131 (65) 479 (69) Unclear 1 (2) – 1 (< 1) – – – 2 (< 1) Study size (Median = 150) > 150 46 (76) 28 (47) 74 (62) 81 (40) 74 (43) 115 (58) 344 (50) ≤150 13 (22) 32 (53) 45 (38) 119 (60) 97 (57) 85 (42) 346 (50) Unclear 1 (2) – 1 (< 1) – – – 1 (< 1) Number of primary outcome(s) 0 – – – – 1 (1) – 1 (< 1) 1 44 (73) 44 (73) 88 (73) 152 (76) 122 (71) 133 (67) 495 (72) > 1 (range 2– 36) 16 (27) 16 (27) 32 (27) 48 (24) 48 (28) 67 (33) 195 (28) Study phase drug trialsf Total 41 (68) 15 (25) 56 (47) 93 (47) 93 (54) 93 (47) 335 (48) 2 9 (15) 5 (8) 14 (12) 23 (12) 23 (13) 23 (12) 83 (12) 3 20 (33) 7 (12) 27 (22) 45 (23) 45 (26) 45 (23) 162 (23) 4 12 (20) 3 (5) 15 (13) 25 (13) 25 (15) 25 (13) 90 (13) Study phase non-drug trialsg Total 19 (32) 45 (75) 64 (53) 107 (53) 78 (46) 107 (53) 356 (52) A – 9 (15) 9 (7) 15 (7) 11 (7) 15 (7) 50 (7) B 16 (27) 33 (55) 49 (41) 82 (41) 43 (25) 82 (41) 256 (37) C 3 (5) 3 (5) 6 (5) 10 (5) 24 (14) 10 (5) 50 (7) a Several trials were registered in more than one trials registry, i.e. Identification of corresponding publications numbers do not sum up to the total numbers (100%); bDRKS: German Clinical Trials Register; cISRCTN: International Standard Randomized Controlled Trials Number registry; dEudraCT: European Union Drug Regulating Authorities Clinical Trials Database; eStatus as of 24 April 2020; f15 drug trials of phase 2–3 were counted as phase 2; 24 non-drug trials of phase A-B were counted as phase A; gIn the sub-cohort “Commercial Germany”, we included all non-drug trials available in the study registries, resulting in slightly differing distributions of study phases among the 4 sub-cohorts Page 5 of 20 Page 5 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 February 2020. For each identified clinical guideline, we retrieved the full text and verified the citations. Identification of secondary research articles citing primary published articles To assess the research impact of the included trials, we investigated whether or not published articles were cited by secondary research articles, i.e. systematic reviews/ meta-analyses and clinical guidelines. Semi-automatic tool Within this project, one author (KN) developed a semi- automatic tool (called DoiScout) that facilitates large- scale literature searches and citation analyses in order to carry out extensive literature searches based on internet search engines more time-efficiently. DoiScout automatically identifies primary published articles that reference a particular study registry ID (e.g. NCT02179424). Bibliographic information about the identified articles is extracted and presented in a list that is formatted in a way that allows passing on the informa- tion to other software programs for further processing. If a DOI of a citing article was found in Epistemoni- kos, the publication type was verified and the citing art- icle labelled as systematic review. We then manually assessed how the published articles were used and where they were cited in the systematic reviews/meta-analyses: A second feature refers to citation analysis. Search en- gines behind platforms such as PubMed (www.pubmed. gov) and Web of Science (www.webofknowledge.com) can be used to identify other articles, e.g. primary re- search articles, systematic reviews and clinical guidelines that cite a given article. DoiScout extracts the biblio- graphic information of the citing articles and provides it to the user in a workable format. In addition, DoiScout can be used to identify articles citing the citing articles of the original source. This can be done for any pre- specified citation depth, thus providing a comprehensive overview of the extent of a project’s academic impact. General information or methods of the published article were used and cited in the systematic review, e.g. in the introduction or discussion section, Study results reported in the published article were included in the systematic review/meta-analyses or Study results reported in the published article were not included in the systematic review/meta-analyses, e. g. not meeting eligibility criteria. The program of the DOIScout and a manual describ- ing the features in more detail are available via the GitHub platform [29]. Data collection We extracted the following information about the publi- cations into an Access database: 1) whether or not study results were reported in study registries, 2) bibliographic information of included publications and content (method article or result article), 3) bibliographic infor- mation of citing systematic reviews/meta-analyses, and 4) bibliographic information of citing guidelines. Clinical guidelines The ultimate step for a successful implementation of tri- al’s results in medical practice is their inclusion in clin- ical practice guidelines. To identify these, we manually searched in the clinical guidelines databases TRIP [26], NICE evidence search [27] and AWMF (Association of the Scientific Medical Societies) [28]. We searched for clinical guidelines citing the trial publications. As search terms, we used (parts of) the title and the name of the first author of the published articles as well as the corre- sponding systematic review/meta-analysis; to identify guidelines citing results published in registries, we searched with the register identification number. The search period for guidelines citing the published articles was between December 2018 and March 2019, for guidelines including systematic reviews between April and August 2019, and for the registry identifier in Systematic reviews For each published article, we downloaded all references listed under the functions “Cited by” in PubMed and “Times Cited” in Web of Science. To identify the sys- tematic reviews and meta-analyses among the citing arti- cles, we matched their Digital Object Identifier (DOI) with the record-DOIs included in the database Episte- monikos, which can be considered as the “largest source of systematic reviews relevant for health-decision mak- ing” [25]. Epistemonikos includes references of four cat- egories: broad syntheses, systematic reviews, structured summaries and primary studies. In our project, we fo- cused on references classified as systematic reviews or broad syntheses. Both categories are hereinafter referred to as “systematic reviews” (SRs). Proportion of published trials For our whole cohort, 576 (83%) of the 691 trials in- cluded were published as a method article or a result article in a medical journal and/or the trial results were made available in study registries; results were available for 555 (80%) of the trials (Fig. 1). For 107 (19%) trials, results were solely published in a registry. Definitions The term “published trials” is used when “method arti- cles”, “result articles” or “results in registers” are avail- able. Results of a trial can be published as “result article” in a journal or as “results in registries”, while methods are always published as “journal article”. We first de- scribed the proportion of publication types for the total cohort and then for the different sub-cohorts. If not mentioned otherwise, all percentages of trials given for the entire cohort are calculated on the basis of the in- cluded 691 trials. Percentages given for the sub-cohorts are based on the number of trials in each sub-cohort. Minor differences in summed percentages derive from rounding to full integer. For 98% (438 of 448) of the trials with published re- sults, the pre-defined primary outcome was reported in the result article. The publication frequency, i.e. the number of pub- lished articles per trial, is shown in Additional file 4. Many trials (284, 60%) were published solely in one jour- nal article. In the remaining trials, multiple publication was highly represented. For example, only 8% of the tri- als generated 29% of the publications, resulting in an average publication frequency of 7.0 (median 6) publica- tions per trial. Sub-cohorts For the sub-cohorts, the proportion of tri- als published varied between 77 and 87% (Fig. 2). Com- pared to the sub-cohort Public Germany (77%), the probability of a trial to be published is higher for the sub-cohorts Public International (87%), Commercial Germany (86%) and Commercial International (82%) (Table 2). The publication of results ranged between 63% for Public Germany (58% for Public Germany gov and 67% Public Germany other) and 86% for Commer- cial Germany. Data analysis d We used queries in MS Access 2010TM and tabulation in Microsoft Excel 2010 to obtain standard descriptive statistics. Multivariable logistic regression was used to determine the association of study characteristics with the probability of a trial to be published, cited by sys- tematic reviews and included in guidelines. Based on the reference sub-cohort Public Germany, it was carried out for the other sub-cohorts, for study phase, number of participants, and number of primary outcomes. For time to publication, multivariable Cox regression was used to account for study characteristics. For distinguishing be- tween first publication in a journal or in a registry, a competing risk model was used and Aalen-Johansen Page 6 of 20 Page 6 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 estimates of the cumulative incidence functions are pre- sented [30]. article for 76 (11%) of the trials. For three trials with a method article, results were published only in registries. No results were published for 21 (3%) trials with a method article, neither in a journal article nor in a registry. Trials published as journal article Obvious differences exist between the sub-cohorts re- garding the type of publication. IITs were more often published as a journal article than ISTs (Table 2). Espe- cially method articles were more present for IITs (Public Germany 38%, Public International 21%) than for the IST-sub-cohorts (Commercial Germany: 6%; Commer- cial International: 2%). Compared to the German sub- cohorts, results were more often published as a journal article for Public International trials and less often for Cohort For 472 (68%) of our 691 trials, we identified 947 corresponding published journal articles (Table 2, Additional file 3). Out of 448 (65%) trials, 843 result arti- cles were published. For 100 (15%) trials, 104 method ar- ticles, without presenting any trial results, were found. For 372 (54%) trials, only a result article was available. We found both, a method article as well as a result Fig. 1 Proportion of published trials and type of publication for the whole cohort (n = 691). Please refer also to Table 2 ig. 1 Proportion of published trials and type of publication for the whole cohort (n = 691). Please refer also to Table 2 Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 7 of 20 Commercial International trials. Looking at the number of publications per trials, multiple publications were more common in IITs (Germany: 33%, International: 32%) compared to ISTs (Germany: 17%, International 14%). Trial information available in study registries Cohort For 293 (42%) of the 691 included trials, at least one reference to a corresponding journal article was re- ported in the study registry and/or a link to the original publication source or a database was provided. This means that 62% (293 of 472) of all published journal ar- ticles could be found in study registries (Table 2). article was reported. For 132 (19%) trials, both sources were present and for 144 (21%) solely a reference of a result article. Sub-cohorts The proportion of trials with a reference or link to the journal article was with 58 and 52% higher in the Public sub-cohorts than in the Commercial sub- cohorts with 33 and 32% (Table 2). Results in registries ranged between 4 and 67%. The proportion of IST with results in registries was higher than for IITs. For Public Germany, only 4% of the trials had results in registries. Trials published as journal article For 132 (19%) trials, both sources were present and for 144 (21%) solely a reference of a result article. Commercial International trials. Looking at the number of publications per trials, multiple publications were more common in IITs (Germany: 33%, International: 32%) compared to ISTs (Germany: 17%, International 14%). Sub-cohorts The proportion of trials with a reference or link to the journal article was with 58 and 52% higher in the Public sub-cohorts than in the Commercial sub- cohorts with 33 and 32% (Table 2). Results in registries ranged between 4 and 67%. The proportion of IST with results in registries was higher than for IITs. Trials published as journal article This small percentage can be explained by the fact that most of those trials derived from the DRKS register (summarized data, see Table 2 for more details), Table 2 Proportion of published trials per sub-cohort and type of publication (total: n = 691) IIT Public Germany gov No. of trials (%) IIT Public Germany other No. of trials (%) IIT Public Germany (total) No. of trials (%) IIT Public International No. of trials (%) IST Commercial Germany No. of trials (%) IST Commercial International No. of trials (%) Total No. of trials (%) Total trials 60 60 120 (100) 200 (100) 171 (100) 200 (100) 691 (100) Proportion of published trials Published 48 (80) 44 (73) 92 (77) 174 (87) 147 (86) 163 (82) 576 (83) 95% CI 68–88 60–84 68–84 82–91 80–91 75–87 80–86 Not published 12 (20) 16 (27) 28 (23) 26 (13) 24 (14) 37 (19) 115 (17) Type of publication; trials published as Journal article 48 (80) 42 (70) 90 (75) 169 (85) 113 (66) 100 (50) 472 (68) 95% CI 68–90 57–81 66–83 79–89 59–73 43–57 65–72 Method article 31 (52) 15 (25) 46 (38) 41 (21) 10 (6) 3 (2) 100 (15) 95% CI 38–65 15–38 30–48 15–27 3–11 0–4 12–17 Result article 34 (57) 38 (63) 72 (60) 163 (82) 113 (66) 100 (50) 448 (65) 95% CI 43–69 50–75 51–69 75–87 59–73 43–57 61–68 Results in registries 3 (5) 2 (3) 5 (4) 65 (33) 101 (59) 134 (67) 305 (44) 95% CI 1–14 0–12 1–10 26–40 51–67 60–74 40–48 Published results 35 (58) 40 (67) 75 (63) 170 (85) 147 (86) 163 (82) 555 (80) Combinations Result as article AND in registries 2 (3) 0 2 (2) 58 (29) 67 (39) 71 (36) 198 (29) Method AND Result article 17 (28) 11 (18) 28 (23) 35 (18) 10 (6) 3 (2) 76 (11) Method article, no published results 13 (22) 4 (7) 17 (14) 4 (2) 0 0 21 (3) Trial information in registries Publ. ref. total 35 (58) 35 (58) 70 (58) 104 (52) 56 (33) 63 (32) 293 (42) 95% CI 50–71 45–71 49–67 45–59 26–40 25–38 39–46 Publ. ref. of result article 27 (45) 30 (50) 57 (48) 102 (51) 55 (32) 62 (31) 276 (40) 95% CI 32–58 37–63 38–57 44–58 25–40 25–38 36–44 article was reported. Study characteristics associated with publication of results Study characteristics associated with publication of results Study characteristics associated with publication of results The multivariable analysis confirmed our findings re- garding publication probability for the sub-cohorts. It also showed that additional study characteristics are as- sociated with the probability to be published: drug trials were published more often than non-drug trials, larger trials more often than smaller trials and trials with more than one primary outcome more often than trials with one primary outcome (Additional file 6). Compared to Public Germany trials, results were pub- lished earlier for trials of the other sub-cohorts (Table 3, Fig. 4). Furthermore, drug trials were published earlier than non-drug trials and larger trials earlier than smaller. In our cohort, we did not find an association of time to publication with the number of primary out- comes (1 or more than 1). Each trial was allocated to one of 23 pre-defined med- ical fields (Additional file 6). In our cohort, the median number of trials per medical field was 25 and ranged be- tween 2 (anaesthesiology) and 104 (surgery), the propor- tion of trials published ranged between 87 and 25%. Statistically significant differences were only found for medical fields with a sufficient number of trials (≥39): higher publication rates were found for neurology (87%) and psychiatry/psychotherapy (84%), lower for surgery (64%) and ophthalmology (25%). Due to the limited number of trials per medical field, an analysis for signifi- cant differences was not appropriate for the sub-cohorts. Further details on publication and impact are presented in the chapter “Overall impact”. Multivariable analysis with estimated covariate effects for sub-cohorts, type of intervention, study size, number of primary outcomes. Hazard ratio with 95% confidence intervals. The intercept stands for the combination of IIT Public Germany, drug trial, n ≤150 and one primary outcome. Trial information available in study registries Cohort For 293 (42%) of the 691 included trials, at least one reference to a corresponding journal article was re- ported in the study registry and/or a link to the original publication source or a database was provided. This means that 62% (293 of 472) of all published journal ar- ticles could be found in study registries (Table 2). For Public Germany, only 4% of the trials had results in registries. This small percentage can be explained by the fact that most of those trials derived from the DRKS register (summarized data, see Table 2 for more details), where results cannot directly be entered. Information on results was available for 449 (65%) tri- als. For 305 (44%) trials, results were directly included in a study registry and for 276 (40%), a reference to a result For the three other sub-cohorts, between 29 and 39% of the trials have results published in both registries and Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 8 of 20 Fig. 2 Proportion of published trials and type of publication per sub-cohort. Please also refer to Table 2 as journal articles and for 20 and 32% of the commercial sub-cohorts, results were solely available in registries. 5.19 years (95% CI: 4.83–5.82); if only result articles were counted, the median was 6.09 years (95% CI: 5.66–6.62). We analyzed the time to first publication of study re- sults in a journal or in a registry also in the framework of a competing risk model. This was visualized as Aalen- Johansen estimators in a stacked probability plot (Fig. 3). The result shows that for the majority of studies (about 52%) the first publication was found in a journal, while about 28% of studies were first published in a study registry. Time to publication One measure of impact of a trial is the citation of their journal articles by systematic reviews. In 2631 systematic reviews, 599 of 947 (63%) published journal articles de- riving from our trials were cited. Overall, we identified 3429 citations in the reviews, as reviews cited more than one of our journal articles (Additional file 7). The median time to any publication as a journal article or in a study registry, including method papers, was 4.07 years (95% CI: 3.79–4.33). If only counting result papers, the median time was longer (4.67 years, 95% CI: 4.36–5.03). The median time for any type of article (in- cluding method papers) to be published in a journal was Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 9 of 20 Fig. 3 Cumulative incidence functions (Aalen-Johansen estimates) Cumulative incidence functions (Aalen-Johansen estimates) Cohort The 599 articles cited by systematic reviews cor- responded to trials (Table 4). Out of those, 27% were cited by only one systematic review; 73% by more than one. It is notable that 15% of the published articles were cited by 10 or more systematic reviews (Additional file 8). The median number of citing systematic review(s) per trial was 4 (range 1 to 99; mean = 4.1). Cohort The 599 articles cited by systematic reviews cor- responded to trials (Table 4). Out of those, 27% were cited by only one systematic review; 73% by more than one. It is notable that 15% of the published articles were cited by 10 or more systematic reviews (Additional file 8). The median number of citing systematic review(s) per trial was 4 (range 1 to 99; mean = 4.1). used (excluded, included or used otherwise). As publica- tions included in secondary research articles are more likely to influence clinical practice than excluded publica- tions, this analysis is important for the assessment of the impact of trials. Of the citations in systematic reviews, 69% (2374 from 3429) were included and correspond to 45% (309 of 691) trials (Table 4), 6% (190 of 3429) were excluded and 25% (865 of 3429) were used otherwise. Nevertheless, 69 of the 87 trials with excluded publica- tions in reviews had included publications in other re- views. For the remaining 18 trials, only exclusions were found. Time to publication Frequently stated reasons for the exclusion of pub- lications were that cohorts failed to meet the eligibility cri- teria and did not report the data of interest. Similar proportions were found for the subgroup of re- sult articles (529 of 843; 63%) and the corresponding tri- als (335; 48%). Of the 104 method articles, 70 (67%) method articles corresponding to 70 (10%) trials were cited by a systematic review. We not only examined whether retrieved publications were cited in systematic reviews but also how they were Table 3 Time from study start to publication of results, either in a registry or journal Covariates Hazard ratio 95% CI p-value Intercept 1 IIT Public International 2.243 1.703–2.956 P < 0.001 IST Commercial Germany 2.343 1.770–3.112 P < 0.001 IST Commercial International 2.332 1.761–3.072 P < 0.001 Non-drug trials versus drug trials 0.838 0.707–0.992 P < 0.05 Study size: n > 150 versus n ≤150 1.215 1.023–1.442 P < 0.05 Number of primary outcome(s): > 1 versus 1 1.141 0.939–1.387 n.s. Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 10 of 20 Fig. 4 Kaplan-Meier estimates of the cumulative distribution function for time to publication of results, grouped by sub-cohort Fig. 4 Kaplan-Meier estimates of the cumulative distribution function for time to publication of results, grouped by sub-cohort Study characteristics associated with citation by systematic review Study characteristics associated with citation by systematic review Sub-cohorts For the public sub-cohorts and for Com- mercial Germany, citation by systematic reviews ranged between 52 and 63% and was higher than in Commercial International with 38% (Fig. 5 and Table 4). This differ- ence might be explained by the lower proportion of tri- als published as journal articles in ISTs (compare Fig. 2). Furthermore, a relevant proportion of articles cited by systematic reviews were method articles, which were rare in ISTs but mainly present in IITs. The multivariable analysis confirmed the significantly lower representation of Commercial International trials in systematic reviews compared to the other sub-cohorts. Both, the type of intervention and the number of primary outcomes are not associated with the inclusion probabil- ity, whereas larger trials are significantly more often in- cluded in reviews than smaller trials (Additional file 9). Table 4 Proportion of trials (n = 691) cited by systematic reviews per sub-cohort and type of publication Trials cited by SR IIT Public Germany gov No. Time to publication of trials (%) IIT Public Germany other No. of trials (%) IIT Public Germany (total) No. of trials (%) IIT Public International No. of trials (%) IST Commercial Germany No. of trials (%) IST Commercial International No. of trials (%) Total No. of trials (%) Total trials 60 60 120 (100) 200 (100) 171 (100) 200 (100) 691 (100) Trials in SR 41 (68) 30 (50) 71 (59) 125 (63) 89 (52) 75 (38) 360 (52) 95% CI 55–80 37–63 50–68 55–69 44–60 31–45 48–56 Trials with method article in SR 25 (42) 7 (12) 32 (27) 31 (16) 5 (3) 2 (1) 70 (10) Trials with method article only in SR 12 (20) 4 (7) 16 (13) 8 (4) 1 (1) 0 25 (4) Trials with result article in SR 29 (48) 26 (43) 55 (46) 117 (59) 88 (51) 75 (38) 335 (48) 95% CI 35–62 31–57 37–55 51–65 44–59 31–45 45–52 Trials with method AND result article in SR 13 (22) 3 (5) 16 (13) 23 (12) 4 (2) 2 (1) 45 (7) Use in SR Trials included in SR 31 (52) 21 (35) 52 (43) 107 (54) 84 (49) 66 (33) 309 (45) 95% CI 38–65 23–48 34–53 46–61 41–57 27–40 41–48 Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 11 of 20 Fig. 5 Proportion of trials cited by systematic reviews Trials included in clinical guidelines directly in 226 of 427 (53%) different guidelines via the citation of 262 result articles. In total, 93% (166 of 178) of the inclusions in guidelines come from result articles via a direct or indirect pathway. 6% (10 of 178) of the trials were included in 12 of 427 (3%) different guide- lines via citation of 12 method articles. 4% (7 of 178) of the trials were included in 6 (2%) different guidelines via citation of seven registry information (Table 5). Cohort We found 574 citations of 178 trials (26%) in guidelines (Fig. 6). Some of the guidelines included in- formation from several of our trials. These corresponded to 427 unique guidelines. On average, each of our trials was cited 3.2 times (574/178) in guidelines. This “guide- line inclusion factor” ranged between 2.9 and 3.7 for the sub-cohorts. Sub-cohorts In Fig. 7 / Table 5 it is shown that for the sub-cohorts the inclusion of trials in guidelines ranged between 17 and 31%. For the subgroup Public Germany gov, even 45% (27 of 60) of the trials were cited in guide- lines. Compared to Public Germany trials, the propor- tion of trials included in a guideline is similar to Public International and Commercial Germany trials, whereas One trial can be included in one guideline via several pathways, namely via a published article or via a system- atic review. The following analysis shows via what publi- cation type trials were included in guidelines: 69% (122 of 178) of the trials were included in 285 of 427 (67%) different guidelines via the citation of 382 systematic re- views. 58% (104 of 178) of the trials were included Fig. 6 Proportion of trials cited by guidelines, shown by type of publication Fig. 6 Proportion of trials cited by guidelines, shown by type of publication Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 12 of 20 Table 5 Proportion of trials (n = 691) cited by clinical guidelines per sub-cohort and type of publication Trials cited by guideline IIT Public Germany gov No. of trials (%) IIT Public Germany other No. of trials (%) IIT Public Germany (total) No. of trials (%) IIT Public International No. of trials (%) IST Commercial Germany No. of trials (%) IST Commercial International No. of trials (%) Total No. Trials included in clinical guidelines of trials (%) Total trials 60 60 120 (100) 200 (100) 171 (100) 200 (100) 691 (100) Trials in guidelines 27 (45) 8 (13) 35 (29) 61 (31) 50 (29) 32 (16) 178 (26) 95% CI 32–58 6–25 21–38 24–37 23–37 11–22 23–29 Direct Trials with method articles in guideline 7 (12) 0 7 (6) 2 (1) 0 1 (< 1) 10 (1) Trials with result articles in guideline 19 (32) 4 (7) 23 (19) 36 (18) 27 (16) 18 (9) 104 (15) Trials with register ID in guidelines 3 (5) 0 3 (3) 2 (1) 2 (1) 0 7 (1) Trials with any direct citation 25 (42) 4 (7) 29 (24) 38 (19) 29 (17) 18 (9) 114 (16) Indirect Trials in guidelines via review 16 (27) 8 (13) 24 (20) 43 (22) 35 (20) 20 (10) 122 (18) Direct AND indirect Trials in guidelines via review AND result article 12 (20) 8 (13) 20 (17) 42 (21) 35 (20) 20 (10) 117 (17) Direct: guidelines cite the original published article(s); Indirect: guidelines cite systematic review(s) that include the original published article(s) Fig. 7 Proportion of trials with research impact per sub-cohort (n = 691). Trials included in a guideline via citation of a published article, of results published in registries or of a systematic review citing the trial Page 13 of 20 Page 13 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 commercial International trials are less often included in guidelines. trials were included in systematic reviews and 178 (26%) in guidelines. trials were included in systematic reviews and 178 (26%) in guidelines. Overall impact Sub-cohort Commercially funded trials, especially Com- mercial International trials, less often gain an impact by inclusion in systematic reviews (52%; 39% for SRs, and 29%; 31% for guidelines) than publicly sponsored trials or guidelines (59%; 63% for SRs, and 29 and 17% for guidelines). The distribution of the three “impact-pro- portions” concerning inclusion in reviews and/or guide- lines showed only minor differences between the sub- cohorts (Fig. 10). Lifecycle of trials Figure 8 shows the fate of the trials included in our cohort from registration to publication and to their impact on clinical practice. During their life- cycle from registration to impact in clinical practice, the number of relevant trials decreases with each step. 17% of the trials have no published results. Of the 576 (83%) published trials, 15% (107 of 691) have their results only published in registries and therefore might have less awareness and a limited impact in the scientific community. Study characteristics associated with inclusion in a guideline Cohort Of all trials, 274 (40%) generated no impact: 115 (17%) of the trials were not published and of the pub- lished trials, 160 (23%) were not cited by either a system- atic review or a guideline (Fig. 9). Similar to the inclusion in reviews, the multivariable analysis confirmed a significantly lower representation of Commercial International trials in guidelines compared to the other sub-cohorts and demonstrated that type of intervention and number of primary outcomes are not associated with the inclusion in guidelines. Larger trials are about twice as often included in guidelines than smaller trials (Additional file 10). Used by secondary research articles were 417 (60%) trials: 361 (52%) were cited by a systematic review, and 178 (26%) by a guideline. Out of those, 123 (18%) were cited by both, a systematic review and a guideline. This means that more than half (52%) of the trials were cited in a systematic review and that about a quarter (26%) reached an impact in a clinical guideline. Medical fields Trials published as journal article(s) (472; 68%) have a good chance to be cited in reviews or guidelines. Never- theless, in our cohort, a relevant percentage did not find an inclusion in clinical practice: only 309 (45%) of the For our cohort, we found clear differences regarding the publication and impact for the main medical fields (number of trials ≥39). The high proportion of guide- lines in psychiatry and psychotherapy, cardiovascular Fig. 8 Impact on clinical practice. Total number of trials, published articles and systematic reviews (SRs) and guidelines, citing the published articles Fig. 8 Impact on clinical practice. Total number of trials, published articles and systematic reviews (SRs) and guidelines, citing the published articles Page 14 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 Fig. 9 Publication and impact of trials Fig. 9 Publication and impact of trials and did not gain an impact on clinical practice. We were also interested in trial characteristics that were associated with impact. Systematic reviews have shown that several study factors are associated with publication of clinical trial results, e.g. direction of study findings, study size and duration [4, 31]. Those factors could also have an influence on the impact measures that go beyond publication, and also study characteristics such as phase of clinical research (study phase), medical field, type of funding or spon- sorship, and place of conduct (country of study sites) could play a role. An increased awareness of the risk factors could improve the feasibility and efficiency of future trials and the validity of trial results. Trials at high risk of having no impact could be adjusted be- forehand to ensure a successful trial progress. disease and neurology is related to a high proportion of publications and systematic reviews for these fields (Fig. 11). When publication is low, this results in fewer reviews and guidelines (ophthalmology, surgery). Interpretation of findings could be shown [38]. Subgroup analyses of this project also confirmed our results, that larger trials are more often published than smaller trials. A relatively high publication proportion of 73% was also found for com- pleted academic drug trials approved by the Danish Medicines Agency [39]. However, academic trials ap- proved by an Ethics committee in Spain had a consider- able lower publication proportion of 39%, whereas 64% of the commercially sponsored trials were published in a peer-reviewed scientific journal [40]. For comparison, the publication proportion of research projects beyond clinical trials, e.g. basic research, funded by a medical faculty in Germany, was 65% for publication in a peer- reviewed journal, and 73% if also other publications were counted [41]. We compared IITs with ISTs because they often focus on different clinical questions and pursue different aims and objectives. IITs play a crucial role in academic clin- ical research whereas ISTs usually focus on commercial interests, mainly of pharmaceutical companies, whose primary aim is to develop and approve drugs or other medical treatments [32]. In IITs, an academic investiga- tor is responsible for the conduct of the clinical trial, which includes planning, registration and publishing the results of the study [33]. IITs are often conducted to ex- pand product knowledge, including safety, and to iden- tify new ways of using existing treatments, which might lead to the improvement of patient health [34]. IITs complement ISTs regarding the medical field, such as physio- and psychotherapy, behavioral changes as well as complementary medicine. Even though there are several advantages for posting results in registries, e.g. results can be presented fast and concisely, they are directly attached to the registry rec- ord, and provide information about the trial methods as well as references and links to further trial information, the publication proportion is still relatively small. In a cross sectional study across academic medical centers in the United States, the publication proportion of com- pleted trials that were registered in ClinicalTrials.gov was analyzed. Across the medical centers, 10.8 to 40.3% of the trials were published within 24 months of study completion, and for 1.6 to 40.7%, results were reported on ClinicalTrials.gov [42]. Compared to previous research, in our project a high proportion of trials (83%) were published. Systematic re- views and retrospective research projects investigating the publication proportion of RCTs resulted in consider- ably lower proportions of 60 to 71% [4, 35–37]. Discussion In the current project we assessed the research im- pact on clinical practice of publicly sponsored trials and commercially sponsored trials conducted in Germany in comparison to those conducted inter- nationally. By using a prospective strategy that followed the lifecycle of a defined set of trials from their registration up to their inclusion in systematic reviews and clinical guidelines, we have collected and analyzed data not only for those trials that were ‘suc- cessful’, but also for trials that were not published Fig. 10 Impact of trials per sub-cohort Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 15 of 20 Fig. 11 Fate of trials per medical fields: surgery (S), psychiatry and psychotherapy (PP), cardiovascular disease (CD), endocrinology, diabetes, and metabolism (EDM), Ophthalmology (O), respiratory system (RS), neurology (N)8 Fig. 11 Fate of trials per medical fields: surgery (S), psychiatry and psychotherapy (PP), cardiovascular disease (CD), endocrinology, diabetes, and metabolism (EDM), Ophthalmology (O), respiratory system (RS), neurology (N)8 Interpretation of findings Only 37% of RCTs presented as conference abstracts were published in full as journal articles [5]. The relatively high proportion of published trials and trial results in our cohort can be attributed to the fact that we also con- sidered a trial as published, when its results were re- ported in a study registry. Results in registries were also considered in a recently published project that investi- gated the publication proportion of trials conducted at German university medical centers. Also in this project, a publication proportion of 83% for completed trials In our cohort, on average, for 45% (range of sub- cohorts: 36–68%) of the trials we found results in study registries. This finding is in line with the results of a re- cent study, investigating the compliance with the Food Blümle et al. BMC Medical Research Methodology (2021) 21:182 Page 16 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 The reasons for these observed differences are unclear and future analyses would be worth to compare the characteristics and results of those trials published solely in registries with those published as journal articles, e.g. regarding publication bias. One explanation could be that for publicly funded trials publication of results in the form of a journal article is often demanded by the funding organization and is part of the funding condi- tions. Advantages of publishing results as a journal art- icle ideally are a quality-assured peer-review, trial methods and results are considered and discussed in the context of the existing evidence and can be commented by other researches e. g. via response letters. For a great proportion of trials in our cohort, results were published as journal articles (81%), and for more than half of the trials results were included in a registry. and Drug Administration Amendments Act of 2007 (FDAAA) concerning reporting of trial results. The re- searchers found, that due to report results under the FDAAA only about 40% of all applicable trials reported their results in ClinicalTrials.gov within the 1 year dead- line after study completion [43]. The possibility to add results to a study record in registries, is certainly an important step to improve transparency in clinical research. However, limited, in- complete or expired trial information in registries often make it difficult to get a complete picture of the trial and to appraise and interpret the results. Interpretation of findings Several initia- tives such as AllTrials and TranspariMed work on the improvement of a trial’s reporting by requiring clinical trials to be registered and to report their full methods and summary results [14, 22]. Disclosure of detailed trial methods of a trial is essential with respect to the critical appraisal and interpretation of the results, and is the basis to enable other researchers to reproduce the trial and verify its results, which is a basic requirement for later implementation in medical practice. While in an original journal research article both methods and results of a trial are described, it is becoming more common to publish articles only describing the detailed methods of a trial and not the results. In our cohort, this was the case for 14% of the trials, for 3% only a method article could be identified. Moreover, it is important to point out that most of the method articles derive from publicly funded trials (87%), of which most of the German IITs were from Public Germany gov (67%). In scientific re- search it is not unusual to publish results of one study in more than one article. One reason for this could be that in academia the reward system is often built on quantity of research output [47]. Scientific success, such as reputation, career advancement, as well as successful applications for research funding, is directly associated with the publica- tion output of a researcher. In our project, multiple publi- cation was the case for 188 (40%) trials. They were more common for IITs (Germany: 33%, International: 32%) than for ISTs (Germany: 17%, International 14%). The trial with the highest number of identified publications (n = 21) was a phase 4 study, conducted in the field of cardiovascular disease, funded by the DFG. For this trial, one method article, two result articles and 18 sub-studies and sec- ondary analysis were published. This trial and also the other high-frequently published trials (25 with more than 5 published articles) were conducted in academia. For this publication frequency, measured as the number of published articles per trial, we found a remarkable phenomenon: about one third of all published articles corresponded to only 8 % of the trials. Even though this aforementioned reward system and its consequences have been in the focus of criticism for several years, structures have still not changed [48]. Interpretation of findings Against the background that still not all trials are registered, our trial cohort might be a “positive” selection compared to those con- ducted worldwide. Therefore, there is a potential risk that our cohort is biased, resulting in a limited external validity of our project results. Typical reasons for not publishing trial results as pre- sented in a systematic review are lack of time and/or re- sources, non-completion of study, publication was not an aim, or only had low priority [50]. Further reasons stated by sponsors of Danish academic clinical drug tri- als were negative or not statistically significant results [39]. Possible explanations for published trial results not being included in systematic reviews are that no review related to the research question has been conducted or updated after the date of publication. Reasons reported for non-inclusion of published articles in the systematic reviews of our study cohort were that the eligibility cri- teria were not fulfilled, e.g. wrong patient group, inter- vention, comparator, outcome measure, or study type. Even though all studies were included in at least one study registry, for some studies information in registries was scarce and detailed study protocols were only rarely available. Therefore, for some trials it was difficult to find out whether a published article corresponded to the trial. We also had to rely on the information reported in registries. Data of prospectively registered studies can in- clude preliminary study information, for example infor- mation about study start and completion date. This may have influenced our findings. For inclusion of trial results in guidelines, the same reasons as for systematic reviews could apply. However, in guidelines, in addition to publications, systematic re- views are also a relevant pathway for inclusion of trial results. A detailed investigation of the systematic reviews that have not been included in guidelines (56%) would be useful, e.g. to find the reasons for lack of guidelines and to be able to further improve the transfer of import- ant trial findings into medical practice. g We tried to assess actual data and included trials that started in 2005 or later and were completed by the end of 2016, for which we searched for corresponding publi- cations in 2018/2019. For trials completed late during this time period, there might not have been sufficient time for publication and inclusion in systematic reviews and guidelines. Interpretation of findings In our cohort, of the trials with published results, 19% (107 of 556) were solely available in study registries. This has serious implications for the search process to iden- tify relevant studies, i.e. which sources need to be searched, especially for systematic reviews and clinical guidelines. A search strategy should not only focus on journal articles, but should be accomplished by an add- itional search in study registries- This has already be- come mandatory for conducting Cochrane intervention reviews [44, 45]. To improve the findability of trial re- sults, the registries themselves should improve their searchability. They should be constructed in a standard- ized format so that they are easily and reliably search- able, e.g. similar to biomedical databases by title, author, keywords and abstracts. Looking at our sub-cohorts, we found a significant difference between IITs and ISTs. For publicly sponsored trials, only 2–4% were solely published in registries, whereas this was the case for 20 to 32% of the commercially sponsored trials. A similar relation was found in a project investigating more than 30,000 clinical trials registered in the EU Clinical Trials Register (EUCTR) [46]. Of those trials that were due, which means that 12 months to publish the results had passed, about 50% reported results in this register; 68% of the commercial trials and only 11% of the non- commercial trials. This higher proportion of results in registries compared to our results could be explained by the fact that in EUCTR only trials investigating medi- cinal product are included and that for those trials dis- closing of their results has been required by the European Medicines Agency (EMA) since 2004. A higher proportion of trials with results in registries were also shown for clinical trials sponsored by the pharma- ceutical industry trials compared to non-commercially sponsored trials in Spain [40]. It must be noted that in this project the commercial trials were registered signifi- cantly more often in ClinicalTrials.gov than the aca- demic trials Page 17 of 20 Page 17 of 20 Page 17 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 Blümle et al. BMC Medical Research Methodology (2021) 21:182 Blümle et al. BMC Medical Research Methodology (2021) 21:182 In contrast, in industry the main (financial) interest lies in the results, i.e. efficacy and safety of the tested treatment, whereas the study protocol and methods used are often confidential to protect commercial interests. Interpretation of findings This is also shown by the public availability of the study protocols: for 40 trials of our cohort, the original study protocol could be identified, 30 belonged to the IIT sub- cohorts and 10 to the IST sub-cohorts. extraction form following a written manual. All data ex- tractors were trained prior to the data extraction. We captured all relevant information available in any study registry. Discrepancies in different sources were dis- cussed and resolved. The identification of systematic re- views citing the original study report was conducted semi-automatically by using a self-developed program. The search for clinical guidelines was done manually fol- lowing predefined standardized rules. A Health Technology Report was conducted to evalu- ate the impact of Cochrane Reviews published by 20 Cochrane Review Groups, on health care, patient out- comes and value for money [49]. Therefore, a random sample of 20 Cochrane Reviews and 40 selected reviews, more likely to have had an impact, were selected. Of the 60 included reviews, a considerable proportion of 67% had been cited in clinical guidance and 15 had influ- enced further primary research. Another strength was that we controlled for possibly biasing factors by design, i.e. by balancing important study characteristics to Public Germany as the reference sub-cohort. A limitation arising from this was the limited number of studies in the sub-cohorts Public Germany and Com- mercial Germany. The number of trials meeting our in- clusion criteria for the Public Germany gov (reference sub-cohort) was fixed to 60. For the sub-cohort Com- mercial Germany, a balancing for non-drug trials was not fully possible: only 171 could be identified in DRKS and ClinicalTrials.gov registries instead of the pre- planned 200 studies per comparison sub-cohort. How- ever, it is not expected that this difference of 29 trials have a relevant influence on our results. We found that more than half of the trials are repre- sented in systematic reviews and more than a quarter in guidelines. To further improve this knowledge transfer from research into practice, several issues have to be considered. The first issue is to understand, why 17% of the trials have not been published. The second issue is how to improve the transfer of the 28% of the trials that were published but reached no impact. Our cohort was composed of trials that were included in study registries and, partially, also in databases main- tained by funding organizations. Interpretation of findings Our results, however, indicate that this only concerns a few trials because since 1) compared to literature, the publication rate of our cohort was rela- tively high, and 2) the stacked probability plot (Fig. 3) also indicates that only few first publications are to be expected. Nevertheless, in such projects there will always Implications for policy, practice and research Implications for policy, practice and research Further efforts are needed to ensure that the results of all trials conducted are published. Publication of all re- sults of all trials should become mandatory, e.g. by legal regulation and by requirement of funding organizations. The proportion of published results of commercial trials is comparable to those of academic trials, but they ap- pear less often in reviews and guidelines. Further re- search is needed to investigate the reasons for this phenomenon. In this project, the only criterion for measuring impact of trials on medical practice was in- clusion or exclusion of their results. A more detailed quantitative analysis of the “value” of their contribution to the overall body of evidence and on medical practice would be helpful to identify “valuable” trials. Considering their study characteristics in the planning of future trials, could increase the impact of clinical research. Specific- ally for Germany, IITs funded by governmental bodies reached an impact comparable to international IITs and ISTs that is respectable. Thus, funding of high-quality IITs by governmental bodies is worth the effort, should be continued and further encouraged. The full text of some clinical practice guidelines from the United Kingdom identified via NICE or TRIP were only accessible to people located within the country, so that we were not able to verify the citation for those. Therefore, we did not consider them for our project. Conclusion To the best of our knowledge, our project provides the first comprehensive and comparative evaluation of investigator-initiated trials and industry-sponsored trials with regard to their impact on clinical practice. It com- prises not only publication of trial results in journals as well as in study registries but also examines the different factors that can be used as selective inclusion criteria of results in systematic reviews, meta-analyses and clinical guidelines. Previous investigations have focused on only one aspect of clinical impact, mostly on the publication of results. They also have been concentrated on a spe- cific medical field or were based on studies conducted at a single institution. An encouraging result of this project is that with 83% a high proportion of clinical trials were published, which is a significant improvement compared to previous investigations. A reasonable percentage of trials were used in systematic reviews (52%) as well as in clinical guidelines (26%). IITs performed comparably or not significantly worse than ISTs with respect to the three IMPACT-aspects investigated. For publishing study results, study registries have become an important alternative or complement to journal articles. Neverthe- less, there still were a certain proportion of trials that were not published and/or had no impact which needs to be decreased to increase the efficiency and to reduce waste in medical research. As a first step, special atten- tion could be given to smaller trials and non-drug trials, which were significantly less often published or cited by systematic reviews or clinical guidelines than larger trials and non-drug trials. For statistical reasons, larger trials had a higher probability to generate statistically signifi- cant results. Nevertheless, trials with a smaller number of participants, e.g. as is the case in early phase studies Abbreviations AWMF: Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften (Association of the Scientific Medical Societies). https:// www.awmf.org/leitlinien/leitlinien-suche.html; BMBF: Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research). https:// www.bmbf.de; Cochrane Library: https://www.cochranelibrary.com/; DFG: Deutsche Forschungsgemeinschaft (German Research Foundation). https://www.dfg.de/; DOI: Digital Object Identifier; DRKS: Deutsches Register Klinischer Studien (German Clinical Trials Register). https://www.drks.de; Epistemonikos: Collaborative, multilingual database of health evidence and largest source of systematic reviews relevant for health-decision making, and of other types of scientific evidence. https://www.epistemonikos.org/; EUCTR: EU Clinical Trials Register. https://www.clinicaltrialsregister.eu/; EudraCT: European Union Drug Regulating Authorities Clinical Trials Database. https://www.clinicaltrialsregister.eu/ctr-search/search; EMA: European Medicines Agency. https://www.ema.europa.eu; GEPR IS: German Project Information System. Online database made available by the DFG that provides information on current DFG-funded research projects. https://gepris.dfg.de/gepris/OCTOPUS; Google scholar: Web search engine providing scholarly literature. https://scholar.google.com; IITs: Investigator Initiated Trials; ISRCTN registry: International Standard Randomized Controlled Trials Number registry. http://www.isrctn.com; ISTs: Industry Sponsored Trials; LIVIVO: Interdisciplinary search engine for literature and information in the field of life sciences, run by ZB MED – Information Centre for Life Sciences. https://www.livivo.de; Medline: Medical Literature Analysis and Retrieval Strengths and limitations of the study A strength of our study was that all trials were registered in study registries so that for all of them basic study in- formation was available. Study characteristics were double-extracted independently in a pre-piloted Page 18 of 20 Page 18 of 20 Blümle et al. BMC Medical Research Methodology (2021) 21:182 Blümle et al. BMC Medical Research Methodology be a compromise between presenting actual data with respect to the timeframe of included studies and leaving enough time for studies to be published and have an impact. or in case of limited scientific resources, are also justified and should be available for inclusion in the total body of evidence. Further research is needed to identify the rea- sons and risk factors for non-publication or delayed pub- lication of registered trials and for non-citation in reviews and guidelines. A standardized reporting system, implemented in the lifecycle of studies, that requests the reasons in these cases could be an approach to reach that goal. Such a system could also reveal, why published results of commercial trials appear less often in reviews and guidelines than academic trials. p An important result of our study was that for 15% of the trials, results were solely available in study registries and were not published as journal article. In such cases, we could only search for guidelines citing the trial by using the registry identification number, but this was not possible for systematic reviews. To identify citing sys- tematic reviews, we used the “cited by”- or “times cited”- functions of PubMed and WoS. These functions only consider journal articles, so that we were limited to the published journal articles. Acknowledgements ld l k h 1. Sackett DL, Rosenberg WM, Gray JA, Haynes RB, Richardson WS. Evidence based medicine: what it is and what it isn’t. BMJ. 1996;312(7023):71–2. https://doi.org/10.1136/bmj.312.7023.71. 1. Sackett DL, Rosenberg WM, Gray JA, Haynes RB, Richardson WS. Evidence based medicine: what it is and what it isn’t. BMJ. 1996;312(7023):71–2. https://doi.org/10.1136/bmj.312.7023.71. We would like to thank our student assistant Svenja Becker for her help with data extraction and Layla Nieden for the final proofreading of the manuscript. 2. Wilkinson MD, Dumontier M, Aalbersberg IJJ, Appleton G, Axton M, Baak A, et al. The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016;3:160018. 2. Wilkinson MD, Dumontier M, Aalbersberg IJJ, Appleton G, Axton M, Baak A, et al. The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016;3:160018. Availability of data and materials y The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. We will publish the data in the institutional repository of the University of Freiburg “FreiDok Plus” (https://freidok.uni-freiburg.de) in due course. The DoiScout – an automatic tool for gathering information about registered clinical trials and resulting publications is available on GitHub: https://github. com/kainitschke/doiscout. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. We will publish the data in the institutional repository of the University of Freiburg “FreiDok Plus” (https://freidok.uni-freiburg.de) in due course. The DoiScout – an automatic tool for gathering information about registered clinical trials and resulting publications is available on GitHub: https://github. com/kainitschke/doiscout. Author details 1 1Institute for Evidence in Medicine (for Cochrane Germany Foundation), Faculty of Medicine and Medical Center, University of Freiburg, Breisacher Str. 86, 79110 Freiburg, Germany. 2Clinical Trials Unit, Faculty of Medicine and Medical Center, University of Freiburg, Elsässer Straße 2, 79110 Freiburg, Germany. 3Cochrane Hungary, Clinical Centre of the University of Pécs, Medical School, University of Pécs, Pécs, Hungary. 4Institute of Medical Biometry and Statistics, Faculty of Medicine and Medical Center, University of Freiburg, Stefan-Meier-Str. 26, 79104 Freiburg, Germany. Additional file 9:. Study characteristics associated with citation by systematic reviews. Additional file 9:. Study characteristics associated with citation by systematic reviews. Received: 9 November 2020 Accepted: 24 June 2021 Additional file 10:. Study characteristics associated with citation by guidelines. Additional file 10:. Study characteristics associated with citation by guidelines. Abbreviations AWMF A b Abbreviations AWMF: Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften (Association of the Scientific Medical Societies). https:// www.awmf.org/leitlinien/leitlinien-suche.html; BMBF: Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research). https:// www.bmbf.de; Cochrane Library: https://www.cochranelibrary.com/; DFG: Deutsche Forschungsgemeinschaft (German Research Foundation). https://www.dfg.de/; DOI: Digital Object Identifier; DRKS: Deutsches Register Klinischer Studien (German Clinical Trials Register). https://www.drks.de; Epistemonikos: Collaborative, multilingual database of health evidence and largest source of systematic reviews relevant for health-decision making, and of other types of scientific evidence. https://www.epistemonikos.org/; EUCTR: EU Clinical Trials Register. https://www.clinicaltrialsregister.eu/; EudraCT: European Union Drug Regulating Authorities Clinical Trials Database. https://www.clinicaltrialsregister.eu/ctr-search/search; EMA: European Medicines Agency. https://www.ema.europa.eu; GEPR IS: German Project Information System. Online database made available by the DFG that provides information on current DFG-funded research projects. https://gepris.dfg.de/gepris/OCTOPUS; Google scholar: Web search engine providing scholarly literature. https://scholar.google.com; IITs: Investigator Initiated Trials; ISRCTN registry: International Standard Randomized Controlled Trials Number registry. http://www.isrctn.com; ISTs: Industry Sponsored Trials; LIVIVO: Interdisciplinary search engine for literature and information in the field of life sciences, run by ZB MED – Information Centre for Life Sciences. https://www.livivo.de; Medline: Medical Literature Analysis and Retrieval Page 19 of 20 Page 19 of 20 (2021) 21:182 Blümle et al. BMC Medical Research Methodology (2021) 21:182 System Online. Bibliographic database of life sciences and biomedical information. Accessible e.g. via the search engine PubMed; NCT: National Clinical Trial (number); PubMed: Free search engine accessing primarily the MEDLINE database. https://www.ncbi.nlm.nih.gov/pubmed/; RCT: Randomized controlled trial; SR(s): Systematic review(s); TRIP: Turning Research Into Practice. https://www.tripdatabase.com/; Web of Science: https://apps.webofknowledge.com Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions 3. Higgins J, Savović J, Page M, Elbers R, Sterne J. Chapter 8: assessing risk of bias in a randomized trial. In: Higgins JP, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA, editors. Cochrane handbook for systematic reviews of interventions version 6.1 (updated September 2020): Cochrane; 2020. Available from www.training.cochrane.org/handbook.2020. 3. Higgins J, Savović J, Page M, Elbers R, Sterne J. Chapter 8: assessing risk of bias in a randomized trial. In: Higgins JP, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA, editors. Cochrane handbook for systematic reviews of interventions version 6.1 (updated September 2020): Cochrane; 2020. Available from www.training.cochrane.org/handbook.2020. AB and MS designed the project and developed the methods. AB, KB, KW, PK, SL and EN extracted data and searched for publications. JZ obtained the full text of the systematic reviews and determined the usage (citation) of the published articles in the systematic reviews. AB wrote the manuscript. GR and AB analyzed the data. GR conducted the statistical analyses. KN developed the semi-automatic tool “DOIScout”. GR and MS substantially re- vised the manuscript. All authors read and approved the final version of the manuscript before submission. 4. Schmucker C, Schell LK, Portalupi S, Oeller P, Cabrera L, Bassler D, et al. Extent of non-publication in cohorts of studies approved by research ethics committees or included in trial registries. PLoS One. 2014;9(12):e114023. https://doi.org/10.1371/journal.pone.0114023. 4. Schmucker C, Schell LK, Portalupi S, Oeller P, Cabrera L, Bassler D, et al. Extent of non-publication in cohorts of studies approved by research ethics committees or included in trial registries. PLoS One. 2014;9(12):e114023. https://doi.org/10.1371/journal.pone.0114023. 5. Scherer RW, Meerpohl JJ, Pfeifer N, Schmucker C, Schwarzer G, von Elm E. Full publication of results initially presented in abstracts. Cochrane Database Syst Rev. 2018;(11). Art. No.: MR000005. https://doi.org/10.1002/14651858. MR000005.pub4. Accessed 18 Aug 2021. Competing interests Competing interests The authors declare that they have no competing interests. Declarations The online version contains supplementary material available at https://doi. org/10.1186/s12874-021-01359-x. The online version contains supple org/10.1186/s12874-021-01359-x. Ethics approval and consent to participate Not applicable. Additional file 1:. PRISMA flowcharts. Additional file 2:. Sources where published articles were identified. Additional file 3:. Number of published articles. Additional file 4: Publication frequency: Proportion of trials (total: n= 472) with n published method and results articles (total: n=947). Additional file 5:. Study characteristics associated with publication of trial results. Additional file 6:. Medical Fields. Additional file 7:. Number of published articles cited by systematic reviews and/or by clinical guidelines per sub-cohort and type of publication. Additional file 8: Citation frequency for published articles (n=599) by systematic reviews (n=2631). Additional file 9:. Study characteristics associated with citation by systematic reviews. Additional file 10:. Study characteristics associated with citation by guidelines. Additional file 1:. PRISMA flowcharts. Additional file 2:. Sources where published articles were identified. Additional file 3:. Number of published articles. Additional file 4: Publication frequency: Proportion of trials (total: n= 472) with n published method and results articles (total: n=947). Additional file 5:. Study characteristics associated with publication of trial results. Additional file 6:. Medical Fields. Additional file 7:. Number of published articles cited by systematic reviews and/or by clinical guidelines per sub-cohort and type of publication. Additional file 8: Citation frequency for published articles (n=599) by systematic reviews (n=2631). Additional file 9:. Study characteristics associated with citation by systematic reviews. Additional file 10:. Study characteristics associated with citation by guidelines. Funding Thi j This project is supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, grant BL 1395/2–1). The funding body has no role in the study design, the collection, analysis, and interpretation of data as well as in writing this manuscript. AB and KB report grants from the DFG during the conduct of the study. SL reports financial support from the Hungarian Academy of Sciences for EBM-related methodological research (János Bolyai Research Scholarship, BO/00498/17/5) and from the Alexander von Humboldt Foundation to support a research stay at the Institute for Evi- dence in Medicine, University of Freiburg, Germany during the conduct of the study. Outside the scope of the current work, SL participated in a re- search project on health effects of inulin-type fructans, supported by Beneo GmbH (Germany), and received a small expense allowance (honorarium) for her participation in a scientific work on the health effects of arachidonic acid intake as an expert in the International Life Sciences Institute (ILSI) Europe’s Nutrient Intake Optimisation Task Force. KW, PK, EN, KN, JZ, GR, and MS have nothing to disclose. Open Access funding enabled and organized by Projekt DEAL. 6. Moorthy V, Karam G, Vannice K, Kieny M-P. 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https://openalex.org/W3167362300	https://geusbulletin.org/index.php/rapggu/article/download/7775/13645	English	null	Lithostratigraphic framework of the Upper Proterozoic and Lower Palaeozoic deep water c1astic deposits of North Greenland	Rapport	1,982	cc-by	8,083	Grønlands Geologiske Undersøgelse (The Ge%gIA>ll Sruvey o/ G~enlmrd) Øster Voldgade IO. DIC-1350 Copenba.... K Grønlands Geologiske Undersøgelse (The Ge%gIA>ll Sruvey o/ G~enlmrd) Øster Voldgade IO. DIC-1350 Copenba.... K Reports D.kr.65.00 p Palaeootology and stratJaraphy øf GreeøIand: sbort contributions. 1980. Db. 65.00 No. 101 Palaeootology and stratJaraphy øf GreeøIand: sbort contributions. 1980. D No. 102 TbennaI maturaliOD øf orgaoic maller by a tbick baaItic si11 in tIpper Creta<eous sbales. Svattenhuk Halvø "Dttal West GreeDIand 1981 by E J Scbiener ol: l Pen palll No. 102 TbennaI maturaliOD øf orgaoic maller by a tbick baaItic si11 in tIpper Creta<eous sbales. Svattenhuk Halvø. "Dttal West GreeDIand. 1981 by E. J. Scbiener ol: l. Pen,palll. No. 103 Tbe DIm""soaq introsion. Soutb 0reenIand. A _ report OD seoloøY, mioeraIo&Y. øeoc:bemislIY and economic geo1osY. 1981. D.kr. 105.00 No. 103 Tbe DIm""soaq introsion. Soutb 0reenIand. A _ report OD seoloøY, mioeraIo&Y. øeoc:bemislIY and economic geo1osY. 1981. D.kr. 105.00 g No. 104 Pleeamblian metamotpbic complexosla the Bast GreeDland Ca1edonides (72"-74"N) - tbelr relationsblps to the Eleonore Bay Gtoup, and CaledoDian <>fOØCIIC8is. 1981 Ily A. K. Higins et øL, tmd Isotopic studies iD tb. Balt GteeD1and CaledoDicles (72°-74"N) - Pralambrian and CaIedoDlan ages. 1981 by D. C. Res'" A. R. Oledhill. Db. 75.00 No. 104 Pleeamblian metamotpbic complexosla the Bast GreeDland Ca1edonides (72"-74"N) - tbelr relationsblps to the Eleonore Bay Gtoup, and CaledoDian <>fOØCIIC8is. 1981 Ily A. K. Higins et øL, tmd Isotopic studies iD tb. Balt GteeD1and CaledoDicles (72°-74"N) - Pralambrian and CaIedoDlan ages. 1981 by D. C. Res'" A. R. Oledhill. 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No. 109 Distribution øf char8cterislic e1emeots iD the radioacdve rocks øftbe nortbenapartøfK....... 1jeId.1Ilmaussaq introsion, Sontb GreeD1aDd. 1982 by H. KUIIlltIIldmf, P. r--__..:::G~R~Ø~N:.=;LANDSGEOLOGISKE UNDERSØGELSE G E U S RAPPORT Nr. 107 Report file no. 22444 The GeologicoJ Survey of Greenland Report No. 107 Lithostratigraphic framework of the Upper Proterozoic and Lower Palaeozoic deep water c1astic deposits of North Greenland by J. D. Friderichsen, A. K. Higgins, J. M. Hum, S. A. S. Pedersen, N. J. Soper and F. Surlyk KØBENHAVN 1982 r--__..:::G~R~Ø~N:.=;LANDSGEOLOGISKE UNDERSØGELSE G E U S RAPPORT Nr. 107 Report file no. 22444 The GeologicoJ Survey of Greenland Report No. 107 Reports No. 85 Repott ofactMtles, 1976. 1977. No. 85 Repott ofactMtles, 1976. 1977. D.kr.40.00 No. 87 Project Westmar - A shaUow marine SCOPbysical_ OD the Weat GreeD1aDd oontinental sbeIf. 1979 by C. P. Brett cft E. F. K. Zarudzki. 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D No. 93 StnIcturaI and straIiaJnpbic framework of the Nortb GreeDland føI4 belt in Johan Jensen Land, Peary Land. 1979 by P. R. Dawes '" N. J. Soper. D.k p Jensen Land, Peary Land. 1979 by P. R. Dawes '" N. J. Soper. D.kr.36.0 i y y p No. 94 Hydroloøi<a1 basins in West Greeo1and. 1980 by A. Weidick '" O. B. Olesen. D tieS ro y y No. 95 Repott of tieS. 1978. 1979. D ro _ No. 96 Tbe SlalUll of th. NeogIaciaI in ro Gt_land. 1980 by M. Keny. D.k _ No. 97 Tbe thoIeiilic and komatiilic affinities of the MaIeøe metavolcau1c alllJlblbol1tes bom mdr- toq. southem West Greentand. 1980 by Ro P. Ha11. D.kr. 25.00 _ No. 97 Tbe thoIeiilic and komatiilic affinities of the MaIeøe metavolcau1c alllJlblbol1tes bom mdr- toq. southem West Greentand. 1980 by Ro P. Ha11. D.kr. 25.00 No. 99 Repott OD the 1979 geo1ogica1 expedItion to the Peuy Land regioo, Nortb 0reenIand 1980. Q.kr.90.00 No. 99 Repott OD the 1979 geo1ogica1 expedItion to the Peuy Land regioo, Nortb 0reenIand. 1980. Q.kr.90.00 No. 100 Repott øf aetivities. 1979. 1980. No. 100 Repott øf aetivities. 1979. 1980. D No. 100 Repott øf aetivities. 1979. 1980. Reports Nyegaar4 &: B. L. N'telseD. N 110 R 1981 1982 No. 110 Repott øf _ ...... 1981. 1982. No. 110 Repott øf _ ...... 1981. 1982. GRØNLANDS GEOLOGISKE UNDERSØGELSE RAPPORT Nr. 107 Lithostratigraphic frarnework of the Upper Proterozoic and Lower Palaeozoic deep water clastic deposits of North Greenland by J. D. Friderichsen, A. K. Higgins, J. M. Hurs!, S. A. S. Pedersen, N. J. Soper and F. Surlyk by by 1982 Abstraet The deep water clastic and subordinate carbonate sediments of the Franklinian Basin of North Greenland are assigned to 6 lithostratigraphic groups. The lowest, Skagen Group (new), consists of structureless quartzitic sandstones and phyllitic mudstones of ?late Precambrian to Cambrian age. The folIowing Paradisfjeld Group (revised) is dominated by lime mudstones and is probably of Cambrian age. Redeposited limestone conglomerates characterise thc top parts of thc group. Thc subscqucnt Polkorridoren Group (revised) consists of thick sandstone turbidite and mudstone units of Cambrian age. The overlying Vølvedal Group (new) consists of cherts, mudstones, turbiditic sandstones and resedimented chert and carbonate conglomerates and is of Cambrian to earliest Ordovician age. The folIowing Amundsen Land Group (new) is dominated by cherts and mudstones with resedimented limestone conglomerates, and is of early Ordovician to early Silurian age. The youngest, Peary Land Group, is dominated by turbiditic sandstones and mudstones and ranges in age from earliest Silurian to possibly earliest Devonian. The 'Sydgletscher Group' is disbanded: the component parts are placed in the Polkorridoren Group, Vølvedal Group, Amundsen Land Group and Peary Land Group. Authors' addresses: J.D.F., A.K.H., J.M.H., ES., Geological Survey of Greenland, øster Voldgade 10, DK-1350 Copenhagen K. Authors' addresses: J.D.F., A.K.H., J.M.H., ES., Geological Survey of Greenland, øster Voldgade 10, DK-1350 Copenhagen K. N. J. S., Department of Geology, University of Sheffield, Mappin Street, Sheffield SI 3JD, England. N. J. S., Department of Geology, University of Sheffield, Mappin Street, Sheffield SI 3JD, England. S. A. S. P., Geologisk Centralinstitut, øster Voldgade 10, DK-1350 Copenhagen K. Fig. 1. Some previous lithostratigraphic schemes covering the deep water sediments of North Greenland. Note that 'formation B' of Dawes & Soper (1979) corresponds to the Amundsen Land Group of this article. However, Dawes & Soper (1979) also considered 'formation C' to approximate to the Polkorridoren Group of Dawes & Soper (1973) as well as the un-named Silurian formations of Christie & Peel (1977). This correlation cannot be clearly depicted on one figure. CONTENTS Introduction 5 Lithostratigraphy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 Skagen Group 5 Paradisfjeld Group 6 Polkorridoren Group 8 Yølvedal Group '" 11 Amundsen Land Group 13 Peary Land Group 15 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 18 References 18 Frankl (1955) Dawes & Soper (1973) Christie & Peel (1977) Dawes & Soper (1979) this article Un-named Silurian 3e Calc - sandstone flysch formation Peary Land Syd91etscher Sandstones (Syd91etscher sandstone ) Un-named Silurian Group black shale formation Flysch Lower and Upper 3d Calcareous and 9raphitic Amundsen Land Nysne Gletscher slates (Upper Sydgletscher shales) Graphitic Slates Sydgletscher 3c Black quartzite (Sydgletscher quartzite) Group Group 3b Calcareous and graphitic Vølvedal Brown Series slates (Lower Sydgletscher shales) Group Frigg Fjord Mudstones 3a Purpie and green mudstones (Nysne Gletscher mudstones) Formation C Polkorridoren Polkorridoren Series Polkorridoren 2b Arkosic psammite (Polkorridoren Group Grønnemark Sandstones psammite) andShales Group 2a Rusty , green quartz phyllite Malcantone Gletscher Marbles ld Yellow limestones Formation B and Slates ; Paradisfjeld Paradisfjeld Green calcareous phyllite Paradisfjeld lc Marbles and Phyllites; Group Formation A Nordgletscher Marbles ; lb Dark gray limestone Group ? Ulvebakkerne Marbles la Graphitic and calcareous phyllites Hundeskrænten carbonates and elastics Skagen Group Fig. 1. Some previous lithostratigraphic schemes covering the deep water sediments of North Greenland. Note that 'formation B' of Dawes & Soper (1979) Fig. 1. Some previous lithostratigraphic schemes covering the deep water sediments of North Greenland. Note that 'formation B' of Dawes & Soper (1979) corresponds to the Amundsen Land Group of this article. INTRODUCTION The Upper Proterozoic and Lower Palaeozoic deep-watersediments of North Greenland and northem Ellesmere Island, Canada, were deposited in the Franklinian Basin (Trettin & Balkwill, 1979). The deep-water facies ofNorth Greenland are predominantly clastic and up to 8 km thick. The Upper Proterozoic - Ordovician part of the sequence outcrops mainly in a broad zone along the north coast of Greenland, within the present North Greenland fold belt, of which the eastern halt is shown in Plate 1. In the Silurian the deep-water flysch basin expanded southwards for considerable distances, and areas as far south as Kronprins Chri- stian Land ean be envisaged to have formed part of the enlarged Franklinian Basin (fig. 8). A full discussion of previous geological work in the clastic facies, and North Greenland in general, has been given by Dawes (1971, 1976) and Dawes & Soper (1973, 1979). This article is prompted by the need to rationalise the numerous stratigraphic terms which have been formally and informally applied to some of the sediments. In particular, the litho- stratigraphies erected north (Dawes & Soper, 1973) and south (Dawes & Soper, 1979) of the Harder Fjord fault zone (fig. 1) ean now be integrated (Hurst & Surlyk, 1980; Surlyk, Hurst & Bjerreskov, 1980). The whole sequence is divisible into lithostratigraphic units of mainly formational rank, the descriptions of which are in preparation. The present paper is limited to the formal definition (and redefinition) of lithostratigraphic units on group level. Distribution of the groups is shown on Plate 1 and fig. 8. Consequently, a basic comprehen- sive lithostratigraphic nomenclature is provided to cover the whole deep-water clastic sequ- ence of North Greenland. The Upper Proterozoic and Lower Palaeozoic deep-watersediments of North Greenland and northem Ellesmere Island, Canada, were deposited in the Franklinian Basin (Trettin & Balkwill, 1979). The deep-water facies ofNorth Greenland are predominantly clastic and up to 8 km thick. The Upper Proterozoic - Ordovician part of the sequence outcrops mainly in a broad zone along the north coast of Greenland, within the present North Greenland fold belt, of which the eastern halt is shown in Plate 1. In the Silurian the deep-water flysch basin expanded southwards for considerable distances, and areas as far south as Kronprins Chri- stian Land ean be envisaged to have formed part of the enlarged Franklinian Basin (fig. 8). CONTENTS However, Dawes & Soper (1979) also considered 'formation C' to approximate to the Polkorridoren Group of Dawes & Soper (1973) as well as the un-named Silurian formations of Christie & Peel (1977). This correlation cannot be clearly depicted on one figure. Fig. 1. Some previous lithostratigraphic schemes covering the deep water sediments of North Greenland. Note that 'formation B' of Dawes & Soper (1979) corresponds to the Amundsen Land Group of this article. However, Dawes & Soper (1979) also considered 'formation C' to approximate to the Polkorridoren Group of Dawes & Soper (1973) as well as the un-named Silurian formations of Christie & Peel (1977). This correlation cannot be clearly depicted on one figure. ostratigraphic schemes covering the deep water sediments of North Greenland. Note that 'formation B' of Dawes & Soper (1979) ndsen Land Group of this article. However, Dawes & Soper (1979) also considered 'formation C' to approximate to the Dawes & Soper (1973) as well as the un-named Silurian formations of Christie & Peel (1977). This correlation cannot be clearly INTRODUCTION g g g A full discussion of previous geological work in the clastic facies, and North Greenland in general, has been given by Dawes (1971, 1976) and Dawes & Soper (1973, 1979). This article is prompted by the need to rationalise the numerous stratigraphic terms which have been formally and informally applied to some of the sediments. In particular, the litho- stratigraphies erected north (Dawes & Soper, 1973) and south (Dawes & Soper, 1979) of the Harder Fjord fault zone (fig. 1) ean now be integrated (Hurst & Surlyk, 1980; Surlyk, Hurst & Bjerreskov, 1980). The whole sequence is divisible into lithostratigraphic units of mainly formational rank, the descriptions of which are in preparation. The present paper is limited to the formal definition (and redefinition) of lithostratigraphic units on group level. Distribution of the groups is shown on Plate 1 and fig. 8. Consequently, a basic comprehen- sive lithostratigraphic nomenclature is provided to cover the whole deep-water clastic sequ- ence of North Greenland. Skagen Group new group History. This is the lowest group recognised in the North Greenland part of the Franklinian Basin (fig. 1). It corresponds to the 'Unnamed quartzite group' of Soper et al. (1980) and Higgins et al. (1981). Rocks referred to this group were observed by J. P. Koch (1916) who reported the area around Skagen to consist of "steel-grey granite" (p. 344); these are structureless quartzitic sandstones. Brief mention is also made by Christie & Ineson (1979, p. 65) in their description of a "third sedimentary sequence, north-east of Depotbugt". White quartzites, cut by dolerite intrusions, south of Depotbugt (Dawes, 1976, p. 277; Dawes & Soper, 1979, p. 10; Christie & Ineson, 1979, p. 64) have been correlated with the Proterozoic Independence Fjord Group (Collinson, 1980, p. 23); they were deposited in a different sedimentological environment from the Skagen Group, and are thought to be older in age. 6 Name. After the peninsula Skagen on the north-east coast of Peary Land (Plate l). Type area. The peninsula Skagen and the area immediately to the west of it (Plate l) Thickness. The full thickness is not known as the lower boundary of the group is not exposed. It is estimated to be at least 500 m thick. Lithology. Throughout the outcrop, the group is tightly folded, and a detailed stratigraphy has not been established from the few localities examined. There appear to be three main divisions. The lowest unit comprises thick beds of structureless, quartzitic sandstones. This succession is overlain by dark coloured, predominantly phyllitic mudstones. The top of the group is forrned by a further sequence of thick bedded, structureless quartzitic sandstones, with thin phyllitic mudstone interbeds and very rare pebble conglomerates; on the north side of Frederick E. Hyde Fjord this upper unit appears to be more thinly bedded. Boundaries. The lower boundary is not exposed. The upper boundary is gradational and is defined at the top of the last massive unit of quartzitic sandstone (c. 40 cm thick), and at the base of dark lime mudstones (calcareous phyllites) of the Paradisfjeld Group. In the known outcrops the boundary strata are strongly folded, but the contact is apparently conformable. Distribution. The group occurs in the area north-east of Depot Bugt, north-eastern Peary Land, and on the north side of Frederick E. Hyde Fjord in easternmost Johannes V. Jensen Land. Skagen Group new group An isolated structural inlier crops out on the western tip of Johannes V. Jensen Land between Moa ø and Lockwood ø (Plate l). The group mayaiso be represented in the highly deformed northern part of the fold belt between Benedict Fjord and Kap Morris Jesup, but as sedimentary structures are here no longer visible, stratigraphical relationships cannot be demonstrated (see also below, under Polkorridoren Group). Geological age. The age of the group is unknown. It underlies the Paradisfjeld Group, the top of which is Cambrian. The Skagen Group could be late Proterozoic, Cambrian, or late Proterozoic to Cambrian in age. Subdivisions. Three, as yet un-named, formations are recognisable, corresponding to the lower structureless quartzitic sandstones, the middle phyllitic mudstone unit and the upper structureless quartzitic sandstones. Paradisfjeld Group History. The term 'Paradisfjeld Marbles and Phyllites' was intrbduced by Frankl (1955) for a succession exposed in Polkorridoren, the north-south trending, glacier-filled valley crossing central Johannes V. Jensen Land (fig. 1). The terms 'Malcantone Gletscher Marbles and Slates' and 'Nordgletscher Marbles' were proposed for comparable rocks exposed in the same area (Frankl, 1955). It was shown by Dawes & Soper (1973) ihat all these units could be correlated and the term Paradisfjeld Group was introduced to cover the sequence. Our field work shows that the 'Ulvebakkerne Marbles' and part of the 'Sortevæg Marbles and 7 N s Fig. 2. Outcrop stylc af thc lime mudstones assigllcd to tile Pllflldisfjcld Grour in central Hazen Land (Plalc I). Thc light colourcd unit is near the lop Clf the Par::ldisfjcld Group. Nole the intense folding wilh slight northwards vcrgcncc. Polkorridoren Group sediments (thin-bcddcd lurbidilcs) occur in lhc corcs of Ihe synclincs. Cliff hcighl approximalely 800 111. s N Fig. 2. Outcrop stylc af thc lime mudstones assigllcd to tile Pllflldisfjcld Grour in central Hazen Land (Plalc I). Thc light colourcd unit is near the lop Clf the Par::ldisfjcld Group. Nole the intense folding wilh slight northwards vcrgcncc. Polkorridoren Group sediments (thin-bcddcd lurbidilcs) occur in lhc corcs of Ihe synclincs. Cliff hcighl approximalely 800 111. Phyllites' (Frankl, 1955) arc also part of thc Paradisfjeld Group (Sopcrer af., 1980; Higgins ,,' al., J981). Name, From thc mountain Paradisfjeld in central Johannes V. Jensen Land (Plalc I). Type areas. Tile castcrn tip of Johannes V. Jensen Land, Paradisfjeld, Gertrud Rask Land and Nansen Land (PJale I). Type areas. Tile castcrn tip of Johannes V. Jensen Land, Paradisfjeld, Gertrud Rask Land and Nansen Land (PJale I). Thickness. Tile precise thickness is difficult to estimate due to intense deformation in most areas, but is thought to be at least 1 km and probably mueb more. Thickness. Tile precise thickness is difficult to estimate due to intense deformation in most areas, but is thought to be at least 1 km and probably mueb more. Dominant Jitl101ogy. The Paradisfjeld Group is dominateJ by thiek scquences af dark grey, impure lime mudstanes, while thick beds af light grey limestone eonglomcratcs are charac- tcristie for the top parts of tbe group (fig. 2). Other varietics include laminated pak grey, orange and yellaw limestones, orange calcareous siltstoncs and oceasional dark grey to black non-calcareous muJstoncs (now shalcs ar phyllites). Paradisfjeld Group Low grade metamorphism and defor- mation has resultcd in recrystallisation and oblileralion of original slructures of rock types in northern areas, and these were usually described in the field as limcslones, marbles ar calcareous phyllitcs. Baunclaries. The lower gradalional baundary is plaeed wherc tbe earbonate rocks af lhe group overlie the quartzites af lhe Skagen Group (see Skagen Group). The upper boundary 8 iscomparatively sharp where the carbonate lithologies give way to the terrigenous mudstones and turbiditic sandstones of the Polkorridoren Group. In the environs of eastern Frederick E. Hyde Fjord, the top of the Paradisfjeld Group is placed at the top of re- sedimented limestone conglomerates, of which the last bed characteristically has a sandy matrix. Between Moa ø and Nansen Land, the conglomerates are overlain by up to 15 m of orange weathering lime mudstones (calcareous shales) which here form the top strata of the group. In northern Johannes V. Jensen Land, the top of the group is placed at the top of the last marble or calcareous phyllite bed. Distribution. The Paradisfjeld Group is widely distributed throughout Johannes V. Jensen Land, Nansen Land and the archipelago in between. Outcrops also occur in the cliffs of Hundeskrænten, north-east of Depotbugt (Plate 1). Geological age. The top of the group has yielded inarticulate brachiopod fragments and a broken phosphatic internal mould of a spicule of Chancelloria, suggesting a Cambrian age for that part of the sequence (Peel & Higgins, 1980). an regional stratigraphic considera- tions, Surlyk et al. (1980) concluded that the group was not younger than Cambrian. It is not known if the whole group is contained within the Cambrian or whether it extends down into the late Proterozoic. If it extends into the Proterozoic, then the underlying Skagen Group must be of wholly Proterozoic age. Subdivisions. The group is characterised by strong north-south variation of facies which may reflect an original broad zonation parallel to the southern platform. In central Johannes V. Jensen Land, around Paradisfjeld, a four-fold formation scheme ean be recognised (la-d of Dawes & Soper, 1973), while new formations will eventually be introduced for the eastern and western outcrops. Polkorridoren Gronp History. The group corresponds to the Polkorridoren Group of Dawes & Soper (1973) except for the upper boundary which is redefined to include the variegated, red and green 'Frigg Fjord Mudstones' (Frankl, 1955) and its equivalent, the 'Nysne Gletscher mudstone' (Dawes & Soper, 1973). The latter was originally placed by Dawes & Soper (1973) in their 'Sydgletscher Group' (fig. 1). The Polkorridoren Group includes the 'Polkorridoren Series', 'Grønnemark Sandstones' and 'Grønnemark Shales' of Frankl (1955). It probably also includes most of Frankl's 'Nunatak Quartzitic Slates', 'Kap Morris Jesup Quartzphyllites' and 'Sands Fjord Quartzphyllites', but in northern parts of the fold belt deformation and metamorphism have obliterated stratigraphical relationships and these units may thus also include parts of the Skagen Group (cf. Soper et al., 1980; Higgins et al., 1981). It is convenient at this juneture to indicate why the 'Sydgletscher Group' is disbanded and the component parts placed in the Polkorridoren, Vølvedal, Amundsen Land and Peary Land Groups. Firstly, the original Sydgletscher name was introduced by Frankl (1955) for a unit of formational rank. Later Dawes & Soper (1973) included this unit in their 'Sydgletscher Group'. The formation is readily recognisable and is being maintained, thus the name has priority for the formation and a new group name is required. Secondly, the 9 Fig. 3, Typical outcrop stylc af thick bedded sandstone lurbiditcs assigned to tlle Polkorridoren Group. Note intense deformation and nat lyillg folds. Cliff height approximatcly 1200 m. Snuth end af Hun! Fjord, an tile eastern side. Fig. 3, Typical outcrop stylc af thick bedded sandstone lurbiditcs assigned to tlle Polkorridoren Group. Note intense deformation and nat lyillg folds. Cliff height approximatcly 1200 m. Snuth end af Hun! Fjord, an tile eastern side. original definition af thc ·Sydglelscher Group" did not cover a cobercnt group af sediments \Vith sirnilar lithologics; these sediments are nQw rcfcrred to the Polkorridoren, Vølvedal, Amundsen Land and PeafY Land Groups. original definition af thc ·Sydglelscher Group" did not cover a cobercnt group af sediments \Vith sirnilar lithologics; these sediments are nQw rcfcrred to the Polkorridoren, Vølvedal, Amundsen Land and PeafY Land Groups. Name. After the glacicr~filled rass, Polkorridoren (Pi<lte 1). Type areas. Polkorridoren, Grønnemark. MacMillan ø. H. H. Benedict Bjerge (Plate I). Type areas. Polkorridoren, Grønnemark. MacMillan ø. H. H. Benedict Bjerge (Plate I). Thickness. Polkorridoren Gronp Tlle precise thickncss is not know'll due to structural complcxity but is estimated to be at Ieast 2 km. Thickness. Tlle precise thickncss is not know'll due to structural complcxity but is estimated to be at Ieast 2 km. Dominant lithology. Alternating, thick sandstane and ITIlIdsl0nc units. The thickness af these units is of the order uf SOIllC tens to a few hundred metres. The sandstone units comprise brown weathered, gradcd or non-gradcd tllrbidites \Vith thin muJstonc intcrbcds, whilc the mudstone units are dark grey or variegated (rcd, purpie or green) and cuntain scattcrcd thin. finc-graincd silty turbidites. The whole group compares well with the concept of ·ciassical f1ysch' (rig, n Boundaries. The lower boundary is placcd where the carhonatc lithologies af the Paradistjcld Group give way to terrigenous, sandy turbidites and mudstones of tile Pulkor- Boundaries. The lower boundary is placcd where the carhonatc lithologies af the Paradistjcld Group give way to terrigenous, sandy turbidites and mudstones of tile Pulkor- 10 Fig. 4. Cliff scclion in thc Vølvedal Group norlh af thc central P<lfl af Vølvcdal (Pl<:llc 1). Sediments in thc forcground are Frigg Fjord Mudstoncs (M) \vhich <lfC assigned IO thc Polkorridoren Group. Note thrust (1) fl:pc<lting succession. Thc Vølvcdal Group hcre consists prcdominanlly of mudstone and thin-bcddcd sillslone turbiditcs. Cliff hcighl approximalely 600 m. Fig. 4. Cliff scclion in thc Vølvedal Group norlh af thc central P<lfl af Vølvcdal (Pl<:llc 1). Sediments in thc forcground are Frigg Fjord Mudstoncs (M) \vhich <lfC assigned IO thc Polkorridoren Group. Note thrust (1) fl:pc<lting succession. Thc Vølvcdal Group hcre consists prcdominanlly of mudstone and thin-bcddcd sillslone turbiditcs. Cliff hcighl approximalely 600 m. ridorcn Group (see seelion Oll Paradisfjeld Group). The upper boundary is placed wherc the variegatcd Frigg Fjord Mudstones are overlain by dark muJstoncs, eherts and turbiditcs af the Yølvedal Gmup. The boundary is transitional. varying over tens of metres and is placcd at the top 01' the laS1 variegated unit. Distriburion. Widcly cxposcd throughout Johannes V. Jensen Land, Nansen Land and the archipelago in between (Platc 1). Geu/ogica/ agl'. No age-diagnoslic fossils are known from the group. Thc top af the under- Iying P<lT8disJjeld Group contains fossils suggesting a Ci:lmbrii:ln age. Grapw!itcs from the top part af thc ovcrlying Vølvedal Group indicatc an carlicsl Ordovician age. TllUs it is reasonablc to concludc that the Pol korridoren Group is cuntained within the Cambrian. Polkorridoren Gronp but a more precisc ag(' dd'inilion is not yet possib1c (cf. Surlyk er al., 1980). Subdivisions. Seven mapping divisions have becn recognised in central and eastcrn Johannes V. Jensen Land (Soper et al., 1980). In the western Olllerop area, betwecn Moa ø and Nansen Land, another scven mapping divisions ean be distinguished. A clear correlation between the two successions cannot be established due to ascarcily 01' mappabJe mudstone divisions in the alpine terrain ofwestern Johannes Y. Jensen Land, and due to the structural compkxity. However, red and green mudsloncs form the uppermosl unit in bOlh regions, and in thc Frigg Fjord region are referred \l) as the Frigg Fjord Mudstones. w 1I E Fig. 5. C"a:~tal cliff sCl:tion uf Ih..: Vølvctlal Group 011 thl,.' north side (lf Frederick E. Hydc Fjord (Plalc I). Thc profile is oricntnh..:d approximalciy pcrpcndicular to thc fold axes of the anlidines associatr.:d with Ihc Ihru,,! sh(:cts (Pedersen. 1980). Thc whilc band is a conglomcrrtlc unit in thc top part of thc group. Cliff hcighl approximalcly 500 m. 1I E w E Fig. 5. C"a:~tal cliff sCl:tion uf Ih..: Vølvctlal Group 011 thl,.' north side (lf Frederick E. Hydc Fjord (Plalc I). Thc profile is oricntnh..:d approximalciy pcrpcndicular to thc fold axes of the anlidines associatr.:d with Ihc Ihru,,! sh(:cts (Pedersen. 1980). Thc whilc band is a conglomcrrtlc unit in thc top part of thc group. Cliff hcighl approximalcly 500 m. Vølvedal Group new grotlp new grotlp 1-lislUry. The group (fig. I) includes the -Brown Series' af Frankl (1955) and its equivalcnt [unnalion 'Jb' ol' Dawes & Sara's (197.1) 'Sydglctschcr GrOllp'. Il ",Iso inc!udes 'formation A' uf Dawcs & Soper (J 979). The arguments for disbanding the 'Sydgk:tscher Group' are discusscd under the history section af thc Polkorridorcll Group. Name. Arter the valley Vølvedal in tlle southern part of Johannes V. Jensen Land, where the total thickness af the group is cxposcd (Plale I). Type area. Vølvcdal. southern Johannes V. Jensen Land (Platc I). ThieklIw'. 600--700 m. ThieklIw'. 600--700 m. Dominant Iilll%gy. Thc Yølvcdal Group is essentiaIly a turbiditic facics associalion com- prising cherlificd fine-grained, silty distal turbiditcs, white-wcalhering chertified. caJcarcous turbidites, fine IO medium bcddcd sandslone turbiditcs, subordinatc black hedded cherts. black mudstoncs and redeposited congiomerates (figs 4-6). Thc group encompasscs a erude upwards eoarscning unit. Mudstones and eherts dominalc Ihe lowcr part of tIle group, \\ hilst tlle middlc and upper parts are dominatcd by turbiditcs and the IOP is characteriscd by rcdcpositcd conglomerates dominatcd by chert and carbonatc dasts. Thc different rock Iypes are chcrtificd Io varying degrees. masking original scdimentary structurcs. As aresult the sediments supcrficially resemblc the ovcrlying mudstonc and ehert dominalcd Amundsen Land Group. especiaIly in structurall) compk:x areas. Fig. 6. Top uf thc Yølvcdal Group (Y). Amundsen Land Group (A) and the basc of thc Pc'lry Land Group (P) on the north side of O. B. Bøggild Fjord (Platc l). Note thc thrust (I) repeating thc Amundscn Land Group. ThruSIS also occur in the base of tlle Pcary Land Group, bUl lhey are nol shown. Cliff hcight approximatcly 1000 m. Here th\..' Yølvedal Group consists of quanzilic IUrbidites and rcscdimented limestone conglol1lcrates. The Amunuscn Land Group is dominatcd by JlludS{Qncs and cherts wi!h !he lighter bands n.:presenting thin rcscdimemc<l conglomcrittcs. Sillstoncs and finc-gmincd sandSlunl' !urbiditcs constilU!C thc Pcary Land Grollp. Fig. 6. Top uf thc Yølvcdal Group (Y). Amundsen Land Group (A) and the basc of thc Pc'lry Land Group (P) on the north side of O. B. Bøggild Fjord (Platc l). Note thc thrust (I) repeating thc Amundscn Land Group. ThruSIS also occur in the base of tlle Pcary Land Group, bUl lhey are nol shown. Cliff hcight approximatcly 1000 m. Here th\..' Yølvedal Group consists of quanzilic IUrbidites and rcscdimented limestone conglol1lcrates. new grotlp The Amunuscn Land Group is dominatcd by JlludS{Qncs and cherts wi!h !he lighter bands n.:presenting thin rcscdimemc<l conglomcrittcs. Sillstoncs and finc-gmincd sandSlunl' !urbiditcs constilU!C thc Pcary Land Grollp. Fig. 6. Top uf thc Yølvcdal Group (Y). Amundsen Land Group (A) and the basc of thc Pc'lry Land Group (P) on the north side of O. B. Bøggild Fjord (Platc l). Note thc thrust (I) repeating thc Amundscn Land Group. ThruSIS also occur in the base of tlle Pcary Land Group, bUl lhey are nol shown. Cliff hcight approximatcly 1000 m. Here th\..' Yølvedal Group consists of quanzilic IUrbidites and rcscdimented limestone conglol1lcrates. The Amunuscn Land Group is dominatcd by JlludS{Qncs and cherts wi!h !he lighter bands n.:presenting thin rcscdimemc<l conglomcrittcs. Sillstoncs and finc-gmincd sandSlunl' !urbiditcs constilU!C thc Pcary Land Grollp. 13 Boundaries. The lower boundary is placed where the variegated Frigg Fjord Mudstones of the Polkorridoren Group are overlain by dark mudstones, cherts and turbidites (see bound- ary definition under Polkorridoren Group). The upper boundary is placed where medium bedded turbidites and redeposited conglomerates are overlain by black bedded cherts and mudstones. The boundary is sharp and occurs some 40 m above a prominent redeposited conglomerate, throughout southern Johannes V. Jensen Land. Boundaries. The lower boundary is placed where the variegated Frigg Fjord Mudstones of the Polkorridoren Group are overlain by dark mudstones, cherts and turbidites (see bound- ary definition under Polkorridoren Group). The upper boundary is placed where medium bedded turbidites and redeposited conglomerates are overlain by black bedded cherts and mudstones. The boundary is sharp and occurs some 40 m above a prominent redeposited conglomerate, throughout southern Johannes V. Jensen Land. Distribution. Widely distributed in south-central and south-western Johannes V. Jensen Land, and between inner J. P. Koch Fjord and O. B. Bøggild Fjord (Plate 1). Distribution. Widely distributed in south-central and south-western Johannes V. Jensen Land, and between inner J. P. Koch Fjord and O. B. Bøggild Fjord (Plate 1). Geological age. The age of the base of the group is unknown. In the upper part of the group in western Amundsen Land, the graptolites Dictyonema sp. and Anisograptus sp. probably indicate an earliest Canadian (earliest Ordovician) age (M. Bjerreskov, personal communi- cation, 1981). Ten metres below the top of the group on the north coast of B. O. Bøggild Fjord, the presence of ?Adelograptus probably indicates a late Early Canadian (Early Ordovician) age (M. new grotlp Bjerreskov, personal communication, 1981). Thus, the upper part of the group is of Early Canadian (Early Ordovician) age and it probably extends well down into the Cambrian. Subdivisions. Three formations can be recognised in the type area, but in some areas the intense chertification makes a subdivision impossible. new group History. The group (fig. 1) essentiaily corresponds to the '3c' and '3d' formations of the 'Sydgletscher Group' of Dawes & Soper (1973) and 'formation B' of Dawes & Soper (1979). It approximately corresponds to Frankl's (1955) 'Nysne Gletscher Graphitic Slates'. The arguments for disbanding the 'Sydgletscher Group' are discussed under the history section of the Polkorridoren Group. Name. After the south-western peninsula of Johannes V. Jensen Land (Plate 1). Type area. Amundsen Land (Plate 1). Type area. Amundsen Land (Plate 1). Thickness. 350-500 m. Dominant lithology. Black bedded chert and laminated mudstone, often chertified, are characteristic of the group. Chertified thin bedded turbidites and greenish chertified siltstones are subordinate. Some horizons are bioturbated. In some areas, especially in the Nordpasset - Kap Mjølner region (Plate 1), the succession is characterised by thick redepo- sited chert and limestone conglomerates interbedded with thick calcareous turbidites (figs 6 & 7). & 7). 14 Fig. 7. MIldstones and resedimenled conglorncratcs (lighter bands) nf the uppcr pan of thc Amundsen Land Group (A) overlain by Lhe Peary Land Group (P) in lhc northcrn part af Lauge Koch Land (fig. R). Cliff ho.:igh! approxirnately 400 m. Fig. 7. MIldstones and resedimenled conglorncratcs (lighter bands) nf the uppcr pan of thc Amundsen Land Group (A) overlain by Lhe Peary Land Group (P) in lhc northcrn part af Lauge Koch Land (fig. R). Cliff ho.:igh! approxirnately 400 m. Boundarics. Tbc lower boundary is placcd wherc file medium beddcd turbidites of the Vølvcdal Group are overlaitl hy hlack, bcddcd eherts and mudstoncs. The tipper bOllndary is placed where 1he brown to yella\\' weathering 'classical f1ysch' turbidites of the Pear)' Land Group overlie the dark finc~graincd sediments af the Amundsen Land Group. The houlld- ary is diachronous and whi1c it is very sharp in some ar(;:as, such as amund SydgJetscher (Plate l), il is of a transitionai nature in other areas, such as O. B. Bøggild Fjord (PJalc l). Disrribulion. The group is widely exposed in south-central and south-western Johannes Y. Jensen Land, bctween iIlIler J. P. Koch Fjord and O. B. Bøggild Fjord, northel'l1 Lauge Koch Land, Freuchcn Land, probably Nares Land, and \Vulff Land (Plate l; fig. 8). Geologieni agt'. Poorly prcserved graptolites, including Ciollograprus sp. sevcraJ metres above the base of the gru up indicatc a Jate Early Canadian age (M. Bjerreskov, personal communicatinn, 11)81), similar to the top af the Yølvedal Group. new group Thc top nf the group is diachronous. In Amundscl1 Land Ciimacograptus miserabilis, and Orrhograptus sp. rcscmb- Jing o. quadrimucrO!1fl/lIs, from the very top af !hc group indiCafe a Ja!es! Ordovician (Cincinllatian) age (Surlyk er al., 1980). Same 142111 from the top of the group in the SydgJdschcr area (Platc I), the graptolites Climacograptus rectangularis and Atavugraptus af[. A. alavflS indicate the atuvus to cyphus Zones in the Lowcr Llandovcry (Sijurian). Thus, the top of the group is diaehronous het\veen the Late Ordovician and Early SiJurian (Surlyk Cl al., 1980). 15 Ic.'''" ". ". Fig. 8. Distribution uf lhc Pcary Land Group in NurLh Greenland. ParI of thc distribulion af the gruup in Kronprins Chrislian Lanu from J. S. Peel (personal communiculion, 1981). Washington Land (I). Hall Land (2), Nycboe Land (3). Warming Land (4), Hcmlriks ø (5), Wulff Land (6). Narcs Land (7). Frcuchcn Land (8). Lauge Koch Land (1). Pcary Land (IO) and Kronprins Christian Land (11). Ic.'''" ". ". Fig. 8. Distribution uf lhc Pcary Land Group in NurLh Greenland. ParI of thc distribulion af the gruup in Kronprins Chrislian Lanu from J. S. Peel (personal communiculion, 1981). Washington Land (I). Hall Land (2), Nycboe Land (3). Warming Land (4), Hcmlriks ø (5), Wulff Land (6). Narcs Land (7). Frcuchcn Land (8). Lauge Koch Land (1). Pcary Land (IO) and Kronprins Christian Land (11). Subdivisiolls. Thc group is divisiblc into two mainly finc-graincd formations corrcsponding lU lhe beddcd chcrt and laminaced muds!one units, and a souchern rescdimcnrcd con- glomcratic formation. Subdivisiolls. Thc group is divisiblc into two mainly finc-graincd formations corrcsponding lU lhe beddcd chcrt and laminaced muds!one units, and a souchern rescdimcnrcd con- glomcratic formation. Pcary Land Group I-JislOry. Hurst (1980) erected the Peary Land Group. Thc group encompasscs thc 'Cape Rawson Beds' of Feilden & De Rance (1878) and the 'Cape Ra'...·son Group' in the sense af Dawcs (1966, 1971) and Dawc, & Soper (1973). Thc group also includes thc sedimcnlS in North Greenland that were referred to the Imina Group of Ellcsmcrc Island, Canada (ef. Dawes, 1971, 1976). Also inc1udcd are the: 'Un-namcd Silurian black shaie formation' and the 'Un-named Silurian nysch formation' of Christie & Peel (1977), 'formation C' of Dawcs & Soper (llJ79) and lhe 'Sydglctscher Sandstones' (Frank I, 1955) af rlle 'SydgfcIschcr Group' (Dawes & Soper, 1973). The arguments for disbanding the 'Sydgietseber Gruup' are discussed under the history scction of the Polkorridorcn Group. The Profilfjeldct Shale af Kronprins Christian Land (Nielsen, 1941; Frankl, 1954) and the KjuvcslcLtc Sandstones (FrankI, 1956) are also includcd in (he group, as well as the Hendrik conglomcrate of Dawes (1976). The recent I)' redefined Cape Schucert Fonnaliun together with the newly erccted Lafaycttc Bugt Formation (Hurst, 1980) are a1so assigned to the gcoup in Washington Land. The extent of the 'Polans Harbour Formation' of Koch (1929) is now known (Dawcs & Haller, 1979) and (hese strata are also includcd in thc Pcary Land Group; however, use of this formatiun name has been discontinued (Lane er al., 1980). 16 Fig. 9. Fining-upward cycles in the Peary Lmd Group at the north-clIstcrn end of Es- rum Elv, north Peary Land (Pllltc I). Cliff hcight ap- proximately 800 m. Name. After Peary Land where the group forms the uppermost stratum in most af the afca (lig 8). Fig. 9. Fining-upward cycles in the Peary Lmd Group at the north-clIstcrn end of Es- rum Elv, north Peary Land (Pllltc I). Cliff hcight ap- proximately 800 m. Name. After Peary Land where the group forms the uppermost stratum in most af the afca (lig 8). Name. After Peary Land where the group forms the uppermost stratum in most af the afca (lig 8). Type area. The conslituent forrnations have type areas in North Greenland, from Kronprins Christian Land to Washington Land (fig. 8). Type area. The conslituent forrnations have type areas in North Greenland, from Kronprins Christian Land to Washington Land (fig. 8). Thickness. The group rcaches ils rnaxirnum thickness af aboul 3 km in Pcary Land. Fig. 9. Fining-upward cycles in the Peary Lmd Group at the north-clIstcrn end of Es- rum Elv, north Peary Land (Pllltc I). Cliff hcight ap- proximately 800 m. Subdivisiolls. The group is subdivided into eighl formations. scveral af which arc lateral cquivalents (Hurst & Surlyk. in press). Pcary Land Group 111 the western end af the ollterep belt in \Vashington Land il has dccrcascd to abouI 500 m. Thickness. The group rcaches ils rnaxirnum thickness af aboul 3 km in Pcary Land. 111 the western end af the ollterep belt in \Vashington Land il has dccrcascd to abouI 500 m. Dominant lirhology. The bulk af the group comprises yellow ro brown weathering turbidites af 'classical flysch' appearance (figs 9-11); the weathering colour is due to significant amounts of carbonate material in Ihe cement or matrix. A major mudstone unit occurs in the middle nf the group and the proportion af mudstone increases to the west. In tlle lower half of the group along the south coasl af Frederick E. Hyde Fjord and in \Vashington Land, a sequence of redcposited limcSlone conglomnates occurs. Thc upper part of the group is characterised by abundant ehert pebble conglomeratcs. [7 N s Fig. IO. Mudsloncs and lurbidiles of the Peary Land Gruup (P) o\'l.::r1ying platform carbonatcs (c) Oll Ihc easl side of Lauge Koch Land (Plate l). s N Fig. IO. Mudsloncs and lurbidiles of the Peary Land Gruup (P) o\'l.::r1ying platform carbonatcs (c) Oll Ihc easl side of Lauge Koch Land (Plate l). 8ou/ldarie.~. The lowcr boundary is. placed wherc tile black eherts and ITIudstoncs af the Amulldsen Ll1nd Group are overlain by buf[ wcathcring turbiclites af the Pcary Land Group. Along lhe soulncrn rnargin of the outcrop belt (fig. 8), the grO\.lp overlaps maillly Silurian platform carbonulCs. The group forms the top Slratum in the western part af the fold belt. In the Peary Land region il is overiain, wirh angular unconfOTlllity, by Luc Paiaeozoic con- glomeratcs and sandstones in red-bcd facies of the Mallemuk Mountain Group (Håkansson, 1979). Dislribwiofl. The group is widespread in North Greenland, from Kronprins Christian Land in thc cast to Washington Land in the west (fig. 8). Ceological agt!. Thc base of the group is diachronous between thc lalest Ordovician and Early Silurian (scc scction Oll Amundsen Land Group). The youngcst graptolites occur in western North Greenland where MOllograprus cf. M. rraflsgradil!lls occurs ncar the top ofthe sequencc. indicating a Latc Silurian (Pridoli) age. There is no dcfinitc c\'idence of Devonian strata. but it is possibie that the top af the group may prove to reach into thc carlicst Devonian (cf. Surlyk er al., 1980). 18 Fig. ll. Pcary Land Group Thin beddcd mudslone and sihstone turbiditcs uf thl: Peary Land GnJup nt~ar the lIorthern CO:lst uf H~lll Land (fig. 8). Cliff heighl approxim3tcly 600 m. Fig. ll. Thin beddcd mudslone and sihstone turbiditcs uf thl: Peary Land GnJup nt~ar the lIorthern CO:lst uf H~lll Land (fig. 8). Cliff heighl approxim3tcly 600 m. Acknowlcdgcmcnts Eckan H~k<lnsson discusscd various aspccts uf the manuscript. Thc critical comments uf thTee rdeT- ces are gratefully acknowledged. Dawcs, P. R. & Haller, J. 1979: Historicai aspects in thc geological invcsligalion of norlhcrn Green- land. Meddr Grønland 200(4), 38 pp. References Christie. R. L. & Incson, J. R. 1979: Prccambrian-$ilurian geology of the G. 8. Schley Fjord region, castern PeaTY Land. Nonh Greenland. Happ. Gron/ands geo/. Unders. 88.63-71. Christie. R. L. & Peel, J. S. 11.)77: Cambrian-5ilurian stratigraphy of Børglum Elv. Pcary Land. caSh.:rn ;-.sonh Gn.:cnland. Rapp. Grotllalld~ ge%~~ Unders. 82,48 pp. Christie. R. L. & Peel, J. S. 11.)77: Cambrian-5ilurian stratigraphy of ;-.sonh Gn.:cnland. Rapp. Grotllalld~ ge%~~ Unders. 82,48 pp. Collinsun. J. D. 1980: Slratigraphy uf the lndependem:e Fjmd Group (Proterozoic) uf eaSlern ~()r1h Greenland. Rapp. Gronlands geol. Unders. 99. 7-23. Orm·es, P. R. ]1)66: Lower Palacowic geolog)' of Ihe weslern part of thc Norlh Greenland fold bell. Rapp. (;f(Jnl(lIld.~ geo/. Unders. Il. 11-15. Da\\t:s, P. R. 1971: The North Greenland fold belt and cnvirons. Bul/. geo/. StK. Denmark 20. 197-231). Dawes, P. R. 1976: Prcc:mlbrian to Teniary ef norlhern Grccnland.11I Esch Geolog)' o[GreMland, 248-303. Copenhagcn: Geol. Surv. Greenland. Dawes, P. R. 1976: Prcc:mlbrian to Teniary ef norlhern Grccnland.11I Escher. A. & Wan, W. S. (edit.) Geolog)' o[GreMland, 248-303. Copenhagcn: Geol. Surv. Greenland. Dawcs, P. R. & Haller, J. 1979: Historicai aspects in thc geological invcsligalion of norlhcrn Green- land. Meddr Grønland 200(4), 38 pp. 19 Dawes, P. R & Soper, N. J. 1973: Pre-Quaternary history ofNorth Greenland. In Pitcher, M. G. (edit.) Arctic Geology. Mern. Arner. Ass. Petrol. Geo!. 19,117-134. awes, P. R & Soper, N. J. 1979: Structural and stratigraphicframework of the North Greenland f belt in Johannes V. Jensen Land, Peary Land. Rapp. Grønlands geol. Unders. 93, 40 pp. Feilden, H. W. & De Rance, e. E. 1878: Geology of the coasts of the Arctic lands visited by the late British Expedition under Captain Sir George Nares, RN., K.e.B., ERS. Quart. II geol. Soc. Land. 34, 556-567. Frankl, E. 1954: Vorlaufige Mitteilung tiber die Geologie von Kronprins Christians Land (NE-Gron- land). Meddr Grønland 116(2), 85 pp. Frankl, E. 1955: Rapport tiber die Durchquerung von Nord Peary Land (Nordgronland) im Sommer 1953. Meddr Grønland 103(8), 61 pp. 1953. Meddr Grønland 103(8), 61 pp. Frankl, E. 1956: Some general remarks on the Caledonian chain of East Greenland. Meddr Grønland 103(11), 43 pp. A. K., Friderichsen, J. D. & Soper, N. J. 1981: The North Greenland fold belt between central Higgins, A. K., Friderichsen, J. D. & Soper, N. J. 1981: The North Greenland fold belt between central Johannes V. Jensen Land and eastern Nansen Land. Rapp. Grønlands geol. Unders. References 106, 35-45. gg , , , p , Johannes V. Jensen Land and eastern Nansen Land. Rapp. Grønlands geol. Unders. 106, 35-45. Hurst, J. M. 1980: Silurian stratigraphy and facies distribution in Washington Land and western Hall Land, North Greenland. Bul!. Grønlands geol. Unders. 138, 95 pp. Hurst, J. M. 1980: Silurian stratigraphy and facies distribution in Washington L Land, North Greenland. Bul!. Grønlands geol. Unders. 138, 95 pp. Hurst, J. M. & Surlyk, F. 1980: Notes on the Lower Palaeozoic clastic sediments of Peary Land, North Greenland. Rapp. Grønlands geo!. Unders. 99,73-78. Hurst, J. M. & Surlyk, F. in press: Stratigraphy of the Silurian turbitite sequence of North Greenland. Bul!. Grønlands geol. Unders. 145. Håkansson, E. 1979: Carboniferous to Tertiary development of the Wandel Sea Basin, eastern Greenland. Rapp. Grønlands geo!. Unders. 88, 73-83. pp g Koch, J. P. 1916: Survey of Northeast Greenland. Meddr Grønland 46(2), 79-468. Koch, L. 1929: Stratigraphy of Greenland. Meddr Grønland 73(2), 2, 205-320. Lane, P. D., Dawes, P. R & Peel, J. S. 1980: A new Silurian Hemiarges (Trilobita) from North Greenland and the question of the Polaris Harbour Formation. Rapp. Grønlands geol. Unders. 101, 45-53. Nielsen, E. 1941: Remarks on the map and the geology of Kronprins Christian Land. Meddr Grønland 126(2), 34 pp. Pedersen, S. A. S. 1980: Regional geology and thrust fault tectonics in the southern part of the North Greenland fold belt, north Peary Land. Rapp. Grønlands geol. Unders. 99,79-87. Peel, J. S. & Higgins, A. K. 1980: Fossils from the Paradisfjeld Group, North Greenland fold belt. Rapp. Grønlands geol. Unders. 101,28 only. Soper, N. J., Higgins, A. K. & Friderichsen, J. D. 1980: The North Greenland fold belt in eastern Johannes V. Jensen Land. Rapp. Grønlands geo!. Unders. 99, 89-98. Surlyk, F., Hurst, J. M. & Bjerreskov, M. 1980: First age-diagnostic fossils from the central part of the North Greenland foldbelt. Nature 286, 800-803. Trettin, H. P. & Balkwill, H. R 1979: Contributions to the tectonic history of the 1nnuitian Province, Arctic Canada. Can. l. Earth Sd. 16, 748-769. Plate 1 Distribution of the Skagen Group, Paradisfje1d Group, Polkorridoren Group, Vølvedal Group and Amundsen Land Group in eastern North Greenland. Place names mentioned in text are indicated here and on fig. 8. Rapp. Grønlands geu\. Unders. 107 ds geu\. Unders. 107 83'30' Report File no. 22444 Enclosure (1/1) G E U S 24' Paradisfjeld Group Amundsen Land Group Vølvedal Group Peary land Group Faults Skagen Group Boundary , inferred Pol korridoren Group / Boundary , observed 4 4 Major thrusts in north ..,...,."" ......... -"'y Minor thrusts in south >- CJ c=J DI EE2J ? Ordovician Silurian Cambrian precambrian ?----- ?-----? <-----~-~-~--I I 20 km O HANS EGEDES LAND 30' ? ? LAND PEARY 36' _.,:- ,. Land o Borup ø ~ Inge ø '-----'-, Halen 42' ~ NANSEN V ---'O""'oc-c LAND ,II 36' 42' Paradisfjeld Group Amundsen Land Group Vølvedal Group Peary land Group Skagen Group Pol korridoren Group CJ c=J DI EE2J ? Ordovician Silurian Cambrian precambrian ?----- ?-----? ~ NANSEN V ---'O" LAND ,II HANS EGEDES LAND HANS EGEDES LAND Skagen Group Faults Boundary , inferred / Boundary , observed 4 4 Major thrusts in north ..,...,."" ......... -"'y Minor thrusts in south >- Faults
https://openalex.org/W3082606012	https://link.springer.com/content/pdf/10.1007/s10787-020-00751-1.pdf	English	null	Alzheimer’s disease: natural products as inhibitors of neuroinflammation	Inflammopharmacology	2,020	cc-by	10,844	REVIEW REVIEW REVIEW Abstract Alzheimer’s disease (AD) is the most common form of dementia and affects 44 million people worldwide. New emerging evidence from pre-clinical and clinical investigations shows that neuroinflammation is a major pathological component of AD suggesting that anti-inflammatory strategies are important in delaying the onset or slowing the progression of the disease. However, efforts to employ current anti-inflammatory agents in AD clinical trials have produced limited success. Conse- quently, there is a need to explore anti-inflammatory natural products, which target neuroinflammatory pathways relevant to AD pathogenesis. This review summarises important druggable molecular targets of neuroinflammation and presents classes of anti-neuroinflammatory natural products with potentials for preventing and reducing symptoms of AD. Keywords Alkaloids · Marine natural products · Polyphenols · NF-κB · Terpenes Alzheimer’s disease: natural products as inhibitors of neuroinflammation Olumayokun A. Olajide1 · Satyajit D. Sarker2 Received: 14 May 2020 / Accepted: 28 August 2020 / Published online: 15 September 2020 © The Author(s) 2020 Received: 14 May 2020 / Accepted: 28 August 2020 / Published online: 15 September 2020 © The Author(s) 2020 * Olumayokun A. Olajide o.a.olajide@hud.ac.uk 2 Centre for Natural Products Discovery, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, UK Inflammopharmacology (2020) 28:1439–1455 https://doi.org/10.1007/s10787-020-00751-1 Inflammopharmacology (2020) 28:1439–1455 https://doi.org/10.1007/s10787-020-00751-1 Inﬂammopharmacology 1 Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, UK Introduction strong links between neuroinflammation and AD pathogen- esis. Increasing evidence from several studies revealed that inflammatory responses in the brain are a major contributor to the pathogenesis of AD (Heppner et al. 2015; Fu et al. 2019). In fact, high levels of pro-inflammatory mediators have been detected in the brain of AD patients (Hesse et al. 2016). Furthermore, neuroinflammation which is character- ised by activation of brain-resident macrophages with the resultant hyper-secretion of pro-inflammatory cytokines and chemokines such as interleukin-1beta (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNFα), interleukin-8 (IL-8), transforming growth factor-β (TGF-β) and mac- rophage inflammatory protein-1α (MIP-1α) is a well-doc- umented consequence of high levels of insoluble forms of Aβ (Akiyama et al. 2000). Consequently, anti-inflammatory strategies have the potential to delay the onset or slow the progression of the disease. Alzheimer’s disease (AD) is the most common form of dementia. AD has been reported to affect about 44 million people globally, and is estimated to triple by 2050 due to general population ageing (Prince et al. 2014). The main pathological features of AD include extracellular amyloid- beta (Aβ) plaques and intracellular neurofibrillary tangles (Bloom 2014). Studies have established that there is a strong correlation between symptoms of AD and the accu- mulation of these plaques and tangles due to their ability to induce neurodegeneration that mediates loss of memory and cognition. Interestingly, new emerging evidence continues to dem- onstrate that neuroinflammation is also a major pathologi- cal component of AD (Webers et al. 2020). Several reports of animal experiments and clinical studies have provided It is now well-established that the transcription factor nuclear factor-kappa B (NF-κB) plays a major role in neu- roinflammation-mediated AD. NF-κB is a master regulator of inflammatory gene transcription and has been shown to be expressed in the brains of AD patients (Boissière et al. 1997; Liao et al. 2016). NF-κB has also been proposed as a molecular mechanism underlying the development of some sporadic cases of AD (Chen et al. 2012). These reports link- ing NF-κB to AD strengthen the role of neuroinflammation in AD. (0121 3456789) 3 1440 O. A. Olajide, S. D. Sarker multi-faceted nature of AD pathology. The multi-target approach is an alternative strategy in evaluating anti-inflam- matory drugs for effectiveness in slowing the progression of AD. A number of molecular mechanisms and cellular signal- ling pathways have been proposed to contribute to neuroin- flammation in AD. Introduction Some of these mechanisms are known to be under the direct influence of NF-κB, while others have been reported to cross-talk with this transcription factor in a manner that makes them a molecular target for drug action in AD therapeutics. Some natural products are able to produce multi-target anti-inflammatory activity in AD through modulation of multiple signalling pathways. It has been proposed that the multi-pharmacological actions of black and green tea poly- phenols could be valuable in the treatment of neurodegen- erative disorders including AD (Mandel et al. 2011, 2012). Inhibition of neuroinflammation by natural products has also been linked to their ability to produce anti-amyloid effect. Apigenin is an anti-inflammatory natural product which showed effects on APP processing and preventing Aβ bur- den through down-regulation of BACE1 levels, the relief of Aβ deposition, and the decrease of insoluble Aβ levels (Zhao et al. 2013). It is worth evaluating other anti-inflammatory natural products with multi-target anti-inflammatory activity as potential candidates for AD therapeutics. The signalling pathways involving the mitogen activated protein kinases (MAPKs) have been strongly linked to neu- roinflammation and AD. Of the MAPKs, the p38 MAPK has been implicated in neuroinflammation. Evidence linking p38 MAPK to neuroinflammation was put forward by Kim and Choi (2015), who suggested that exposure of microglia to Aβ induces microglial activation with the subsequent production of neurotoxic pro-inflammatory cytokines and reactive oxygen species, which in turn activate p38 MAPK signalling. Further reports show that Aβ-induced oxidative stress results in the activation of p38 MAPK with the result- ant tau hyperphosphorylation (Giraldo et al. 2014). Recent reports have also suggested that p38 MAPK plays a role in neuroinflammation and AD due to its ability to activate NF-κB (Kheiri et al. 2018), thus making it a potential molec- ular target for novel AD treatment. Natural product inhibitors of neuroinflammation The nuclear factor E2-related factor 2 (Nrf2) is a tran- scription factor that regulates phase II antioxidant response mechanisms in response to oxidative stress. Emerging evi- dence links activation of the Nrf2 protective mechanism to anti-inflammatory effects involving NF-κB (Nair et al. 2008; Sandberg et al. 2014). Specifically, NF-κB is a known nega- tive regulator of Nrf2 (Liu et al. 2008; Kim et al. 2010a, b; Yu et al. 2011). Rojo et al. (2010) demonstrated that an acti- vation of the microglia in Nrf2-deficient animals is accom- panied by increased levels of pro-inflammatory cyclooxy- genase-2 (COX-2), inducible nitric oxide synthases (iNOS), IL-6, and TNFα. To confirm these observations, Ramsey et al. (2007) reported that brains from AD patients have decreased levels of Nrf2 in the hippocampus. Consequently, there is an increasing interest in pharmacological activa- tors of Nrf2 to activate or restore its protective mechanisms (Sandberg et al. 2014).il Several natural products have been reported to produce anti- neuroinflammatory activity through mechanisms involving inhibition of microglia activation, reduction of the release of pro-inflammatory cytokines from activated microglia, or through inhibition of NF-κB and p38 MAPK activation. Other natural products produce marked activation of Nrf2, a mechanism which has been shown to contribute at least in part to their anti-neuroinflammatory activity. This review will highlight some of our investigations and those reported by other investigators on the main classes of natural prod- ucts with promising therapeutic potentials for inhibiting neuroinflammation. 3 Alkaloids Alkaloids are pharmacologically-active secondary metabo- lites consisting of nitrogen, often as an integral part of the ring (Ziegler and Facchini 2008; Nahar and Sarker 2019) and exist as either proto-alkaloids (nitrogen-containing but not heterocyclic in structure) or true alkaloids (nitrogen- containing heterocyclic compounds) (Rosa et al. 2007). Alkaloids (Fig. 1) have been linked to a wide variety of phar- macological activities, including anti-inflammatory effects.l Research findings implicating neuroinflammation in AD have resulted in pre-clinical and clinical investigations of NSAIDs and other anti-inflammatory agents as potential therapeutic strategies for AD (McGeer et al. 2016; Cuello, 2017). However, efforts to use current anti-inflammatory agents in AD clinical trials have not been successful. Inves- tigations showed that anti-inflammatory drugs failed to delay or reduce the pathological symptoms of patients with mild cognitive impairment or AD (Fu et al. 2019). Most of the anti-inflammatory drugs which have been investi- gated in clinical trials for AD are known to target specific single inflammatory mechanisms. It was therefore not sur- prising that these drugs failed in clinical trials, given the lf With regards to anti-inflammatory activity in the CNS, our investigations revealed that the alkaloid cryptolepine found in Cryptolepis sanguinolenta produced a reduction in the levels of TNFα, IL-6, IL-1β, NO, and PGE2 in LPS- stimulated rat microglia. There are currently no studies to determine demonstrating anti-inflammatory effects of 3 Alzheimer’s disease: natural products as inhibitors of neuroinflammation 1441 Cryptolepine Tetrandrine Fig. 1 Examples of some anti-neuroinflammatory alkaloids Cryptolepine Fig. 1 Examples of some anti-neuroinflammatory alkaloids catechins, flavones, flavonols, flavanols, flavanones, and isoflavonoids. They are found in abundance in flowers, fruits, barks, roots, stems, tea, wine and vegetables (Nahar and Sarker 2019). Several flavonoids and polyphenolic compounds have been shown to possess anti-neuroinflam- matory activity (Fig. 2).l cryptolepine on microglia stimulated with either amyloid beta. We also observed reductions in protein and mRNA levels of COX-2 and iNOS and further demonstrated that the effects of the compound are mediated through blocking activation of NF-κB, p38 MAPK in the microglia (Olajide et al. 2013). The effects of cryptolepine in animal models of neuroinflammation or AD are yet to determined. However, we have shown that this alkaloid produced anti-inflamma- tory activity in animal models of peripheral inflammation (Olajide et al. 2009). Alkaloids A major limitation in the development of this alkaloid in the treatment of AD is related to its cyto- toxicity due to its ability to cause DNA damage (Gopalan et al. 2011). y y g Kaempferol is a flavonol which has been shown to inhibit neuroinflammation by reducing LPS-induced production of pro-inflammatory mediators in BV2 microglial cells through mechanisms involving NF-κB and p38 MAPK (Park et al. 2011). A related glycosidic flavonoid to kaempferol, tiliro- side (contained in plants such as rosehip, linden and straw- berry) was demonstrated in our studies to inhibit neuroin- flammation in BV-2 microglia through multiple mechanisms including attenuation of NF-κB and p38 MAPK, in addi- tion to activating Nrf2 (Velagapudi et al. 2014, 2018a, b). Neither kaempferol nor tiliroside has been investigated for clinical efficacy in humans. Apigenin (4′,5,7-trihydroxy- flavone), structurally similar to kaempferol with just one less –OH group at C-3 is a flavone found in chamomile, celery and parsley, and many other plants. Studies reported by Rezai-Zadeh et al. (2008) showed that treatment of inter- feron gamma-activated microglia with apigenin resulted in a decrease in the production of pro-inflammatory IL-6 and TNFα through mechanisms involving STAT1. Further evi- dence of the anti-inflammatory activity of this flavonoid was provided in investigations showing reductions in iNOS/NO and PGE2/COX-2 in activated microglia (Choi et al. 2014). Animal studies have also suggested that the anti-inflamma- tory activity of apigenin may contribute to its neuropro- tective activity in models of AD. Treatment of mice with apigenin improved spatial learning and memory following amnesia induction with Aβ25–35 (Liu et al. 2011). It is widely known that one of the mechanisms involved in Aβ-induced neurodegeneration involves neuroinflammation (Ralay Rana- ivo et al. 2006), suggesting that the neuroprotective activity In similar fashion, tetrandrine (a bisbenzylisoquino- line alkaloid isolated from Radix Stephania tetrandra) has shown promising NF-κB-mediated anti-inflammatory activ- ity in BV-2 microglia activated with fibrillar amyloid beta and reduced hippocampal neuroinflammation by inhibiting NF-κB activation in a rat model of AD induced by amyloid beta (He et al. 2011a, b). While there are no studies dem- onstrating the clinical efficacy of tetrandrine in AD, it has been suggested that this alkaloid is able to permeate the blood–brain barrier to provide benefits in stroke, due to its lipophilic nature (Chen et al. 2011). Alkaloids A potential limitation to developing tetrandrine for clinical use is related to its ability to produce significant unwanted effects in the cardiovascu- lar system. Tetrandrine is a known calcium channel blocker (King et al. 1988) and has a potential to induce reduction in peripheral resistance as well as decreasing heart rate and cardiac contractility, all of which could result in a reduction in blood pressure and induction of arrhythmias. 3 Flavonoids and other polyphenols This stilbene is reputed with various pharmacological activities and has been widely studied as potential treat- ment for diverse disorders. Several pharmacological studies have demonstrated anti-neuroinflammatory/neuroprotective effects of resveratrol in in vitro and in animal models. The first indication of inhibition of neuroinflammation by resver- atrol was reported by Candelario-Jalil et al. (2007) who pro- vided evidence that this compound inhibited PGE2 produc- tion and free radical formation in LPS-activated primary rat microglia. This study further revealed that resveratrol was the first known inhibitor which specifically prevents micro- somal prostaglandin E synthase-1 (mPGES-1) expression without affecting COX-2. Subsequent studies by Abraham and Johnson (2009) demonstrated that resveratrol consump- tion resulted in reduction of LPS-induced IL-1β in plasma and IL-1β mRNA in the hippocampus of aged mice, as well as in cultured BV-2 microglia. Other studies over the last few years have increased the evidence demonstrating inhibi- tion of neuroinflammation and neuroinflammation-mediated neuronal damage by resveratrol (Lu et al. 2010; Zhang et al. 2013; Potter et al. 2013; Wang et al. 2015; Yao et al. 2015). It is noteworthy that a recent study by Sun et al. (2019) provided a new evidence linking inhibition of neuroinflam- mation by resveratrol to its ability to rescue tau-induced Human trials to evaluate the efficacy of EGCG in improv- ing cognitive function have not reflected results achieved in vitro and in animal models of AD. For example, admin- istration of 300 mg EGCG to healthy volunteers was shown to increase cerebral activity (as evidenced by an increase in alpha, beta and theta activities in the brain) without a cor- responding effect on task performance (Scholey et al. 2012). Furthermore, a double‐blind, placebo‐controlled, crossover investigation of a single oral dose of 135 mg EGCG on cog- nitive performance, mood and cerebral blood flow (CBF) in healthy human adults did not show any significant effects on mood and cognition, in comparison with placebo (Wight- man et al. 2012). The translational gap between bioactivity of EGCG in vitro and in animal studies and its effects in human trials is possibly associated with low oral bioavail- ability of the compound on the one hand, as well as differ- ent metabolism between animals and humans on the other hand (Mähler et al. 2013). More long-term human trials are necessary to establish the effects of EGCG on cognitive per- formance. Flavonoids and other polyphenols 2016; Yao et al. 2018). Investigations on the pomegranate fruit polyphenol punicalagin revealed that the compound produced signifi- cant inhibition of neuroinflammation in LPS-activated rat primary microglia through interference with mechanisms resulting in activation of NF-κB (Olajide et al. 2014). Inter- estingly, one of its gut-derived metabolites urolithin A, showed similar effects on LPS-activated BV-2 microglia (Velagapudi et al. 2019). Urolithin A has also produced neuroprotection by blocking memory impairment and neu- roinflammation in APP/PS1 mice (Gong et al. 2019) and in a Caenorhabditis elegans model (Yuan et al. 2016; Fang et al. 2019). Based on the published literature no studies in humans have been conducted on pomegranate polyphenols or their gut-derived metabolites with respect to cognitive performance or other therapeutic measures of AD. Epigallocatechin-3-gallate (EGCG) is a flavanol found mainly in green tea (Camellia sinensis). Studies have sug- gested that the anti-amyloidogenic and neuroprotective actions of this flavonoid may be due to its ability to inhibit neuroinflammation. Experiments conducted in vitro showed that EGCG could reduce TNFα, IL-1β, IL-6, iNOS levels in Aβ-stimulated EOC13.31 microglia through mechanisms involving NF-κB (Wei et al. 2016). In animals, Lee et al. (2013) reported that EGCG prevented memory impairment and reduced the levels Aβ generation and neurotoxicity in mice following systemic injection of lipopolysaccharide (LPS). Similar observations were made in studies reported by Seong et al. (2016) who further linked the anti-neuroin- flammatory activity of this compound to inhibition of NF-κB activation. In vivo experiments have also shown that EGCG produced neuroprotection in animal models of AD. For example, intraperitoneal injection of 12-month-old Tg2576 mice with 20 mg/kg EGCG resulted in decreased levels of Aβ as well as plaque load in the brain. Similar observations were made following oral administration of the compound to TgCRND8 (Tg) mice (Walker et al. 2015).fi Mangiferin is a naturally occurring glucosylxanthone found in the stem bark and leaves of mango plant (Mangif- era indica). Investigations in rat primary microglia revealed that mangiferin could inhibit COX-2 expression and pros- taglandin E2 (PGE2) production following activation with LPS (Bhatia et al. 2008). In vivo experiments showed that mangiferin diminished neuroinflammation and improved cognitive deficits in APP/PS1 mice (Infante-Garcia et al. 2017). Based on published literature no human studies have demonstrated the efficacy of mangiferin in therapeutic end- points of AD. Resveratrol is a polyphenol found in grapes and ber- ries. Flavonoids and other polyphenols Flavonoids are naturally occurring phenolic compounds that can be structurally classified as anthocyanins, 1 3 1442 O. A. Olajide, S. D. Sarker Agathisflavone Apigenin R = H Luteolin R = OH Curcumin Epigallocatechin gallate s of some anti-neuroinflammatory flavonoids and polyphenols Apigenin R = H Luteolin R = OH Agathisflavone Agathisflavone Epigallocatechin gallate Fig. 2 Examples of some anti-neuroinflammatory flavonoids and polyphenols 1 3 Alzheimer’s disease: natural products as inhibitors of neuroinflammation 1443 pl Formononetin Kaempferol R = H Quercetin R = OH Mangiferin Punicalagin ed) Formononetin Kaempferol R = H Quercetin R = OH Mangiferin Kaempferol R = H Quercetin R = OH Formononetin Kaempferol R = H Quercetin R = OH Mangiferin Punicalagin Fig. 2 (continued) Fig. 2 (continued) Fig. 2 (continued) Fig. 2 (continued) 1 3 1444 O. A. Olajide, S. D. Sarker Resveratrol Tiliroside Urolithin A Resveratrol Resveratrol Resveratrol Tiliroside Tiliroside Urolithin A Urolithin A Fig. 2 (continued) reported by Liu et al. (2011) may be related to anti-inflam- matory action of the compound. Nrf2 is required for the anti-inflammatory effect of quercetin (Kang et al. 2013). These observations were supported by studies linking inhibition of neuroinflammation by quercetin to potential cross-talk between MAPKs and Nrf2 (Sun et al. 2015).l Quercetin, a similar flavonol to kaempferol with an extra hydroxyl group at C-3′ (B ring) is a ubiquitous anti- inflammatory and antioxidant natural product that is found in many fruits, vegetables, and seeds. Treatment with quercetin reduced iNOS-mediated NO production in LPS-stimulated BV-2 microglia through mechanisms involving suppression of NF-κB activation (Kang et al. 2013). These authors fur- ther demonstrated that the antioxidant transcription factor, The anti-inflammatory activity of quercetin has been linked to its effect on cognitive function in APP/PS1 mouse model of AD. Investigations by Lv et al. (2018) demon- strated that quercetin treatment of APP/PS1 mice signifi- cantly reduced Aβ plaques, p-Tau and neuroinflammation. 3 1445 Alzheimer’s disease: natural products as inhibitors of neuroinflammation the compound needs to be taken into consideration in future clinical development for AD therapeutics. The antioxidant activity of quercetin, through activation of Nrf2 and the subsequent anti-inflammatory effect in the microglia are significant in neuroprotection and therapeutic benefits in AD. There are no studies showing clinical effi- cacy of quercetin in AD. This may be due to its poor permea- tion of the BBB. Similar anti-inflammatory/neuroprotective profiles have been reported for the related flavone luteolin (Burton et al. Flavonoids and other polyphenols Investigations to compare oral bioavailability and BBB penetration of EGCG in mice and human subjects will throw more light on the apparent lack of efficacy in human studies. EGCG consumption has been associated with hepa- toxicity (Navarro et al. 2013; Hu et al. 2018). This effect of 1 3 1446 O. A. Olajide, S. D. Sarker treatment (Cox et al. 2015). These studies suggest that the clinical efficacy of curcumin in AD would be increased by approaches which enhance its bioavailability. cognitive deficits and neuropathology in a mouse model of AD. In that study, treatment with resveratrol rescued cogni- tive deficits, reduced levels of phosphorylated tau, prevented neuroinflammation and synapse loss in the brains of mice. These pre-clinical reports on resveratrol are promising and warrant further clinical evaluation. Our investigations revealed that other polyphenols such as formononetin, an isoflavone in Trifolium pratense (red clover) inhibited neuroinflammation through mechanisms involving attenuation of NF-κB activation in LPS-activated BV-2 microglia (El-Bakoush and Olajide 2018). Similar results were obtained in experiments on agathisflavone, a biflavonoid isolated from Anacardium occidentale (Vela- gapudi et al. 2018a, b). These observations have not been confirmed in animal experiments. It is expected that future animal experiments to establish in vivo activities of these compounds would be valuable in determining their poten- tials for follow-up clinical studies. In a clinical trial, treatment of mild to moderate AD patients with resveratrol resulted in the decline of cerebro- spinal fluid amyloid beta, which is an AD biomarker. This was accompanied by reduction in biomarkers of neuroin- flammation (Moussa et al. 2017). However, a pilot study to study the effects of chronic resveratrol use on cognitive function in elderly subjects revealed selectively improved psychomotor speed without significantly affecting other domains of cognitive function (Anton et al. 2018). These findings providing modest clinical evidence for the efficacy of resveratrol in AD is possibly connected to low oral bio- availability of the compound. Larger placebo-controlled, randomised trials with bioavailable formulations of resvera- trol are required to throw more light on the efficacy of the compound in humans. Flavonoids remain one of the important groups of natu- ral products for inhibiting neuroinflammation in AD due to their fundamental inhibitory actions on pro-inflammatory transcription factors. Furthermore, this group of compounds activate antioxidant/anti-inflammatory transcription factors. Flavonoids and other polyphenols While flavonoids have proven to be promising therapeutic natural products in pre-clinical models of AD, it is important to note that the overall bioavailability of parent flavonoids are usually low. Furthermore, flavonoids do not cross the blood–brain barrier easily due to their high polarity. Curcumin found in Curcuma longa (turmeric) is perhaps the most investigated natural neuroprotective polyphenol for treating AD. It has been linked to a diverse range of pharma- cological activities and therapeutic benefits including anti‐ inflammatory, anticancer, antimicrobial, antioxidant, and wound healing effect (Williams et al. 2011). In particular, curcumin has been widely investigated in cellular and ani- mal models of neuroinflammation. Experiments using BV-2 microglia revealed that this diarylheptanoid inhibited neu- roinflammation in LTA-activated microglial cells through reduction in the production of TNFα, PGE2, and nitric oxide (NO), as well as inhibition of NF-κB and MAPK activation (Yu et al. 2018). Similar observations were made in BV-2 microglia stimulated with LPS (Porro et al. 2019; Zhang et al. 2019). These observations in the microglia have been confirmed by results of experiments in animal models of AD which showed that curcumin treatment ameliorated cogni- tive decline and neuroinflammation following exposure to LPS, and in p25 transgenic mice (Sundaram et al. 2017; Sorrenti et al. 2018). Curcumin also inhibited formation of amyloid beta oligomers and fibrils and reduced amyloid in mouse models of AD (Yang et al. 2005). 3 Terpenes Terpenoids are a large and structurally diverse group of compounds formed biosynthetically from a combination of two or more isoprene units (a five carbon unit, chemically known as 2-methyl-1,3-butadiene) (Nahar and Sarker 2019). Terpenoids (Fig. 3) have been widely reported to inhibit neu- roinflammation in animal models and in vitro. l Parthenolide, a biologically-active sesquiterpene lac- tone present in Tanacetum parthenium has been reported to improve cognitive function and decrease levels of TNF-α and IL-6 in the cortical and hippocampal regions of rats (Khare et al. 2017). Recently, this compound was reported to inhibit neuroinflammation in intracerebral haemorrhage- induced brain injury in rats through TLR4/NF-κB-mediated reduction in the levels of TNF-α, interleukin IL-6, IL-17 in the ipsilateral hemispheres of the brain (Wang et al. 2020). It appears that the NF-κB inhibitory action of parthenolide is responsible for its versatile inhibitory actions in different neuropathologies involving inflammation. Neuroprotection by this sesquiterpene lactone needs to be further confirmed in AD clinical trials. Interestingly, a clinical trial with curcumin did not dem- onstrate efficacy in AD in a 24-week placebo-controlled trial (Ringman et al. 2012). The authors suggested that the lack of efficacy could be related to bioavailability of the prod- uct used or differences in the biology of rodent models of AD and human AD. However, a subsequent randomised, double-blind, placebo-controlled study in healthy older population which employed 400 mg/day of a highly bioavail- able curcumin preparation (Longvida), reported a signifi- cantly improved working memory and mood after a 4-week Artemisinin is a sesquiterpene lactone found in the Chi- nese herb Artemisia annua (Qinghao) of the family Aster- aceae, and was originally developed for the treatment of multi-drug resistant malaria. Recently, this compound and some of its synthetic analogues have been reported 3 Alzheimer’s disease: natural products as inhibitors of neuroinflammation 1447 Artemisinin Carnosic acid Carnosol Parthenolide Ginkgolides Ginkgolide A: R = OH; R’ = R’’ = H Ginkgolide B: R = R’ = OH; R’’= H Ginkgolide C: R = R’ = R’’ = OH Thymoquinone Fig. 3 Examples of some anti- neuroinflammatory terpenoids Artemisinin Carnosic acid Carnosic acid Artemisinin Carnosol Parthenolide Carnosol Carnosol Thymoquinone Thymoquinone Ginkgolides Ginkgolide A: R = OH; R’ = R’’ = H Ginkgolide B: R = R’ = OH; R’’= H Ginkgolide C: R = R’ = R’’ = OH compound was neuroprotective in a mouse model of AD through reduction in the levels of IL-1β, IL-6 and TNF-α in the hippocampus and the cortex (Qiang et al. 2018). Similarly, we have reported that artemisinin analogues, artesunate and artemether inhibited neuroinflammation by targeting NF-κB signalling in BV-2 microglia (Okorji and Olajide, 2014; Okorji et al. 2016). Artemisinin and its syn- thetic derivatives have been shown to cross the blood–brain to possess potential neuroprotective activity in AD, partly through their anti-inflammatory activity. Studies reported by Zhu et al. (2012) revealed that artemisinin inhibited LPS- induced release of TNFα, IL-6, MCP-1 and NO in BV-2 microglia. These authors further suggest that the observed inhibition of neuroinflammation by artemisinin was related to its modulatory effects on the NF-κB signalling path- way in the microglia. Subsequent studies showed that this 1 3 1 3 1448 O. A. Olajide, S. D. Sarker barrier due to their lipophilicity (Navaratnam et al. 2000). However, experiments in animals have suggested that the compounds are neurotoxic (Meshnick 2002; Genovese and Newman, 2008), which may be discouraging their investiga- tion in AD clinical trials. in patients diagnosed with mild to AD or vascular dementia showed that the extract improved cognitive function, neu- ropsychiatric symptoms and functional abilities (Ihl et al. 2012). However, in another study to assess the efficacy of long-term use of Ginkgo biloba extract (120 mg) for the reduction of incidence of AD in elderly adults with mem- ory complaints, the extract did not reduce the risk of pro- gression to AD when compared with placebo (Vellas et al. 2012). A systematic review and meta-analysis of randomised controlled trials of Ginkgo biloba extract in mild cognitive impairment and AD attributed the conflicting outcomes of the trials to limited sample size, inconsistent findings and methodological quality of included trials (Yang et al. 2016). Furthermore, the effectiveness of Ginkgo biloba extract in treating established AD without preventing its incidence warrants further investigation. Our investigations have shown that thymoquinone (the main bioactive constituent of Nigella sativa) is a potent inhibitor of neuroinflammation. Carotenoids from Crocus sativus (Saffron) Crocus sativus (Saffron) is a spice that is widely reputed for a wide variety of therapeutic applications, including neurodegenerative disorders, depression, diabetes mellitus, atherosclerosis and cancer (Leone et al. 2018). Evidence from in vitro experiments, animal models of AD and clinical trials have shown that carotenoids in saffron flowers, crocin and crocetin are neuroprotective natural products with thera- peutic potentials in AD. Studies in BV-2 microglia showed that crocin and crocetin inhibited LPS-induced production of NO/iNOS, TNF-α, IL-1β and ROS in BV-2 microglial cells through mechanisms linked to NF-κB (Nam et al. 2010; Zhang et al. 2018). These anti-inflammatory carotenoids have been reported to produce promising activities in ani- mal models of AD through their ability to improve cognitive function (Hosseinzadeh et al. 2012; Asadi et al. 2015). A study published by Mazumder et al. (2017) appears to pro- vide a link between the anti-inflammatory and antioxidant activities of crocin and its ability to enhance cognitive abili- ties in mice. This hypothesis needs to be further investigated to provide a better understanding of how anti-inflammatory natural products promote cognitive abilities.i Carnosic acid and carnosol are brain-permeable natural diterpenes found in Rosmarinus officinalis, and have shown significant neuroprotective activity (de Oliveira, 2016). Studies by Foresti et al. (2013) showed that carnosol could inhibit neuroinflammation in BV-2 microglia by reducing levels of TNF-α, PGE2 and nitric oxide following activation with either LPS or interferon gamma (IFNγ). In addition, carnosic acid was reported to produce anti-inflammatory effect in paraquat-induced increase in the levels of IL-1β, TNFα, and cyclooxygenase-2 (COX-2) in SH-SY5Y cells by targeting Nrf2/HO-1 and NF-κB signalling pathways (de Oliveira et al. 2018). It appears that inhibition of neuroin- flammation by carnosol and carnosic acid could be related to Nrf2 activation, which is known to result in an anti-inflam- matory outcome (Innamorato et al. 2008). Ginkgolides are pharmacologically-active diterpenes found in Ginkgo biloba (Ginkgoaceae). In a study reported by Zhou et al. (2016), ginkgolides were shown to inhibit neuroinflamma- tion by reducing levels of IL-1β, IL-6, IL-8, TNF-α in BV-2 microglia activated with oxygen–glucose deprivation and re- oxygenation through mechanisms involving TLRs/MyD88/ NF-κB signalling pathways. Anti-inflammatory activity has also been reported to contribute to the neuroprotective actions of ginkgolides in models of cerebral ischemia and reperfusion injury (Gu et al. 2012; Jiang et al. 2014).i There are no clinical studies to demonstrate the benefits of crocin and crocetin in AD. Ginkgolides In LPS-activated BV-2 microglia, thymoquinone treatment resulted in significant reduction in TNFα, IL-6, PGE2, and NO protein and mRNA through mechanisms involving inhibition of the pro-inflam- matory NF-κB and activation of the anti-inflammatory Nrf2 pathways (Velagapudi et al. 2017b). We further showed that inhibition of neuroinflammation by this compound was par- tially related to activation of both sirtuin 1 (SIRT-1) and 5′ adenosine monophosphate-activated protein kinase (AMPK) in the microglia (Velagapudi et al. 2017a). Similar obser- vations showing inhibition of neuroinflammation by thy- moquinone were made in recent investigations reported by Cobourne-Duval et al. (2018). Recently, thymoquinone was shown to improve cognitive decline in a rat model of AD, while decreasing Aβ formation and accumulation, as well as TNF-α and IL-1β (Abulfadl et al. 2018). Carotenoids from Crocus sativus (Saffron) However, results of single- and double-blind, placebo controlled clinical trials on saffron have shown promising effects in patients with moderate to severe Alzheimer’s disease (Akhondzadeh et al. 2010a, b; Farokhnia et al. 2014; Tsolaki et al. 2016). 3 Conclusion and future direction Natural products exhibit promising health-promoting effects in neurodegenerative diseases partly due to their anti-inflam- matory property. The emerging research data on the possi- ble therapeutic effects of natural products as neuroprotective agents is particularly exciting due to the steadily increasing population with AD. In spite of the overwhelming evidence suggesting the potentials of anti-inflammatory natural products in treat- ing AD, further investigations are required to assess their clinical efficacy in properly-controlled human trials. Studies have shown that in vitro data demonstrating efficacy do not always translate into in vivo effects. Furthermore, extrapo- lating data from animal models to humans in the search for new treatment for AD is not reliable due to significant spe- cies differences. To overcome this challenge, pre-clinical investigations on anti-inflammatory natural products need to focus on employing new cutting-edge tools. One of such approaches is the use of a tri-culture including human neu- rons, astrocytes and microglia to evaluate neuroinflammation and neuroprotection in vitro. Marine natural products There are no reports in literature indicating the benefits of ginkgolides in clinical trials for AD treatment. Clinical studies on standardised Ginkgo biloba extracts have shown conflicting results. Results of a randomised controlled trial to evaluate the efficacy of Ginkgo biloba extract (240 mg) The most investigated neuroprotective marine natural product is astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′- dione), a xanthophyll carotenoid found in Haematococcus pluvialis, Chlorella zofingiensis, Chlorococcum, and Phaffia 3 3 Alzheimer’s disease: natural products as inhibitors of neuroinflammation 1449 rhodozyma. In LPS-stimulated BV-2 microglia, astaxanthin was reported to inhibit NO/iNOS and COX-2 (Choi et al. 2008). In a separate study, Kim et al. (2010a, b) showed that the compound attenuated LPS-induced production of IL-6 in BV-2 microglia through mechanisms involving ERK1/2- MSK-1 and NF-κB activation. have the advantage of protecting active compounds (Soares et al. 2018). Solid lipid nanoparticles (SLNs) are a new generation of submicron-sized lipid emulsions in which the liquid lipid (oil) has been substituted by a solid lipid with an ability to penetrate the BBB and produce a pharmacologi- cal action in the CNS (Mutoh et al. 2016). Table 1 summa- rises the novel delivery strategies which have been applied in enhancing the permeability of some anti-inflammatory natural products into the brain. Inhibition of neuroinflammation has also been reported for astaxanthin in animal models of neurodegeneration. In a study reported by Zhou et al. (2015), treatment of diabetic mice with astaxanthin alleviated alleviated cognition deficits with accompanying inhibition in NF-κB mediated neuroin- flammation. Inhibition of neuroinflammation was also pro- posed to be responsible for the neuroprotection by astaxan- thin in in experimental subarachnoid haemorrhage (Zhang et al. 2014). Interestingly, clinical trials on the benefits of astaxanthin in improving cognition have shown promising results (Satoh et al. 2009; Katagiri et al. 2012), suggesting that this marine natural product holds significant promise in the development of novel therapeutics for neurodegenerative disorders such as AD. Challenges in the delivery of natural products to the brain: novel delivery technologies Treatment of AD is challenging partly due to the presence physical barriers such as the blood–brain barrier (BBB) in the brain. The BBB is the critical barrier that needs to be overcome to transport natural compounds from the blood into brain. The challenges posed by the BBB is important in the activity of neuroprotective natural products because their benefit is significantly affected by low bioavailability and sometimes poor pharmacokinetic profile (Manach et al. 2004; Soares et al. 2018).l The bioavailability of some anti-inflammatory natural products is attributed to their metabolism. Curcumin is a widely-studied anti-inflammatory and antioxidant polyphe- nol. However, the pharmacological potential is restricted because of its low bioavailability following oral administra- tion (Aggarwal and Sung 2009; Kumar et al. 2010; Di Meo et al. 2019). Resveratrol is a polyphenol with antioxidant, anti-inflammatory and neuroprotective actions. However, in spite of its lipophilicity resveratrol is extensively metabo- lised and rapidly eliminated resulting in poor bioavailability (Chimento et al. 2019). The poor bioavailability profiles of these polyphenols have triggered the synthesis of more bio- available derivatives. 1 3 Table 1 Nanocarriers for delivering natural products into the brain Natural product Delivery strategy References Curcumin Micelles Hagl et al. (2015) Liposomes Mourtas et al. (2014) Nanoemulsion Sood et al. (2014) Solid lipid nanoparticles Kakkar and Kaur (2011) Nanostructured lipid carriers Puglia et al. (2012) EGCG Nanoemulsion Barras et al. (2009) Lipid nanoparticles Smith et al. (2010) Luteolin Solid lipid nanoparticles Dang et al. (2014) Liposomes Zhao et al. (2011) Thymoquinone Nanoemulsion Ahmad et al. (2016) Solid lipid nanoparticles Ramachandran and Thangarajan (2016) Resveratrol Liposomes Wang et al. (2011) Delivery technologies involving mostly lipid-based nano- carriers have been explored to enhance the bioavailability and BBB penetration of neuroprotective natural products. For example, liposomes are formed by amphiphilic sub- stances such as phospholipids that self-assemble as vesicles which compose of lipid bilayers (Lúcio et al. 2010). Over the years, lipid nanoparticles have become more popular and 1 1450 O. A. Olajide, S. D. Sarker j , Fig. 4 Summary of molecular targets of anti-neuroinflamma- tory natural products action involving the transcription fac- tors NF-κB and Nrf2 Fig. 4 Summary of molecular targets of anti-neuroinflamma- tory natural products action involving the transcription fac- tors NF-κB and Nrf2 Fig. Challenges in the delivery of natural products to the brain: novel delivery technologies 4 Summary of molecular targets of anti-neuroinflamma- tory natural products action involving the transcription fac- tors NF-κB and Nrf2 models which combine stress reduction (neuroinflammation, oxidative stress), neuroprotection, and regeneration. Small molecules for AD must be bioavailable and over- come the challenges posed by the BBB to act in the brain. Published articles have shown the potential values of nano- carriers in delivering natural products to the brain. More research efforts need to focus on new delivery methods to achieve significant therapeutic concentrations of polar phy- tochemicals (such as the anti-inflammatory/antioxidant poly- phenols) in the brain. 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/. With regards to molecular target-driven discovery of novel natural products for AD, focusing on a single gene is not the best disease model; as most pharmacologically- active natural products identified using this approach have not resulted in new treatments, mainly due to the complex mechanisms involved in AD. Investigations need to focus on the two principal transcription factors, NF-κB and Nrf2, which control key molecular players in producing inflamma- tion or anti-inflammation (Fig. 4). 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https://openalex.org/W2931004850	http://eujapa.upol.cz/doi/10.5507/euj.2019.005.pdf	English	null	The effect of a pacer versus no-pacer on submaximal fitness test results among Special Olympics athletes	European Journal of Adapted Physical Activity	2,019	cc-by	4,871	Abstract: Using a pacer when administering fitness tests reduces the practicality of testing. Additionally, presuming that a pacer is needed for all Special Olympics athletes is potentially discriminatory. We examined the need for a pacer to enhance performance and the test retest- reliability of the six-minute walk test administered with a pacer (Criterion-m6MWT) and without a pacer (No-pacer 6MWT). Participants were n=18 Special Olympics athletes (men = 12, Mean age=37 years (SD=10.1) with low support needs. After familiarization, participants completed the Criterion- m6MWT and the No-pacer 6MWT. The order of the tests was randomized. A week later, participants completed these tests again. There were no significant differences between any of the walk distances and both the Criterion-m6MWT and the No-pacer 6MWT had high test-retest reliability, intraclass correlation coefficients =.90 and .93, respectively. The interclass correlation coefficients between the first administration of the Criterion-m6MWT and both of the No-Pacer tests were not as strong (i.e. r=.65 and r=.65) as the relationships between the second administration of the Criterion-m6MWT and both No-Pacer tests (r=.81 and r=.87). These results suggest that adult Special Olympics athletes with relatively low support needs can perform the 6MWT without a pacer if the familiarization process is expanded to include a complete 6MWT. Keywords: Intellectual disability, cardiorespiratory, psychometrics European Journal of Adapted Physical Activity 2019, 12, 5 doi: 10.5507/euj.2019.005 Article The effect of a pacer versus no-pacer on submaximal fitness test results among Special Olympics athletes Viviene A. Temple1, Kendal F. Alston2, Jaymie J. Elder3, and Lynneth Stuart-Hill4 Received: 16th August 2018; Accepted: 11th March 2019; Published: 5th April 2019 Article The effect of a pacer versus no-pacer on submaximal fitness test results among Special Olympics athletes Viviene A. Temple1, Kendal F. Alston2, Jaymie J. Elder3, and Lynneth Stuart-Hill4 Received: 16th August 2018; Accepted: 11th March 2019; Published: 5th April 2019 Introduction Higher levels of cardiorespiratory fitness are associated with lower rates of cardiovascular disease and all-cause mortality (Fogelholm, 2010; Zeno et al., 2010), fewer doctor and hospital visits (Mitchell, Gibbons, Devers, & Earnest, 2004), and better athletic performance (Larsen, Nolan, Borch, & Sondergaard, 2005). Among individuals with intellectual disability, cardiorespiratory fitness is predictive of mobility and daily functioning among older adults (Oppewal, Hilgenkamp, van Wijck, Schoufour, & Evenhuis, 2014) and levels of body fat among adolescents (Salaun & Berthouze-Aranda, 2012). In the field, such as during sports practices and monitoring fitness program results, cardiorespiratory fitness is often assessed using a submaximal test (American College of Sports Medicine, 2017). This type of test is typically administered because measuring cardiorespiratory fitness directly (i.e. using respired gas analysis while a person exercises) is often not feasible or practical. Direct measurement of cardiorespiratory fitness requires expensive equipment, well- trained personnel, considerable time, as well as a participant capable of, and willing to, give maximal effort. Submaximal test protocols using a wide variety of exercise modes have been developed, however, a walking test may be preferred for individuals with intellectual disability as walking is common form of physical activity (Dairo, Collett, Dawes, & Oskrochi, 2016; Draheim, Williams, & McCubbin, 2002; Temple & Walkley, 2003). The six-minute walk test (6MWT, American Thoracic Society, 2002) is a submaximal test that involves walking as quickly as possible on a flat, hard surface, for a period of six minutes. Standard phrases of encouragement are provided at the end of each minute and there is no warm-up. The 6MWT has been widely used as a test of submaximal exercise test among individuals living with many different conditions, including: Alzheimer disease (Ries, Echternach, Nof, & Gagnon Blodgett, European Journal of Adapted Physical Activity 2019, 12, 5 doi: 10.5507/euj.2019.005 eujapa.upol.cz 2 of 8 European Journal of Adapted Physical Activity 2019, 12, 5 doi: 10.5507/euj.2019.005 2009), osteoarthritis (Bingchen et al., 2008), severe heart and lung disorders (Brown & Wise, 2007), Parkinson’s disease (Falvo & Earhart, 2009), cerebral palsy (Fitzgerald, Hickey, Delahunt, Walsh, & O'Brien, 2016), and cystic fibrosis (Gruet, Brisswalter, Mely, & Vallier, 2010). The concurrent validity of the 6MWT with the graded maximal treadmill test has been examined among adults (Guerra-Balic et al., 2015; Nasuti, Stuart-Hill, & Temple, 2013) and older adults (Guerra- Balic et al., 2015) with intellectual disabilities. Introduction 2) What is the test-retest reliability of the No-Pacer 6MWT? Introduction Because task understanding and motivation can affect fitness test results among adults with intellectual disabilities (Kittredge, Rimmer, & Looney, 1994; Lavay, Reid, & Cressler-Chaviz, 1990), Nasuti et al. incorporated several accommodations to enhance comprehension, motivation, and maximal effort without changing the performance requirements of the test. Consistent with previously reported fitness testing protocols for adults with intellectual disability (e.g. Pitetti & Fernhall, 2005; Rintala, McCubbin, & Dunn, 1995), a pacer was provided and participants were familiarized with the protocol, equipment, environment, and staff. The pacer walked 1 – 3 metres ahead of the participant and standardized phrases of encouragement were provided every 15-seconds. This version of the test, referred to as the modified six-minute walk test (m6MWT), has excellent feasibility and test-retest reliability and substantial relationships with peak oxygen uptake (VO2peak) among adults with intellectual disabilities (Nasuti et al., 2013). The strong test-retest reliability and feasibility of the 6MWT when a pacer is used has also been demonstrated among adolescents with intellectual disabilities (Casey, Wang, & Osterling, 2012) and those with severe intellectual and sensory disabilities (Waninge, Evenhuis, van Wijck, & van der Schans, 2011). Using a pacer when administering the m6MWT increases the number of personnel needed to conduct the test and possibly the costs and practicality of testing. In addition, presuming that a pacer is needed for all individuals with an intellectual disability to perform the test is potentially discriminatory (Iacono, 2006). To increase the utility of the 6MWT, we compared participants’ performance on the 6MWT with a pacer (Criterion-m6MWT) and without a pacer (No-Pacer 6MWT). The two versions of the test were defined as: • Criterion-m6MWT: participants walk as far as possible in six minutes. Walking back and forth around the orange cones set 30-metres apart. A research assistant walks a little bit in front of the participant (1:1 pacer) and provided encouragement every 15 seconds. • Criterion-m6MWT: participants walk as far as possible in six minutes. Walking back and forth around the orange cones set 30-metres apart. A research assistant walks a little bit in front of the participant (1:1 pacer) and provided encouragement every 15 seconds. • No-Pacer 6MWT: as per the Criterion-m6MWT, but without the use of a pacer. With a population of adult Special Olympics athletes, the specific research questions addressed in this study were 1) What is the relationship between the No-Pacer 6MWT with the Criterion- m6MWT? Supports Intensity Scale (SIS) The SIS (Thompson et al., 2004) measures support needs in six activity categories: home living, community living, lifelong learning, employment, health and safety, and socializing. The assessment assists parents/guardians, service providers, healthcare professionals, and coaches and the like, tailor supports for individuals with intellectual disabilities. Construct-, content-, and criterion-related validity have been established with intelligence and adaptive behaviour (Thompson et al., 2004). The SIS standard score is the sum of the standard scores from all of the subscales, and represents a general indication of the amount and intensity of the support needed. The higher the reported standard scores and percentiles, the greater the amount of support needed. Participants provided the responses to the SIS alongside a person who knew them well. As per the SIS administration protocol, the SIS was administered by one of the research team with the assistance of parents, care providers, coaches, or employers who knew a participant well. The Physical Activity Readiness Questionnaire (PAR-Q) The PAR-Q (Canadian Society for Exercise Physiology, 2002), completed by the participant if they provided consent or a caregiver if they provided assent, was used to prescreen potential participants to determine if further clearance was needed from a physician before the participant was enrolled in the study. Participants Volunteer participants were recruited through Special Olympics in Victoria, British Columbia, Canada. Special Olympics is a global organization that provides opportunities for sport, competition, health screenings, and health promotion activities to more than five million athletes with intellectual disabilities in 172 countries (Special Olympics, 2016). A person is eligible to participate in Special Olympics if he/she has an intellectual disability as determined by meeting any of the following requirements 1) they have a cognitive delay as determined by standardized measures such as an intelligence quotient (IQ), 2) an agency or professional has determined the person has an intellectual disability in accordance with local policies, or 3) the person has functional limitations in both general learning (such as IQ) and in adaptive skills (Special Olympics Inc., 2012). The exclusion criteria for this study were 1) individuals were not ambulatory, 2) they used an assistive mobility device, or 3) subsequent to Physical Activity Readiness Questionnaire (PAR-Q) (Canadian Society for Exercise Physiology, 2002) screening indicating a need for follow-up, the individual did not receive medical clearance to participate. Approval for this study was granted by the Human Research Ethics Board of the University of Victoria BC, Canada, and by SOBC, protocol number 13-518. Potential participants were shown a short video-clip depicting what was involved in the study at a Special Olympics practice. If the eujapa.upol.cz European Journal of Adapted Physical Activity 2019, 12, 5 doi: 10.5507/euj.2019.005 3 of 8 individual was interested in participating, written informed consent was obtained from each athlete or the participant’s legal guardian. When consent was obtained from a guardian, the athlete provided assent. individual was interested in participating, written informed consent was obtained from each athlete or the participant’s legal guardian. When consent was obtained from a guardian, the athlete provided assent. The Six-Minute Walk Tests The modified six-minute walk test (m6MWT) (Nasuti et al., 2013) was used as the Criterion for this study (i.e. Criterion-m6MWT). Nasuti et al. demonstrated that the m6MWT has adequate concurrent validity (R2 = 0.67) with The Graded Maximal Treadmill Test (Fernhall & Tymeson, 1988) and strong test-retest reliability (ICC = 0.98) among adults with intellectual disability. The test involved walking as quickly as possible for six minutes on an indoor flat and hard surface, along a straight 30-metre path, and around a cone at each end of the path. A pacer (ratio of 1 participant to 1 pacer) walked 1 – 3 meters ahead of the participant and the pacer provided standard phrases of encouragement e.g. “you’re doing well” every 15-seconds. At the end of each minute, the pacer indicated how many minutes to go. The path was marked at 2-metre intervals with floor tape, and at the completion of the test, the research assistant timing and recording laps, placed a piece of tape at the heel of the last step taken by the participant. In this way, total distance walked in meters was counted. The m6MWT also had a brief familiarization phase consisting of 1) a demonstration of two laps (30m x 2) of the test by the pacer, and 2) a practice of two laps by the participant with the pacer (30m x 2). The only changes to the m6MWT for the “No-pacer” condition was the removal of the pacer. The additional verbal encouragement (compared to the original American Thoracic Society (2002) version of the test) and familiarization remained. Data analyses Descriptive statistics (mean, standard deviation, minimum, and maximum) were computed for age, SIS, weight, height, BMI, and distances walked. Reliability analysis (Field, 2013) was used to compute intraclass correlation coefficients (ICC, Cronbach, 1951; Field, 2013) to establish the test re- test reliability of the Criterion-m6MWT and the No pacer-6MWT. Interclass correlation coefficients (Pearson’s r) were used to compute the relationship between the Criterion-m6MWTs and the No- pacer 6MWTs. Analyses of variance (ANOVA) were used to examine differences in the distance walked for each comparison (i.e. Criterion1-m6MWT with No-pacer1 6MWT, Criterion1-m6MWT with No-pacer2 6MWT, Criterion2-m6MWT with No-pacer1 6MWT, and Criterion2-m6MWT with No pacer2 6MWT). All analyses were performed using IBM SPSS Statistics Version 24. Walking Tests One week after the familiarization visit, participants completed the Criterion-m6MWT and the No-pacer 6MWT, separated by a 30-minute rest period with juice and/or water provided. To control for the effect of testing the order of the tests was randomized. The walking tests were repeated a week later in the reverse order. Participant information Additional information, specifically: age and date of birth, years of participation with Special Olympics, and contact information, was obtained from the participants or a parent/guardian. With shoes and excess clothing (e.g. coats and jackets), removed, participant height was then measured using a portable stadiometer to the nearest 0.1cm and body mass was measured on a digital scale to the nearest 0.1kg. eujapa.upol.cz eujapa.upol.cz 4 of 8 European Journal of Adapted Physical Activity 2019, 12, 5 doi: 10.5507/euj.2019.005 Familiarization During participants’ first visit to the University, consent and assent (if applicable) forms were collected, the SIS was administered, and weight and height were measured. Subsequently, participants were then shown the Criterion-m6MWT by a member of the research team, and then they practiced several laps of the test with and without a pacer. Procedures A descriptive correlational design was used to establish the relationship between the Criterion- m6MWT and the No-Pacer 6MWT. The test-retest reliability of the Criterion-m6MWT and the No- Pacer 6MWT were also determined. All testing occurred at the University of Victoria. A long and straight hallway was used for the walking tests and a laboratory space was utilized to measure height and weight and to administer the SIS. Results Relationships (Pearson’s r) between m6MWT distances for tests administered with and without a pacer on two occasions for each test Note. Correlation is significant at the 0.01 level (2-tailed). Results The aim of this study was to examine the need for a pacer to enhance performance and the test retest-reliability of the m6MWT administered with and without a pacer. As can be seen in Table 1, the participants were n = 18 adult Special Olympics athletes (men, n = 12) with a mean age of 37 years. After screening with the PAR-Q, three participants were referred to their doctor; all received medical clearance to participate. Individual support needs as measured by the Supports Intensity Scale (SIS; Thompson et al., 2004) were low, ranging from <1% to 14%. Table 2 provides the Pearson’s correlation coefficients for the relationships between each of the Criterion-m6MWTs and each of the No-pacer 6MWTs. All of the correlation coefficients were significant, ranging from r = .65 to r = .87. There were no significant differences between the comparison pairs as evidenced by the ANOVA results, specifically: Criterion1-m6MWT with No- pacer1 6MWT (p = .54), Criterion1-m6MWT with No-pacer2 6MWT (p = .44), Criterion2-m6MWT with No-pacer1 6MWT (p = .23), and Criterion2-m6MWT with No-pacer2 6MWT (p = .08). Both the Criterion-m6MWT and the No-pacer 6MWT had high test-retest reliability, ICC = .90 and .93, respectively. eujapa.upol.cz eujapa.upol.cz European Journal of Adapted Physical Activity 2019, 12, 5 doi: 10.5507/euj.2019.005 5 of 8 Table 1. Mean and range of scores for participant (n = 18) characteristics and 6MWT distances Variable Minimum Maximum Mean SD Age (years) 19 58 36.6 10.1 SIS Score (%)* <1 14 5.1 4.6 SIS Support Needs Index* 56 84 66.1 8.9 Weight (kg) 49.9 133.8 81.0 20.4 Height (cm) 153.9 185.4 169.6 2.4 BMI (kg/m2) 19.0 39.5 28.1 5.8 6MWT distances Criterion test #1 (m) 465.6 747.0 601.0 80.6 Criterion test #2 (m) 477.3 737.8 605.9 72.9 No Pacer test #1 (m) 457.2 745.2 592.4 75.8 No Pacer test #2 (m) 486.0 767.6 588.6 77.6 Note. Data collected on n = 16 participants. 1. Mean and range of scores for participant (n = 18) characteristics and 6MWT distances Note. Data collected on n = 16 participants. Table 2. Relationships (Pearson’s r) between m6MWT distances for tests administered with and without a pacer on two occasions for each test No_Pacer_01 No_Pacer_02 Criterion_01 .65 .65 Criterion_02 .81 .87 Note. Correlation is significant at the 0.01 level (2-tailed). Discussion Table 2. Conclusions Cardiorespiratory fitness is an important health outcome measure and a sensitive measure of changes in response to physical activity (American College of Sports Medicine, 2017), and the 6MWT has been widely used to assess fitness in community and clinical groups (e.g. Møller et al., 2018; Serra et al., 2015). In addition, versions of the 6MWT have been shown to be feasible among individuals with intellectual disabilities (Boer & Moss, 2016; Guerra-Balic et al., 2015; Waninge et al., 2011). The results of this study suggest that with familiarization, that includes a complete 6MWT and encouragement every 15-seconds, adult Special Olympics athletes with relatively low support needs can complete the 6MWT without a pacer. With or without a pacer, the 6MWT is straightforward, with few time, space, measurement, and equipment requirements. However, the No pacer 6MWT has fewer demands for personnel to administer the test than the m6MWT. Discussion Participants in this study performed the m6MWT and the No-pacer 6MWT twice, and both versions of the test had excellent test-retest reliability. These results suggest that either version of the test could be used to monitor change in the cardiorespiratory fitness level of Special Olympics athletes over time. There were no significant differences when the Criterion-m6MWT (with a pacer) distances walked were compared to distances walked without a pacer. Additionally, the relationships between the Criterion2-m6MWT and both of the No-Pacer tests were strong. However, the relationships between the Criterion1-m6MWT and both of the No-Pacer tests (i.e. r = .65 and r = .65) were not as strong as the relationships between the second administration of the Criterion-m6MWT and the No- Pacer tests (i.e. r = .81 and r = .87). The difference in the strength of the correlation coefficients between the first and second administration of the Criterion-6MWT and the No-pacer versions of the tests, may in part be explained by the greater variability in distances walked the first time participants performed a complete Criterion-6MWT. This finding suggests that the familiarization phase of using either the m6MWT or a No-pacer version of the test with adults with intellectual disabilities should include a complete 6MWT. This complete 6MWT would be in addition to the familiarization steps of observing someone doing the test and completing a several 30-metre laps of the test to be performed (i.e. with or without a pacer). This result is consistent with the findings of Casey et al. (2012) who was investigating the test-retest reliability of the 6MWT with a pacer following children and young adults with Down syndrome. When these researchers administered the 6MWT four times over a two- week period, they found the ICC for the four tests was r = .84, however when the first test was omitted the ICC increased to r = .94. There are certain limitations to the present study. Our sample was small, and participants in this study were healthy, motivated, relatively young adult Special Olympics athletes with low support needs, which limits generalizability. It is possible that older adults and those with higher support needs will not respond in the same way to the removal of the pacer. The findings of this study should be replicated with individuals with greater needs for support. eujapa.upol.cz eujapa.upol.cz European Journal of Adapted Physical Activity 2019, 12, 5 doi: 10.5507/euj.2019.005 6 of 8 Funding: This research received no external funding Acknowledgments: We wish to thank Special Olympics Victoria, BC for their assistance with recruitment of participants. Conflicts of Interest: The authors declare no conflict of interest. Perspectives Using a pacer when administering fitness tests reduces the practicality of testing, and presuming that a pacer is needed for all Special Olympics athletes is potentially discriminatory. We found that adult Special Olympics athletes with relatively low support needs could perform the six-minute walk test (6MWT) without a pacer with a high degree of consistency. Further, the results were strongest after a familiarization process and one complete performance of the 6MWT. Author Affiliations: 1 School of Exercise Science, Physical and Health Education, University of Victoria, British Columbia, Canada 2 Community Supports, BC Ministry of Health; Kendal.Alston@gov.bc.ca 1 School of Exercise Science, Physical and Health Education, University of Victoria, British Columbia, Can 1 School of Exercise Science, Physical and Health Education, University of Victoria, British Columbia, Canada 2 Community Supports, BC Ministry of Health; Kendal.Alston@gov.bc.ca 2 Community Supports, BC Ministry of Health; Kendal.Alston@gov.bc.ca 3 School of Exercise Science, Physical and Health Education, University of Victoria, British Columbia, Canada; jelder@uvic.ca j 4 School of Exercise Science, Physical and Health Education, University of Victoria, British Columbia, Ca ada I tuhilll@u i a 4 School of Exercise Science, Physical and Health Education, University of Victoria, British Columbia, Canada; Istuhilll@uvic.ca Correspondence: vtemple@uvic ca; Tel : +01 250 721 7846 Correspondence: vtemple@uvic.ca; Tel.: +01-250-721-7846 Author Contributions: Conceptualization, VT, KA and LSH.; Methodology, VT, KA, JE, LSH; Formal Analysis, VT, JE; Investigation, VT, KA, JE, LSH; Resources, VT, LSH Data Curation, VT, KA, JE,; Writing-Original Draft Preparation, VT.; Writing-Review & Editing, VT, KA, JE, LSH; Supervision, VT, LSH.; Project Administration, VT, KA, JE. Author Contributions: Conceptualization, VT, KA and LSH.; Methodology, VT, KA, JE, LSH; Formal Analysis, VT, JE; Investigation, VT, KA, JE, LSH; Resources, VT, LSH Data Curation, VT, KA, JE,; Writing-Original Draft Preparation, VT.; Writing-Review & Editing, VT, KA, JE, LSH; Supervision, VT, LSH.; Project Administration, VT, KA, JE. Funding: This research received no external funding References Physical activity levels in adults with intellec disabilities: A systematic review. Preventive Medicine Reports, 4, 209-219. doi:10.1016/j.pmedr.2016.06.00 Draheim, C. C., Williams, D. P., & McCubbin, J. A. (2002). Prevalence of physical inactivity and recommended physical activity in community-based adults with mental retardation. Mental Retardation, 40, 436-444. Falvo, M. J., & Earhart, G. M. (2009). Six-minute walk distance in person with Parkinson’s disease: A hierarchical regression model. Archives of Physical Medicine and Rehabilitation, 90, 1004-1008. Fernhall, B., & Tymeson, G. T. 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https://openalex.org/W2225802856	https://leicester.figshare.com/articles/journal_contribution/The_Cost_Efficiency_of_Water_Utilities_When_Does_Public_Ownership_Matter_/10135760/1/files/18266813.pdf	English	null	The cost efficiency of water utilities: when does public ownership matter?	Local government studies	2,016	public-domain	9,460	∗All authors wish to thank Diego Prior and Germà Bel for helpful and constructive discussion, as well as the comments by participants at the Regional Studies Association Early Career Conference 2013 (Manchester, UK), XXI Encuentro de Economía Pública 2014 (Girona, Spain), and the 37th European Accounting Association An- nual Congress 2014 (Tallinn, Estonia). Emili Tortosa-Ausina acknowledges the ﬁnancial support of Ministerio de Economía y Competitividad (ECO2014-55221-P), Universitat Jaume I (P1.1B2014-17) and Generalitat Valenciana (ACOMP/2014/283 and PROMETEOII/2014/046). †C h May 26, 2016 May 26, 2016 The Cost Efﬁciency of Water Utilities: When Does Public Ownership Matter?∗ via Pazzia, Emili Tortosa-Ausina†b, Meryem Duygunc, and Simona Zambellid aSchool of Management, University of Leicester, Ken Edwards Building, University Road, Leicester, LE1 7RH, UK. Email: sp505@le.ac.uk bDepartment d’Economia and Ivie, Universitat Jaume I and Ivie, Campus del Riu Sec, 12071 Castelló de la Plana, Spain. Email: tortosa@uji.es p j cBusiness School, Hull University, Cottingham Road, Hull, North Humberside, HU6 7RX, UK. Email: M.Duygun@hull.ac.uk d yg dDipartimento di Scienze Aziendali, Università di Bologna, Via Capo di Lucca 34, Bologna, Italy. Email: simona.zambelli@unibo.it dDipartimento di Scienze Aziendali, Università di Bologna, Via Capo di Lucca 34, Bologna, Italy. Email: simona.zambelli@unibo.it Keywords: efﬁciency, geographical location, ownership, size, water utilities. JEL classiﬁcation: H4, H7, H83. Abstract This study explores the impact of different ownership types on the efﬁciency of water utilities. The- ories and evidence have shown a puzzling relationship between ownership and performance. More- over, relatively recent contributions (Andrews et al., 2011) have argued that this relationship can be further convoluted by the effect of organisational and environmental variables. The current study aims to contribute to this literature by providing some empirical evidence for Italy, by proposing a methodology that combines nonparametric efﬁciency estimation and cluster analysis. Our main ﬁnd- ings indicate that privately owned utilities indirectly controlled by a public organisation reach the highest level of efﬁciency but, when size and geographical location enter the analysis, ownership has a stronger signiﬁcant effect on efﬁciency, and mixed utilities gain higher cost efﬁciency. Therefore, we may conclude that administrative reforms about privatisation and the institutional setting should consider a set of variables that characterise each individual organisation. Keywords: efﬁciency, geographical location, ownership, size, water utilities. †Contact author. 1. INTRODUCTION In recent decades waves of administrative reforms have been implemented to improve local public services performance and cope with increasing constraints on ﬁnancial resources. In this scenario, devolution and changes in ownership structure have occurred as a solution to public sector inefﬁciencies (Guy et al., 1996; Pollitt and Bouckaert, 2011; Savas, 2000; Shaw and Munday, 1999). Several scholars have investigated whether and how ownership affects perfor- mance, in order to ﬁnd the most efﬁcient, effective and fair way to deliver public services. The persistence of this issue in the literature can be motivated by different theoretical perspectives, puzzling empirical results and the acknowledgement that the link between ownership and per- formance is further complicated by the existence of ‘moderators’ such as organisational and environmental characteristics of the services provided (Andrews et al., 2011). The extent of the debate about the ownership of public service production has been exac- erbated by a wide acceptance of neo-liberal and New Public Management policies (Osborne and Gaebler, 1992; Hood, 1991) rooted in the Public Choice theory (Niskanen, 1971). Accord- ing to this perspective, competition represents a solution to overcome public overproduction and inefﬁciency. Therefore it is assumed that governments, at any level, should privatise and contract-out services in order to achieve technical and cost efﬁciency. Ultimately this process would shift the ownership of service providers from the public to the private sector. Along with Public Choice theory, other theoretical perspectives have dealt with issues re- garding service delivery choices. First, Williamson (1979, 1999) suggests that transactions cost and monitoring can play an important role in the choice to externalise services. In particu- lar, this approach suggests that when transaction costs are low, privatisation can lead to cost savings. Second, property rights theory (Demsetz, 1967) advocates that private ownership can lead to higher performance, due to better deﬁned property rights and incentives to monitor and control the managers’ behaviour. Third, the theory of incomplete contracts (Hart and Moore, 1990) suggests that privatisation could reduce costs, but without an adequate incentive sys- tem, it can also lower services’ quality. In recent years several studies, such as Bel and Fageda (2010), Warner and Hefetz (2008), and Bel et al. (2014), among others, have highlighted the popularity of alternative ownership structures that combine public and private capital—such as mixed companies and public-private partnerships. JEL classiﬁcation: H4, H7, H83. JEL classiﬁcation: H4, H7, H83. Communications to: Emili Tortosa-Ausina, Department of Economics and Ivie, Universitat Jaume I, Campus del Riu Sec, 12071 Castelló de la Plana, Spain. Tel.: +34 964387168, e-mail: tortosa@uji.es. 1. INTRODUCTION Therefore these new types of organisa- tions can challenge even more the relationship between ownership and performance (Vining and Weimer, ming). 1 1 Empirical evidence on the relationship between ownership and performance has been re- viewed by recent studies, such as Andrews et al. (2011) and Bel et al. (2010). Andrews et al. (2011) review thirty-one studies examine the link between ‘publicness’(Bozeman, 1987) and performance in a wide range of public services. Bel et al. (2010) conduct meta-analysis of twenty-seven studies comparing the costs of public and private production in solid waste ser- vices and water distribution. Both of these extensive reviews reveal that there is no systematic evidence supporting the superiority of either public or private production for delivering public services. These studies suggest that performance and efﬁciency seems to be affected by other factors apart from ownership, such as transaction costs, economies of scale, regulation, gover- nance, or the environment. Andrews et al. (2011) refer to these factors as ‘moderators’ of the relationship between ownership and performance. In light of the literature, this study investigates whether ownership structure has a signiﬁ- cant effect on the cost efﬁciency of water service utilities when ‘moderators’ such as size and geographical features are simultaneously considered. The empirical evidence is based on a sample of Italian water utilities from 2008 to 2011. In this regard, Italy represents an ideal geographical case study given a highly heterogene- ity in the ownership structures, size and environmental features of the water utilities operating in this country. Moreover, attention to Italian water utilities can be further motivated by three main reasons. First, in recent years the Italian water industry has been at the centre of a debate about the possibility of liberalisation (Massarutto et al., 2008). Second, in 2011 the legisla- tor modiﬁed the multilevel governance of the industry by abolishing the so-called “Autorità d’Ambito Ottimale”(“optimal area authority”), more popularly known by their initials, ATOs, in charge of coordinating the service at territorial level. However the current regulation has not yet determined which existing or new authorities are to take their place. Third, it is claimed that the price of water in Italy is one of the cheapest in Europe, but research results ﬁnd that this is not sustainable in the long term (Utilitatis, 2011). In this context, efﬁciency is a neces- sary condition to guarantee this vital service in a fair and equal manner. 1. INTRODUCTION The same concern is shared with previous studies carried out for other European countries, such as Spain and Por- tugal (González-Gómez et al., 2013; Da Cruz et al., 2012). Therefore the current study attempts to provide empirical results that can help policy makers and local governments in countries where the implementation of administrative reforms on ownership structure need to be made in a changing institutional environment and the pressure to provide a fair price for public services is high. 2 The method applied in this paper combines two well-known nonparametric efﬁciency estimators—namely, Data Envelopment Analysis (DEA, Charnes et al., 1978) —with cluster analysis, following O’Donnell et al. (2008) and Balaguer-Coll et al. (2013). The advantage of using DEA is to rank water utilities on the basis of their efﬁciency score without requiring any assumption on the distribution function of the data (Rao et al., 2005). Moreover, by applying statistical clustering techniques the study controls for the effect of the ‘moderators’, which has not been carried out in previous studies. The plan of the paper is as follows. Section 2 provide a brief overview of the studies regarding the efﬁciency of water utilities. Section 3 describes the regulatory framework of the Italian water supply service. Section 4 provides an explanation of the method and data. Section 5 reports the results and Section 6 concludes the paper. 2. REVIEW OF THE RELEVANT LITERATURE Since the early 1970s, several studies have assessed the effect of ownership on water supply service (WSS) efﬁciency. These studies differ in several respects, including the method used to measure their efﬁciency levels. In particular, two groups of studies can be identiﬁed: those using accounting methods, and those applying econometrics and operational research meth- ods. The current study applies an operational research method, namely DEA, to estimate WSS utility efﬁciency. As pointed out by Bogetoft and Otto (2011), the selection of a benchmark- ing approach should ‘reﬂect and respect the characteristics of the industry’. With particular reference to the WSS, Berg and Marques (2011) argue that the lack of knowledge on the pro- duction function in this industry can justify the application of DEA. This method is considered more ﬂexible than parametric approaches, since it does not require any assumption on the distribution function of the data. Moreover, Bogetoft (1994) highlighted the incentive-efﬁcient properties of DEA that can be applied by regulators as it can be seen in England and Wales (Thanassoulis, 2000a,b). The ﬁrst study to apply the concept of Farrell (1957) efﬁciency—on which DEA is based—in this particular context was Byrnes et al. (1986), in an analysis focused on the US. The theoretical perspective on which the study was grounded provided arguments that privately-owned ﬁrms were more efﬁcient than their publicly-owned counterparts. However, the nonparametric tests reveal no evidence that the latter utilities were ‘more wasteful or operated with more slack than privately owned utilities’ (Byrnes et al., 1986, p.341). Following and ‘adjusting’ Byrnes 3 et al.’s (1986) method, several studies have applied DEA to analyse the relationship between ownership and water services’ efﬁciency around the world. In line with the purpose of current study, the following review brieﬂy outlines the research on the effect of ownership on WSS utility efﬁciency, classifying the studies into three groups according to their results: (i) studies that reported no inﬂuence of ownership on efﬁciency; (ii) studies ﬁnding that public ownership improves efﬁciency; and (iii) those ﬁnding better efﬁciency scores for privately owned utilities. One of the most relevant contributions among the ﬁrst group of studies would include Byrnes et al. (1986). More recent research includes García-Sánchez (2006), who measures the technical and scale efﬁciency of Spanish municipalities, distinguishing between those which externalised the water services to privately owned utilities and those which provide the service through public business corporations. 2. REVIEW OF THE RELEVANT LITERATURE The study claims that, in the speciﬁc context analysed, the creation of a quasi-market does not seem to affect efﬁciency. The author suggests that this result can be justiﬁed by the fact that the creation of public business corporations relieves the management of the business from the traditional public sector bureaucratic procedures. In this group of studies we also ﬁnd Peda et al. (2013) who, in an application to Estonian water service utilities, found ‘no difference in efﬁciency between water utilities with different types of ownership’. Their study also found a positive relationship between population size and efﬁciency, corroborating the hypothesis that efﬁciency gains are attributable to scale economies. In the second group of studies, one of the most relevant contributions is the one by Romano and Guerrini (2011) on the efﬁciency of Italian water utilities. To the best of our knowledge, this is the ﬁrst study to apply DEA to Italian water utilities, ﬁnding that publicly-owned utilities obtain higher efﬁciencies than mixed-owned. The authors interpret these results as an indica- tion that publicly-owned utilities are better able to acquire and use their inputs. Moreover, the study considers the effect of size and geographical location on the performance of the water utilities. The results show the existence of economies of scale, since larger companies perform better. Regarding the geographical location issue, utilities located in Central and Southern Italy are more efﬁcient than those operating in the north—although the differences were not statistically signiﬁcant. Finally, the third group of studies ﬁnd superior performance in privately-owned utility ﬁrms. Speciﬁcally, Picazo-Tadeo et al. (2009) ﬁnd that privately-owned utilities are more ef- ﬁcient than their publicly-owned counterparts. The authors claim that this result is due to efﬁciency in the use of labour, pointing out that the inﬂuence of trade unions makes it difﬁcult to adjust the number of employees. González-Gómez et al. (2013), focusing on Spain’s rural 4 areas, ﬁnd that both privately-owned utilities and public-private partnerships are signiﬁcantly more efﬁcient. Notably, the differences in term of efﬁciency between the three forms of owner- ship disappear when environmental variables are considered. As environmental variables the authors suggest the existence of economies of consumer density, the origin of water resources and the seasonality of demand. These are factors that can inﬂuence the efﬁciency but they do not depend on ownership structure. 2. REVIEW OF THE RELEVANT LITERATURE The authors concludes that public-owned utilities oper- ate in a more challenging environment while private utilities avoid it due to low proﬁtability expectations. The authors remark that public-private owned utilities do not perform badly in comparison with the other two kinds of utility ownership. The available empirical evidence suggests that the debate on the links between ownership and performance is still unsettled. In addition, other variables also seem to be relevant in as- sessing performance and institutional choices. Firstly, the efﬁciency of water supply services can be related to their size, as the law of economies of scale would predict, however previous literature also suggests that economies of scale occur only after reaching certain level of output (Walter et al., 2009). Secondly, some studies highlight the effect of regulatory framework and in- centive mechanisms on performance. González-Gómez and García-Rubio (2008) highlight that the greater efﬁciency observed for the private utilities could result from either the ownership features themselves or the regulatory framework within the industry. De Witte and Marques (2010) present a cross-country comparison examining the role of incentive mechanisms in re- lation to efﬁciency levels. The results show a positive effect of incentive mechanisms (such as benchmarking) on efﬁciency. On the basis of these results, the authors conclude that bench- marking could become a tool to create ‘competition by comparison’ in contrast to ‘competition in the market’ or ‘competition for the market’. The importance of regulation, the creation of independent authorities to control the conduct of water utilities and benchmarking initiatives are also highlighted in recent research by Bel et al. (2015). This paper suggests these factors are essential in ensuring a fair water price, especially with respect to privatised utilities. Finally, efﬁciency can be affected by environmental variables, such the hydrographical characteristics of the geographical area in which the utilities are located (Martins et al., 2012). The current research therefore attempts to contribute to this literature by investigating the effect of three variables, namely: ownership types, size and geographical location. These three variables are considered separately in the ﬁrst stage of analysis and by combining their effects in the subsequent stage. This could shed light on the contribution of different ownership structures to assist in mitigating exogenous conditions such as hydrographical characteristics 5 and aid in strategic planning on utilities size. 3. WATER SERVICE IN ITALY About 90 ATOs were identiﬁed according to the political-administrative 6 6 and hydrographical features of each area (Utilitatis, 2011). The main function assigned to the ATOs was to draw up a management plan for the WSS and to designate the WSS provider. In the mid-2000s, Law 196/94 was replaced by the Environmental Code (Decree 152/2006), which retained the two main innovations of the previous law and introduced the European principle of cost recovery for the WSS. Among other norms, article 154 of the Environmental Code stated that the WSS price had to guarantee remuneration for the capital invested. Meanwhile, changes had occurred in the institutional organisation of service providers. Since 1990, inspired by New Public Management, a series of reforms have been introduced to promote externalisation of local public services. The result is that the WSS provider could be a municipality, a municipal corporation, a mixed enterprise or a private entity. Moreover some municipalities have created municipal holdings that invested in private entity providing public service (Grossi and Mussari, 2009), therefore private entities can have a municipality as indirect shareholder. Finally, water supply services were also affected by a series of relatively recent events. First, the ﬁnancial crisis forced governments to cut their budgets. In this context, the Italian legislator suggested eliminating the ATOs by the end of 2011. However, this regulation did not determine which authority should replace the ATOs, a question that still remains unanswered. Second, in 2011 a referendum repealed article 23-bis of Law 113/2008 and article 154 of the Environmental Code. Subsequently, the appointment of the WSS is based only on European legislation, with the result that the service can be provided by municipalities directly, in house, by mixed enterprises without any speciﬁcation of the percentage that must be owned by private partners, or by privately owned enterprises. A further consequence of the referendum was that the tariff should not be set according to the return on capital invested. 3. WATER SERVICE IN ITALY Water supply services (WSS) are generally considered public services provided through a net- work regulated by public authorities, therefore any speculation on the organisation, governance and performance is strongly affected by the regulatory framework within each country. The Italian WSS are regulated by four hierarchical levels of jurisdiction: the European Union, the central government, the regional governments, and the local governments. European legislation classiﬁes water supply services as a ‘service of general economic in- terest’ (European Community Treaty, Article 86(2)). Therefore WSS are economic services that have to be provided to every citizen on a regular basis and at affordable prices, regardless of the ownership of the service provider. Moreover, in 2000 the European Commission issued the Water Framework Directive (WFD), addressing most of the challenges facing the management of this crucial resource. Two of the innovations introduced by the WFD were the cost recovery for water services and the ‘polluter-pays’ principles. These principles aim to create incentives for the sustainable and efﬁcient use of water. As highlighted in the previous section, the last decades have witnessed changes in the own- ership of public service providers. The European Commission lets each Member State decide how it organises the provision of a service of general economic interest so long as the rules on both the internal market and competition are observed. As a result, different approaches to the organisation of WSS can be found among EU Member States. For instance, in The Netherlands and Germany, municipal public enterprises provide water services. Conversely, in England and Wales the service was totally privatised and a regulatory authority established (Bauby, 2012). In Italy, water supply services were traditionally provided by municipalities. In this context, the service was ﬁnanced via public budget, and the tariff was usually insufﬁcient to cover the costs (Massarutto et al., 2008). In order to improve the efﬁciency of the industry, the Law 196/94 was enacted in 1994 to reform the industry. First, the reform recognised the network features of the WSS and introduced the concept of ‘integrated water service’, considering the whole water supply and sewage system. Second, the reform reorganised the WSS by introducing territorial authorities, ATOs, with the aim of exploiting economies of scale in the management of services. Regions were in charge of identifying these ATOs and municipalities could own equity shares in ATOs. 3. WATER SERVICE IN ITALY In conclusion, it can be argued that the main consequences of reforms and counter-reforms of the water supply services are: (i) a multilevel governance structure of the industry, although the levels of this structure are still uncertain regarding the replacement of the ATOs and the role of the regions; and (ii) in the absence of an intermediate authority such as the ATOs, it seems that municipalities could once again be free to choose the delivery mode and appoint the service provider as they did in the past; (iii) changes in the tariff computation, with particular regard to the return on capital invested. 7 7 1As indicated by Rao et al. (2005), the input-oriented efﬁciency addresses the question: ‘By how much can input quantities be proportionally reduced without changing the output quantities produced?’ (Rao et al., 2005, p.137). 4. METHODS AND DATA Our study investigates the effect of ownership and the ‘moderators’, i.e., size and geographical location, on the cost efﬁciency of Italian water utilities. To this end a three-stage methodol- ogy is applied: (i) we measure cost efﬁciency using a nonparametric estimators, namely, Data Envelopment Analysis (DEA); (ii) cluster analysis, building groups based on ownership, size and geographical allocation of the organisations; and (iii) testing for differences in the efﬁcien- cies in each group and each cluster—i.e., nonparametric test is applied to verify whether type of ownership, size, geographical location, or their combination in clusters result in signiﬁcant efﬁciency differences. This methodological approach is similar to the one considered by Balaguer-Coll et al. (2013) in studying the efﬁciency of Spanish municipalities. However it differs from previous relevant work on water utilities, such as (Peda et al., 2013; Romano and Guerrini, 2011), who considered an a priori classiﬁcation of organizations, without considering the combined effect on perfor- mance. Therefore, the procedure carried out in this study allowed the deﬁnition of clusters ex-post instead of ex-ante identifying a combination of factors that can inﬂuence cost efﬁciency and controlling for heterogeneity. For measuring cost efﬁciency we consider Data Envelopment Analysis (DEA). Its origins date back to Farrell’s (1957) approach to frontier estimation, although it was not until 1978 that the term was ﬁrst used (Charnes et al., 1978). Since then, this method has become one of the most popular techniques for benchmarking, with applications from ﬁnancial ﬁrms to public service utilities—including water utilities (Fethi and Pasiouras, 2010). DEA is a mathematical programming technique for the estimation of the best production frontier (or envelopment) and the measurement of the relative efﬁciency of different organisa- tions (Bogetoft and Otto, 2011). This approach assigns a score between 0 and 1 to each decision making unit (in the case that an input orientation and Farrell distance functions are consid- ered), allowing the organisations to be ranked on the basis of an increasing efﬁciency order. The term ‘frontier’ identiﬁes the most efﬁcient organisation that satisﬁes either the input or output-based Farrell efﬁciency condition. In this study, efﬁciency measures are computed on the basis of two assumptions. Firstly efﬁciency scores are input-based and thus measure the level of input to obtain a given amount of output 1. Secondly inputs are expressed in monetary terms allowing the measurement of 8 cost efﬁciency. 4. METHODS AND DATA Formally, the input-oriented DEA is based on the solution of the following linear program- ming problem (Rao et al., 2005; Coelli and Walding, 2006): minθ,λ θ s.t. yi + Yλ ≥0, θxi −Xλ ≥0, N1′λ = 1, λ ≥0. (1) minθ,λ θ s.t. yi + Yλ ≥0, θxi −Xλ ≥0, N1′λ = 1, λ ≥0. (1) (1) where: where: • yi is an M × 1 vector of outputs produced by the ith ﬁrm, • yi is an M × 1 vector of outputs produced by the ith ﬁrm, • Y is the M × N matrix of outputs of the N ﬁrms in the sample, • X is the K × N matrix of inputs of the N ﬁrms, • λ is an N × 1 vector of weights (which relate to the peer ﬁrms) and θ is a scalar measure of efﬁciency, which takes a value between 0 and 1 (inclusive). Further details on this approach are also available in Balaguer-Coll et al. (2007), among others, who propose a very similar program to the one followed in this paper. For a more comprehensive view, see also Cooper et al. (2007) and Färe et al. (1994). This approach seems particularly suitable for the context of the water industry, where utilities are more able to control their inputs rather than their outputs—such as water delivery and population served (Abbott and Cohen, 2009; Coelli and Walding, 2006; Romano and Guerrini, 2011). 4.1. Testing for the equality of distributions of efﬁciency scores In the second stage of the analysis we try to ascertain whether the differences found among the efﬁciency scores of the ﬁrms in each group are statistically different or not. In this regard, a variety of instruments can be considered to test whether the differences between some of the moments that characterise two given distributions differ statistically. Some of these instruments are tests, such as the Wilcoxon test, which have the advantage of being relatively robust to the violation of the normality assumption but have the limitation of restraining the analysis to one moment of the distribution only (in our case, the distribution of efﬁciency scores), namely the 9 median. However, some recent applications (Balaguer-Coll et al., 2010) have considered some tools developed in the ﬁeld of nonparametric statistics such as the Li (1996) test, which tests whether two distributions, not just two summary statistics such as the mean or the median, differ statistically. 2In 2015 Federutilities was merged in Utilitalia. 2In 2015 Federutilities was merged in Utilitalia. 4.2. The sample Only three utilities were classiﬁed as type 4 and six were classiﬁed as type 5. types 2 and 3. Only three utilities were classiﬁed as type 4 and six were classiﬁed as type 5. The size of water utilities is usually measured considering the population served, however, due to a lack of data over the time span analysed, a proxy was used in this study. A possible proxy is total revenue, obtained from utilities’ ﬁnancial statements. This variable shows a strong linear correlation with the population served (92%), suggesting that revenue can be used as proxy of the population served with conﬁdence. Table 1 shows that the sample is mainly characterised in small and medium size utilities, only three are considered to be large. Finally, the third variable considered is geographical location. Most of the utilities in the sample are situated in the Northern of Italy, while 15% and 28% are in the Centre and Southern regions, respectively (Table 1). Italy is characterised by heterogeneous hydrographical features which can affect efﬁciency levels. Northern and Southern regions, saving a few exceptions, are characterized by surface waters that require a more sophisticated puriﬁcation process, leading to higher operational and capital costs (Istat, 2008, 2014). Utilities are further classiﬁed using cluster analysis in an the attempt to maximise the ho- mogeneity of units within the clusters while maximising the heterogeneity among clusters. In the current analysis, ﬁve clusters are identiﬁed. The characteristics of these clasters are shown in Table 2 with their associated descriptions shown in Table 3. All variables were shown to be signiﬁcant with regard to all clusters, with the exception of the fourth type of ownership - mixed owned utilities with a private organisation that owns 50% or more. The cluster analy- sis discriminates between medium size, publicly owned utilities in central and Southern Italy (Cluster 1) and those that are located in the north of the country (Cluster 3). Cluster 2 contains both mixed and privately owned utilities however in both cases, the cluster analysis identiﬁes the main direct or indirect shareholder as a public organisation and but does not discriminate between size and geographical location. Cluster 4 is characterised primarily by small sized, privately owned utilities located in Southern Italy. Finally, Cluster 5 aggregates primarily small sized, publicly owned and mixed owned utilities in Northern Italy. 4.2. The sample As stated above, the empirical evidence presented in this paper focuses on a sample of water utilities operating in Italy from 2008 to 2011. A complete list of Italian water utilities was obtained from Federutilities, an Italian association of public services provider.2 However, the sample is restricted to mono-service utilities with available data and stable ownership structure. Therefore, only utilities for which the percentage of ownership has not changed from 2008 to 2011 are included in the analysis. The ﬁnal sample is comprised of 68 utilities in each of the four years analysed, leading to 272 observations across the four years study (Table 1). The 68 utilities represent 70% of those listed by Federutilities and they served about 45% of the Italian population in 2011. Furthermore, utilities are classiﬁed according to three variables: ownership structure, size and geographical location. As highlighted in the previous section, a water supply service provider could be a munici- pality, a municipal corporation, a municipal holding, a mixed enterprise or a private entity. In this scenario, our study focuses on water services which are externalised by the local govern- ment through a separate entity, namely an utility, with a different type of ownership structure. In our particular sample, ﬁve types of ownership were identiﬁed (Table 1). As demonstrated above, the conventional classiﬁcation of private, public and mixed ownership used by previous research (Guerrini et al., 2011) does not fully reﬂect the complexity of the Italian context or any other national setting where many alternative modes to delivery public services coexist (Tavares and Camöes, 2007; Bel and Fageda, 2010). In addition to the utility ownership models of publicly owned (type 1) and privately owned (type 2), this research distinguishes between two speciﬁc groupings within mixed utilities. The ﬁrst of these groups are utilities which have a public organisation as the controlling shareholder (type 3) and, the second group are utilities which have a private organisation as the controlling shareholder (type 4). Finally, we deﬁne a separate category of private utilities in which the indirect main shareholder is a public organi- sation (type 5). As reported in 1 32 utilities (128 observations over four years), corresponding to 47% of the sample, are publicly owned. The remaining utilities are primarily spread between 2I 2015 F d ili i d i U ili li 10 types 2 and 3. 4.3. Inputs and outputs One of the biggest challenges in the application of DEA was the selection of the input-output variables suitable and available for water utilities. Consistently with the aim to estimate cost efﬁciency scores, operational costs were used. Four operational costs were considered as inputs, namely: cost of materials, cost of services, cost of using third party resources (such as rented or leased plant and equipment), and wages. The most popular measures of outputs are: the amount of water delivered, the population served, and the length of water mains (Coelli and Walding, 2006). The above data are not accessible for all the utilities in the sample and the population served is available only for 2011, therefore revenue is used as a proxy for the variable size. Furthermore, since the analysis is longitudinal and both inputs and outputs are expressed in monetary terms, the data are deﬂated by the Italian consumer price index in order to correct them for inﬂation (Coelli and Walding, 2006). This adjustment is particularly relevant, since the time frame analysed is characterised by a considerable increase in prices (5.5%). Finally, Table 4 reports the deﬁnition of inputs and outputs, and Table 5 their corresponding descriptive statistics for each year under analysis. It is worthwhile noticing that skewness and kurtosis are far from zero, the value that indicates the variables under analysis follow a normal distribution. 4.2. The sample Since we are using a data panel of 68 utilities from 2008 to 2011, a window analysis could also have been considered. However, we consider the approach used in this paper is appropri- ate due to the low likelihood of technical change in the short term in the context of the urban water sector. 11 11 5.1. DEA efﬁciencies Efﬁciency scores for the utilities in the sample over the 4 years computed via DEA are reported in Tables 6 and 7. The tables report DEA efﬁciencies considering the three classiﬁcation criteria both separately (Table 6) and jointly (Table 7). When considering the ex-ante classiﬁcations (ownership, size, geographical location), re- markable differences are perceived among groups within each of the hypotheses considered. In the case of the groups constructed according to their ownership type, the discrepancies are par- ticularly large. As indicated by the efﬁciency scores in Table 6, the discrepancies among average efﬁciencies are quite large, ranging from 49.19% for the most inefﬁcient group (privately-owned utilities) to 90.42% for the least inefﬁcient (privately-owned utilities with a public organisation as the main indirect shareholder). Focusing on the median, in order to isolate the effects of potential outliers, these discrepancies are even higher—the medians are 48.99% and 97.78% for 12 these two groups, respectively. these two groups, respectively. When the ‘moderators’, i.e., size and geographical location, are considered separately, the results vary depending on the hypothesis considered. Regarding size, large ﬁrms show com- paratively higher values—58.33% of them are efﬁcient (see Table 6), whereas small ﬁrms are quite inefﬁcient by comparison as only 17.97% of such ﬁrms are efﬁcient and the median is also quite low (29.30%). This ﬁnding is consistent with previous research that indicated the existence of economies of scale in the water industry (Romano and Guerrini, 2011; Peda et al., 2013). In addition to this, the number of efﬁcient ﬁrms for small, medium and large ﬁrms is 17.97%, 36.36% and 58.33%, respectively, however this ﬁnding was partly to be expected given the assumption of variable returns to scale and the fact that the number of large ﬁrms is lower than the number of smaller ﬁrms. The discrepancies are more modest when analysing results for groups based on their geo- graphical location. The discrepancies among groups’ average efﬁciencies are much lower (Table 6), and the utilities in the centre of Italy are the least inefﬁcient, a ﬁnding that concurs with previous research (Romano and Guerrini, 2011). 5.2. The ‘moderators’ As indicated in the introduction, understanding the link between ownership and performance may be particularly intricate due to the effects of ‘moderators’, among which Andrews et al. (2011) highlight the role of size, geographical location, and governance. This study combines these factors in a clusters to take into account their effect on efﬁciency. The summary statistics for the efﬁciencies corresponding to the ﬁve groups identiﬁed by the cluster analysis are reported in Table 7. The differences betare high, especially when compar- ing the least inefﬁcient groups 2 (mixed ownership with both direct and indirect main public organisation as shareholder) and 3 (publicly owned, medium, in Northern Italy), with clusters 4 (privately owned, small, in Southern Italy) and 5 (publicly owned, small, in Southern Italy). More speciﬁcally, the average efﬁciencies corresponding to groups 2 and 3 are particularly high (81.29% and 86.26%, respectively), analogously to the values for the medians (93.99% and 97.16%, respectively). In contrast, the behaviour is quite the opposite for clusters 4 and 5, whose medians are 52.78% and 24.93%, which suggests that the mix of privately owned and small ﬁrms in Southern Italy may be particularly problematic in terms of efﬁciency. This ﬁnding seems to emphasise the relevance of economies of scale and the importance of public investment in the water industry, especially in areas where the puriﬁcation process needs to be 13 more intense, such as in the southern parts of the country. 5.3. Testing for the differences among WSS efﬁciency scores The analysis in the above paragraphs is based on soley summary statistics and its statistical precision is therefore limited. In this section the methods proposed in section 4.1 are applied to test whether the differences among the efﬁciencies of ﬁrms in the groups formed according to different criteria are signiﬁcant or not. The method employed, as indicated in section 4.1, has the interesting virtue that it does not compare summary statistics but entire distributions of ef- ﬁciency, as well as being fully nonparametric (and, therefore, consistent with the nonparametric DEA estimators). This test compares the densities, estimated via kernel smoothing, for the unconditioned and conditioned relative series of efﬁciencies, where the unconditioned relative efﬁciency se- ries corresponds to each ﬁrm’s efﬁciency, divided by the average corresponding to all ﬁrms (computed yearly), and the conditioned relative efﬁciency series corresponds to each ﬁrm’s efﬁciency divided by its group average. This average will differ depending on the hypothesis considered—ownership, size, geographical location or their combined effect. The densities are displayed in Figure 1. The lines in each sub-ﬁgure correspond to the un- conditioned (solid line) and conditioned (dashed lines) relative efﬁciency series. Regardless of whether the series is unconditioned or conditioned, the amount of multi-modality is remark- able, with pronounced modes well below the mean (which is 1, given we are dividing by the mean). This suggests there are non-negligible pockets of inefﬁcient behaviour which do not vanish after controlling for our three factors—or their combined effects. If the conditioning results in tighter densities and closer to the mean (i.e., unity), this would indicate that the conditioning scheme considered is relevant, i.e., efﬁciencies for all utility ﬁrms in the same group would be similar. This is only the case when conditioning for size and, to a lesser extent, ownership, whereas the effect of geography is negligible as the densities almost overlap. The combined effect (the ‘moderators’) shows the strongest effect, as densities shift leftwards, approaching the mean (see Figure 1.d), corroborating the descriptive analysis carried out in the previous section. Li’s (1996) test provides statistical evidence to support this visual analysis. Results, shown in Table 8, corroborate the analysis stemming from the visual inspection of the densities, since differences are particularly signiﬁcant when considering size alone, or the combined effect of the three hypotheses. 5.3. Testing for the differences among WSS efﬁciency scores In contrast, geographical location the differences does not produce 14 signiﬁcant differences, whereas in the case of the type of ownership the effect is only signiﬁcant at the 5% signiﬁcance level. signiﬁcant differences, whereas in the case of the type of ownership the effect is only signiﬁcant at the 5% signiﬁcance level. 6. CONCLUSIONS This paper foucuses on a key public service, water supply services, and purpose of this study has been to analyse the inﬂuence of local public ownership on the efﬁciency of Italian water utilities. The study was motivated by the puzzling relationship between the different types of ownership and efﬁciency. In addition, the literature has identiﬁed a gap in understanding the effect of ‘moderators’ on the performance of water supply services. We have considered the case of Italy, where these services have traditionally been pro- vided by local governments but changes in regulation and the acceptance of paradigms such as New Public Management have resulted in such services being provided by different organi- sations. The current study has gone beyond the conventional classiﬁcation of three ownership types (public, private and mixed), identifying ﬁve types of ownership and better reﬂecting the complexity of public service organisation in Italy and other countries. In this context, the re- lationship between types of ownership and efﬁciency is further involved due to the disparate sizes and geographical locations of the utilities. Previous studies have considered the effect of ownership type, geographical location and size in isolation, whereas this study explores the combined effect of these three factors on efﬁciency simultaneously. From a methodological point of view, it can be argued that cluster analysis and appropriate nonparametric tests help to better discriminate among the different factors that can affect the efﬁciency of water utilities. Speciﬁcally, we measure efﬁciency by applying Data Envelopment Analysis and tests based on kernel smoothing to ascertain whether the differences between the clusters were signiﬁcant or not. Using these methods the current study has found statisti- cally signiﬁcant differences in efﬁciencies across ownership types. Even stronger results were seen when considering groups based on size or the groups yielded by cluster analysis, which combine all the three factors of ownership type, size and geographical characteristics. Furthermore the results suggest that privately owned utilities which are indirectly con- trolled by public organisations reach the highest level of efﬁciency when size and geographical location are not considered. However, the combined effect of ownership, size and geographical location has a stronger effect on efﬁciency. In this case, mixed-owned water utilities, in which a public organisation has direct or indirect control, are those with the higher efﬁciency levels. 6. CONCLUSIONS Furthermore the results suggest that privately owned utilities which are indirectly con- trolled by public organisations reach the highest level of efﬁciency when size and geographical location are not considered. However, the combined effect of ownership, size and geographical location has a stronger effect on efﬁciency. In this case, mixed-owned water utilities, in which a public organisation has direct or indirect control, are those with the higher efﬁciency levels. 15 Our results suggests that policy makers and regulators should carefully consider the intrin- sic characteristics of each industry in order to achieve better performance for public services. In particular, with respect to the water industry, both public-private partnerships and economies of scale seem to be important aspects to take into consideration, particularly when evaluating them simultaneously. Finally, we draw attention to the need to broaden this line of research to improve the likely implications for regulators and policy makers. Although ownership and efﬁciency are im- portant dimensions which affect the ‘publicness’ and performance, a comprehensive analysis would require to simultaneously consider the impact of ‘control’,‘funding’ and ‘change’ on ef- ﬁciency, effectiveness and equity (Andrews et al., 2011; Bowles et al., 2005; Bozeman, 1987). In a recent paper, (Bel et al., 2015) consider the effect of market concentrations on water service prices. 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Journal of Law, Economics, & Organization, 15(1):306–342. 20 on according to ownership, size and geographical locatio ip # # % 32 128 47% 15 60 22% wns 50% or more (ownership type 3) 12 48 18% owns 50% or more (ownership type 4) 3 12 4% ion as indirect shareholder (ownership type 5) 6 24 9% 68 272 100% # # % 32 128 47.1% 33 132 48.5% 3 12 4.4% 68 272 100% ocation # # % 39 156 57.4% 10 40 14.7% 19 76 27.9% 68 272 100% ly owned util- 0 0% 2 13% 0 0% 10 91% 3 17% owned utili- th public or- ion that owns more 0 0% 6 40% 0 0% 1 9% 5 28% owned utili- th private or- ion that owns more 0 0% 2 13% 0 0% 0 0% 1 6% ly owned util- ith public or- ion as main t shareholders 0 0% 5 33% 0 0% 0 0% 1 6% 3 23% 0 0% 0 0% 11 100% 18 100% m 7 54% 15 100% 11 100% 0 0% 0 0% 3 23% 0% 0% 0% 0% rn Italy 1 8% 9 60% 11 100% 0 0% 18 100% Italy 5 38% 5 33% 0 0% 0 0% 0 0% rn Italy 7 54% 1 7% 0 0% 11 100% 0 0% 13 100% 15 100% 11 100% 11 100% 18 100% er to assign the variables to the clusters. a In thousands of e. References For all the variables the test was signiﬁcant ownership #4 (mixed owned utilities with private organisation that owns 50% or e clusters # of ﬁrms in th cluster 13 n as shareholder 15 11 11 18 68 8 incom s and outputs ome statement ed in the income statement e income statement the income statement operating leasing recorded in the incom Table 5: Italian water service utilities, descriptive statistics for inputs and outputsa Year Variable Mean Std. dev. Median Skewness Kurtosis Year 2011 y1 10,121,335 16,988,394 2,348,111 2.7 8.5 x1 1,083,785 3,111,111 193,655 6.4 45.2 x2 1,738,548 3,050,778 446,292 2.9 9.5 x3 4,405,616 7,292,794 1,016,890 2.3 5.1 x4 1,215,181 5,526,878 56,840 7.4 55.7 Year 2010 y1 10,043,699 17,009,684 2,403,003 2.9 10.4 x1 1,078,828 2,853,741 214,342 6.0 40.5 x2 1,645,831 2,794,543 500,416 3.1 12.1 x3 4,027,370 6,293,973 973,705 2.1 4.5 x4 655,829 1,302,237 37,137 2.2 3.6 Year 2009 y1 10,024,592 16,372,146 2,776,805 2.5 7.4 x1 1,006,826 2,778,297 219,541 6.1 42.1 x2 1,751,179 3,107,051 527,256 3.2 12.2 x3 4,298,332 6,879,418 966,142 2.2 4.7 x4 674,433 1,408,328 29,394 2.4 4.5 Year 2008 y1 9,674,737 16,020,107 3,014,006 2.7 8.2 x1 1,061,385 3,181,462 202,686 6.6 47.2 x2 1,714,371 3,172,536 473,426 3.5 14.9 x3 4,354,073 7,209,273 1,060,797 2.3 5.0 x4 676,034 1,443,635 32,759 2.5 5.7 a In thousands of e 25 Table 6: DEA efﬁciency scores for Italian water service utilities, distribution according to own- ership, size and geographical location Classiﬁcation Group Mean 1st quartile Median 3rd quartile Std.dev. References % efﬁcient ﬁrms Ownership Type 1 0.6567 0.3149 0.7587 1.0000 0.3404 26.56 Type 2 0.4919 0.1532 0.4899 0.8770 0.3634 21.67 Type 3 0.6397 0.2076 0.7183 1.0000 0.3781 39.58 Type 4 0.5774 0.2890 0.5375 0.8670 0.3033 16.67 Type 5 0.9042 0.8035 0.9778 1.0000 0.1224 41.67 Size Small 0.4572 0.1416 0.2930 0.7944 0.3584 17.97 Medium 0.7818 0.6717 0.8737 1.0000 0.2656 36.36 Large 0.9311 0.8871 1.0000 1.0000 0.1196 58.33 Geography North 0.6371 0.2451 0.7382 1.0000 0.3483 28.21 Centre 0.7115 0.4676 0.9037 1.0000 0.3651 35.00 South 0.5929 0.2073 0.6001 1.0000 0.3518 26.32 Total 0.6357 0.2451 0.7478 1.0000 0.3524 28.68 Table 6: DEA efﬁciency scores for Italian water service utilities, distribution according to own- ership, size and geographical location Table 6: DEA efﬁciency scores for Italian water service utilities, distribution according to own- ership, size and geographical location 26 Table 7: DEA efﬁciency scores for Italian water service utilities, distribution according to clus- ters Table 7: DEA efﬁciency scores for Italian water service utilities, distribution according to clus- ters Classiﬁcation Group Mean 1st quartile Median 3rd quartile Std.dev. % efﬁcient ﬁrms Clusters Cluster 1 0.6366 0.3786 0.7589 0.9142 0.3407 19.23 Cluster 2 0.8129 0.7198 0.9399 1.0000 0.2555 41.67 Cluster 3 0.8626 0.7514 0.9716 1.0000 0.1862 47.73 Cluster 4 0.5161 0.1532 0.5278 0.9687 0.3729 25.00 Cluster 5 0.4216 0.1423 0.2493 0.6648 0.3425 15.28 Total 0.6357 0.2451 0.7478 1.0000 0.3524 28.68 27 27 Table 8: Testing the closeness between unconditioned and conditioned relative efﬁciency series (Li, 1996) DEA Null hypothesis T-statistic p-value H0 : f (Unconditioned) = g(Ownership-conditioned) 2.1631 0.0153 H0 : f (Unconditioned) = g(Size-conditioned) 26.1374 0.0000 H0 : f (Unconditioned) = g(Geographical location-conditioned) –1.0027 0.8420 H0 : f (Unconditioned) = g(Combined effect) 22.5473 0.0000 Testing the closeness between unconditioned and conditioned relative efﬁciency series (Li, 1996) 28 28 Figure 1: Kernel density plots of the unconditioned vs. conditioned efﬁciencies, DEA Figure 1: Kernel density plots of the unconditioned vs. conditioned efﬁciencies, DEA 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency a) Unconditioned vs. ownership-conditioned 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency b) Unconditioned vs. size-conditioned 0.0 0.5 1.0 1.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency c) Unconditioned vs. geography-conditioned 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency d) Unconditioned vs. References clusters-conditioned Unconditioned ——— Conditioned –––––– Notes: All ﬁgures contain densities estimated using kernel smoothing for unconditioned and conditioned DEA efﬁciency scores. The vertical lines in each plot represent the average for all series, since we divide by the corresponding (group) mean. A Gaussian kernel and the Sheather and Jones (1991) plug-in bandwidth were chosen. 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency a) Unconditioned vs. ownership-conditioned ownership-conditioned 0.0 0.5 1.0 1.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Density Relative efﬁciency Density Density Notes: All ﬁgures contain densities estimated using kernel smoothing for unconditioned and conditioned DEA efﬁciency scores. The vertical lines in each plot represent the average for all series, since we divide by the corresponding (group) mean. A Gaussian kernel and the Sheather and Jones (1991) plug-in bandwidth were chosen. 29
https://openalex.org/W2905169175	https://europepmc.org/articles/pmc6311726?pdf=render	English	null	Antifibrotic Effect of Marine Ovothiol in an <i>In Vivo</i> Model of Liver Fibrosis	Oxidative medicine and cellular longevity	2,018	cc-by	8,631	Mariarita Brancaccio,1 Giuseppe D’Argenio,2,3 Vincenzo Lembo ,2 Anna Palumbo ,1 and Immacolata Castellano 1 1Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy 2Gastroenterology Unit, Department of Clinical Medicine and Surgery, School of Medicine, Federico II University, Naples, Italy 3IBAF Institute, National Research Council (CNR), via P. Castellino, Naples, Italy Correspondence should be addressed to Immacolata Castellano; immacolata.castellano@szn.it Correspondence should be addressed to Immacolata Castellano; immacolata.castellano@szn.it Received 5 June 2018; Accepted 18 September 2018; Published 17 December 2018 Guest Editor: Anna M. Giudetti Copyright © 2018 Mariarita Brancaccio 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. Liver ﬁbrosis is a complex process caused by chronic hepatic injury, which leads to an excessive increase in extracellular matrix protein accumulation and ﬁbrogenesis. Several natural products, including sulfur-containing compounds, have been investigated for their antiﬁbrotic eﬀects; however, the molecular mechanisms underpinning their action are partially still obscure. In this study, we have investigated for the ﬁrst time the eﬀect of ovothiol A, π-methyl-5-thiohistidine, isolated from sea urchin eggs on an in vivo murine model of liver ﬁbrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver ﬁbrosis and treated with ovothiol A at the dose of 50 mg/kg 3 times a week for 2 months. Treatment with ovothiol A caused a signiﬁcant reduction of collagen ﬁbers as observed by histopathological changes and serum parameters compared to mice treated with control solution. This antiﬁbrotic eﬀect was associated to the decrease of ﬁbrogenic markers involved in liver ﬁbrosis progression, such as the transforming growth factor (TGF-β), the α-smooth muscle actin (α-SMA), and the tissue metalloproteinases inhibitor (TIMP-1). Finally, we provided evidence that the attenuation of liver ﬁbrosis by ovothiol A treatment can be regulated by the expression and activity of the membrane-bound γ-glutamyl-transpeptidase (GGT), which is a key player in maintaining intracellular redox homoeostasis. Overall, these ﬁndings indicate that ovothiol A has signiﬁcant antiﬁbrotic properties and can be considered as a new marine drug or dietary supplement in potential therapeutic strategies for the treatment of liver ﬁbrosis. Hindawi Oxidative Medicine and Cellular Longevity Volume 2018, Article ID 5045734, 10 pages https://doi.org/10.1155/2018/5045734 Hindawi Oxidative Medicine and Cellular Longevity Volume 2018, Article ID 5045734, 10 pages https://doi.org/10.1155/2018/5045734 2. Methods 2.1. Experimental Model of Progressive Fibrosis and Animal Treatment. Male balb-c albino mice (20–25 g) were housed in a room at a mean constant temperature of 22°C with a 12 h light–dark cycle and free access to standard pellet chow and water. The study was approved by Federico II University School of Veterinary Medicine Animal Care N° 104/2015-PR. Liver ﬁbrosis was induced in mice by intraperitoneal (ip) injection of carbon tetrachloride (CCl4) 0.2 mL/100 g body weight (b.w.) in reﬁned olive oil (1 : 1) twice a week for 7 weeks according to a well-established protocol [32]. Two experimental groups were designed as follows: (1) mice receiving CCl4 and ip injection of the disulﬁde form of ovothiol A 50 μg/g b.w. 3 times a week for 7 weeks (n = 7); (2) control group receiving CCl4 and ip injection of vehicle (aqueous solution) alone (n = 7) with the same timing. Ovothiol A was prepared as described by Russo et al., 2014 [31]. The dose of administration was chosen on the basis of a previous work demonstrating that at this posology an ovothiol analogue induced no toxicity in not injured mice and neuroprotection in mice aﬀected by brain injury [29]. A group of 5 normal mice was also included in the study. The animals were then killed under anaesthesia, and their livers were harvested at peak ﬁbrosis (3 days after the ﬁnal injection of CCl4). After harvesting, livers were divided with a minimum of two lobes ﬁxed in formalin for histologic anal- ysis and histochemistry, and the remaining tissue was snap- y y g [ ] Several studies demonstrated the eﬃcacy of diﬀerent nat- ural products and phytochemicals present in foods and used as food extracts (such as sulforaphane, S-allylcysteine, curcu- min, proanthocyanidins, garlic extract, coﬀee, and grape skin or seeds) to prevent or reduce liver ﬁbrosis progression by diﬀerent mechanisms in several animal models [10, 13–17]. However, despite the striking progress in understanding the molecular mechanisms involved in liver ﬁbrosis and cirrho- sis, the antiﬁbrotic therapies are still lacking. In this context, methyl-5-thiohistidines, also called ovothiols, isolated in huge amounts from the eggs of marine invertebrate species, represent promising bioactive compounds [18–20]. First of all, they display unusual antioxidant properties due to the peculiar position of the thiol group on the imidazole ring of histidine [21, 22]. Oxidative Medicine and Cellular Longevity 2 induction of inﬂammatory cytokines, and proliferation of nonparenchymal cells producing ECM, mainly hepatic stel- late cells (HSCs) [5]. Active HSCs are characterized by increased proliferation, migration, and contractility, and a relative resistance to apoptosis. At the molecular level, they show increased expression of α-smooth muscle actin (α-SMA) and procollagen-I; both associated with the ability of the activated HSCs to depose collagens and other matrix proteins in the extracellular space [6]. Indeed, activated HSCs present an altered regulation of matrix remodeling enzymes, such as metalloproteinases (MMPs) and their tis- sue inhibitors (TIMPs), modulating matrix degradation and production, respectively [7]. Another key player in ﬁbrosis development is the pleiotropic cytokine TGF-β1, which is secreted in the latent form and when active, induces the activation of HSCs and modulates the expres- sion and secretion of a number of proteases and their reg- ulators, including MMPs and TIMPs [8]. TGF-β1 can also auto-induce its own production thus subsequently amplify- ing its actions [9, 10]. recently identiﬁed in the green microalga Euglena gracilis, which is a rich source of vitamins and antioxidants [28]. Interestingly, the biosynthetic pathway leading to ovothiols lacks in vertebrates [18]; therefore, mammals do not produce autonomously these molecules. Up to date, a very few studies have focused on the biological activities of ovothiols in human model systems. Several years ago, a synthetic ana- logue of ovothiol was reported as a neuroprotective agent in a murine model of brain injury [29]. Recently, we have claimed the nutraceutical and/or pharmaceutical use of marine ovothiol to relieve pathologies associated with chronic endothelial dysfunction, such as diabetes [30], and suggested a role of the molecule in regulating cell prolifera- tion in human liver tumor cells [31]. In particular, ovothiol A, puriﬁed by sea urchin eggs, was shown to induce autoph- agy in human hepatocarcinoma cell lines. The aim of this study was to evaluate in depth the eﬀect of ovothiol A in an in vivo model of chronic liver inﬂammation, which can silently progress, leading to cirrhosis and eventu- ally to hepatocarcinoma, which is one of the most aggressive tumors with a very poor prognosis. We used a murine model of liver ﬁbrosis in order to test if the administration of ovothiol A in its disulﬁde form could induce the recovery of liver functionality. Oxidative Medicine and Cellular Longevity In order to deﬁne the molecular mech- anism underpinning this process, we tested the gene and pro- tein expression of diﬀerent ﬁbrogenic markers (α-SMA, TGF-β1, and TIMP1) and the activity of the mature membrane-bound GGT form. Overall, our experiments point to evaluate the eﬃcacy of ovothiol A as a novel anti- inﬂammatory and antiﬁbrotic bioactive molecule. g Moreover, liver functionality is ﬁnely regulated by glu- tathione (GSH) levels, the most abundant cellular thiol in the cells. GSH is synthesized inside the cell and partially secreted in the extracellular space along a concentration gradient. In the extracellular space, GSH is hydrolyzed by γ-glutamyl-transpeptidase (GGT), a dimeric enzyme located on the membrane surface, and highly expressed in the liver and kidney [11]. This enzyme is therefore involved in GSH metabolism, amino acids recycling, and detoxiﬁcation mech- anisms. In detail, it catalyzes the hydrolysis of the γ-glutamyl bond in GSH and the transfer of the γ-glutamyl group to amino acids and small peptides. It often catalyzes the γ-glu- tamylation of the administered drugs, allowing the liver and the kidney to detoxify the organism [11, 12]. 1. Introduction marine natural products have inspired several approved pharmaceutical products, which are now in clinical use and/or in various stages of clinical development [2]. In the last decades, the need to face complex challenges, i.e., the supply of sustainable food, human health, and aging pop- ulation has stimulated research eﬀorts to discover new active compounds from natural sources. In particular, the need to develop more eﬃcient products and beneﬁts for mankind, in order to treat or prevent many human disorders and to overcome the side eﬀects of most of the approved drugs, has stimulated a great interest from pharmaceutical and nutraceutical industrial ﬁeld to search for new natural prod- ucts from less explored sources. In the last years, growing attention has been focused on the ocean characterized by a higher biodiversity compared to the earth [1]. In particular, g Liver ﬁbrosis represents a worldwide health problem for its growing incidence and prevalence, and its evolution towards cirrhosis, which is associated with high morbidity and mortality. Thus, there is an urgent need to develop anti- ﬁbrotic treatments that can prevent, halt, or even reverse liver ﬁbrosis or cirrhosis [3]. Liver ﬁbrosis results from chronic liver injury during a long-term wound-healing response, which causes increasing excessive accumulation of extracel- lular matrix (ECM) proteins, leading to ﬁbrogenesis and later cirrhosis [4]. It represents a complex process that includes apoptosis of hepatocytes, inﬁltration of inﬂammatory cells, Oxidative Medicine and Cellular Longevity 2. Methods All histologi- cal analyses were performed by an experienced histopa- thologist in a blinded manner. For each mouse, 64 ﬁelds of a constant raster of 31mm2 were analyzed at 100-fold ﬁnal magniﬁcation. For semiautomated morphometry, a Sony 3CCD (model DXC-950P) video microscope equipped with a motor stage and the Quantimed 500MC (Leica, Germany) software were used. Serum aspartate aminotrans- ferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels were determined to assess liver function by using standard laboratory techniques and equip- ment (Roche Diagnostics, Germany). 2.3. RNA Extraction and cDNA Synthesis. Total RNA was extracted from frozen tissue by homogenization in Trizol Reagent according to the manufacturer’s protocol (Life Tech- nologies). The amount of total RNA extracted was estimated measuring the absorbance at 260 nm and the purity by 260/ 280 and 260/230 nm ratios by Nanodrop (ND-1000 UV-Vis Spectrophotometer; NanoDrop Technologies). The integrity of RNA was evaluated by agarose gel electrophoresis. For each sample, 1200 ng of total RNA extracted was retro- transcribed with iScriptTM cDNA synthesis kit (Bio-Rad), following the manufacturer’s instructions. 2.5. Protein Analysis. Liver samples, about 20/30mg, were homogenized in 1 mL of RIPA lysis buﬀer (1X) containing a 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 0.5% Sodium deoxycholate, 10% SDS, phospha- tase, and protease inhibitor cocktail (Roche). Liver homoge- nates were run on 12% SDS/polyacrylamide gel according to Laemmli. Following electrophoresis, proteins were trans- ferred onto a PVDF (Millipore) membrane (Bio-Rad Trans- Blot Apparatus) and detected using a mouse anti-TGF-β polyclonal antibody (Sigma-Aldrich, USA), mouse anti- GGT monoclonal antibody (Santa Cruz Biotechnology, USA), rabbit anti-α-SMA monoclonal antibody (Santa Cruz Biotechnology, USA), rabbit TIMP-1 polyclonal antibody (Elabscience), and as an internal control mouse anti- GAPDH monoclonal antibody (Santa Cruz Biotechnology, USA). All primary antibodies were incubated at 4°C over- night. The appropriate secondary antibody was added, and immunoreactive proteins were detected using the ECL (Wes- ternBrightTM detection kit ECL, Advansta, USA) according to the manufacturer’s instructions. Protein expression levels were analyzed by means of densitometric analysis using the Image J software. 2.4. Gene Expression by Real-Time qPCR. For real-time qPCR experiments, the data from each cDNA sample were normalized using the mouse housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). In the case of GAPDH, TGF-β, α-SMA, Col1a1, and GGT-1, the speciﬁc primers were designed based on the nucleotide sequences downloaded by NCBI database (accession numbers) using Primer3WEB v.4.0.0. 2. Methods Thanks to their chemical properties, ovothiols can recycle oxidized GSH and play a key role in controlling the cellular redox balance [23, 24]. These mole- cules were found in diﬀerent methylated forms at the amino group of the lateral chain of histidine in several marine inver- tebrates [19, 20, 25–27]. For example, ovothiol A, unmethy- lated on the lateral chain, was isolated from the eggs of the sea urchin Paracentrotus lividus [25] and the sea cucumber Holothuria tubulosa [26]. Ovothiol B, mono-methylated on the lateral chain of histidine, was found in the ovaries of the scallop Chlamys hastata [21]. Ovothiol A has also been Oxidative Medicine and Cellular Longevity 3 frozen for RNA and protein extraction. Serum was also col- lected from each mouse to analyze biochemical parameters. cycle for cDNA denaturation; 95°C for 15sec and 60°C for 1 min, 40 cycles for ampliﬁcation; 72°C for 5 min, one cycle for ﬁnal elongation; and one cycle for melting curve analysis (from 60°C to 95°C) to verify the presence of a single product. Each assay included a no-template control for each primer pair. Speciﬁcity of ampliﬁcation reactions was veriﬁed by melting curve analysis. The eﬃciency of each primer pair was calculated according to standard method curves using the equation E =10–1/slope. Five serial dilutions were set up to determine Ct values and reaction eﬃciencies for all primer pairs. Standard curves were generated for each oligonucleo- tide pair using the Ct values versus the logarithm of each dilution factor, and PCR ampliﬁcations were performed in a ViiATM 7 real-time PCR system (Applied Biosystems) ther- mal cycler using the standard protocol previously reported. To capture intra-assay variability, all real-time qPCR reac- tions were carried out in triplicate. Fluorescence was mea- sured using ViiATM 7 Software (Applied Biosystems). The expression of the genes was analyzed and internally normal- ized against GAPDH using relative expression software tool (REST) software based on the Pfaﬄmethod (2002). Relative expression ratios above two cycles were considered signiﬁ- cant. Experiments were repeated at least three times. 2.2. Histology and Determination of Serum Biochemical Parameters. Formalin-ﬁxed paraﬃn-embedded tissue was cut into 4 μm sections by using routine techniques and mounted onto slides with coverslips. Representative sec- tions of each ﬁxed liver were stained with haematoxylin/ eosin (H&E) for routinely observations. For the detection of collagen content, sections were stained with Sirius red/ Fast green, according to standard protocols. 2. Methods (A) (C) (E) (B) (D) (F) (a) 250 Mice (treatment) 200 150 100 UI 50 0 AST ⁎⁎ ⁎ ALT # ## NT Vehicle + CCI4 Ovo + CCI4 ALP (b) Mice (treatment) (b) (a) Figure 1: (a) Histological analysis. H&E staining and sirius red dye to highlight the collagen ﬁbers of liver section: (A, B) healthy hepatic tissue; (C, D) ﬁbrotic liver tissue induced by CCl4; (E, F) liver tissue with hepatic ﬁbrosis treated with ovothiol A. (b) Evaluation of serum levels of liver enzymes. The levels of AST, ALT, and ALP were determined in the serum from mice aﬀected by liver ﬁbrosis and treated with ovothiol A or control solution. Data are expressed as mean ± SD, n = 7. The signiﬁcance was determined by the ANOVA and post hoc analysis: (∗p < 0 05) and (∗∗p < 0 01) represent signiﬁcance compared to vehicle + CCl4; (#p < 0 05) and (##p < 0 01) compared to nontreated (NT) healthy mice. ANOVA followed by Bonferroni or Tukey’s multiple com- parison tests. Values of p < 0 05 were considered signiﬁcant. determined by a colorimetric test. The assay buﬀer contains 100 mM Tris-HCl pH7.8 or PBS 1X pH 7.4. Each reaction contains 1 mM of γ-glutamyl-para-nitroanilide as a donor substrate and 40 mM glycylglycine as an acceptor substrate. The product formation, p-nitroaniline, was continuously monitored at room temperature at A405 nm using a Bio- Rad 680 microplate reader with Microplate Manager 5.2 (Bio-Rad) software. One unit of GGT activity was deﬁned as the amount of GGT that released 1 μmol of p-nitroani- line/min at room temperature. determined by a colorimetric test. The assay buﬀer contains 100 mM Tris-HCl pH7.8 or PBS 1X pH 7.4. Each reaction contains 1 mM of γ-glutamyl-para-nitroanilide as a donor substrate and 40 mM glycylglycine as an acceptor substrate. The product formation, p-nitroaniline, was continuously monitored at room temperature at A405 nm using a Bio- Rad 680 microplate reader with Microplate Manager 5.2 (Bio-Rad) software. One unit of GGT activity was deﬁned as the amount of GGT that released 1 μmol of p-nitroani- line/min at room temperature. 2. Methods GAPDH primer forward 5′-GGTG AAGGTCGGTGTGAACG-3′, primer reverse 5′- CTCGCT CCTGGAAGATGGTG-3′; TGF-β primer forward 5′- TGC GCTTGCAGAGATTAAAA-3′, primer reverse 5′-CTGCC GTACAACTCCAGTGA-3′; α-SMA primer forward 5′-CT GACAGAGGCACCACTGAA-3′, primer reverse 5′-CATC TCCAGAGTCCAGCACA-3′; Col1a1 primer forward 5′-A CAGTCGCTTCACCTACAGC-3′, primer reverse 5′-TGG GGTGGAGGGAG;TTTACA-3′ GGT-1 primer forward 5′- TGCTCGGTGACCCAAAGTTT-3′, primer reverse 5′-TT CAGAGGATGGCAGTGCTG-3′. A ﬁnal concentration of 1.4 pmol/μL for each primer and 1 FastStart SYBR Green Master Mix (total volume of 10 μL) were used for the reac- tion mix. PCR ampliﬁcations were performed in a ViiATM 7 real-time PCR system (Applied Biosystems) thermal cycler using the following thermal proﬁle: 95°C for 10min, one 2.6. Enzyme Isolation—GGT Activity. Tissues were homog- enized with Potter-Elvehjem tissue homogenizer at 4°C in 5 volumes of 25 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, containing 0.33M sucrose, 1 μM leupeptin, and 1.4 μg/mL aprotinin [33]. The homogenate was centrifuged at 9000 × g for 20 min; the supernatant was spun at 100,000 × g for 1 h to spin down nuclei, mitochondria, and cellular debris. The pellet was homogenized in 25 mM Tris-HCl, pH7.35, 0.5% Triton X-100, 1 μM leupeptin, 1.4 μg/mL aprotinin, and then centrifuged again at 100,000 × g for 1 h. The super- natant was aliquoted and stored at −80°C and then assayed for GGT protein expression and activity. GGT activity was 4 Oxidative Medicine and Cellular Longevity (A) (C) (E) (B) (D) (F) (a) 250 Mice (treatment) 200 150 100 UI 50 0 AST ⁎⁎ ⁎ ALT # ## NT Vehicle + CCI4 Ovo + CCI4 ALP (b) Figure 1: (a) Histological analysis. H&E staining and sirius red dye to highlight the collagen ﬁbers of liver section: (A, B) healthy hepatic tissue; (C, D) ﬁbrotic liver tissue induced by CCl4; (E, F) liver tissue with hepatic ﬁbrosis treated with ovothiol A. (b) Evaluation of serum levels of liver enzymes. The levels of AST, ALT, and ALP were determined in the serum from mice aﬀected by liver ﬁbrosis and treated with ovothiol A or control solution. Data are expressed as mean ± SD, n = 7. The signiﬁcance was determined by the ANOVA and post hoc analysis: (∗p < 0 05) and (∗∗p < 0 01) represent signiﬁcance compared to vehicle + CCl4; (#p < 0 05) and (##p < 0 01) compared to nontreated (NT) healthy mice. 4. Discussion 3.2. Eﬀect of Ovothiol on Gene Expression of Biomarkers of Liver Fibrosis. To evaluate the transcript regulation of speciﬁc liver ﬁbrotic markers, gene expression analysis was carried out by real-time qPCR on TGF-β, α-SMA, GGT, and ﬁbrillar type collagen 1 (Col1a1). In the last decade, studies on the isolation and struc- tural characterization of ovothiols have attracted the attention of many scientists [19, 20]. These molecules are sulfur-containing compounds derived from histidine, which, despite the relative structural simplicity, are likely involved in diﬀerent biological processes in marine organisms [19, 20]. Of particular interest is the ﬁnding that vertebrates lack the biosynthetic pathway leading to ovothiol [18], thus envisag- ing new perspectives on the possible pharmacological appli- cations of the molecule in humans [20, 34]. On this basis, great eﬀorts have been devoted to the chemical synthesis of this class of compounds, lately leading to ovothiols [35, 36] or to 5-thiohistidine, the precursor of ovothiols, unmethy- lated at the imidazole ring [37]. However, the described chemical procedures are somehow cumbersome and not environmentally friendly; thus, the need to develop an eco- sustainable production of the molecule [20]. A signiﬁcant increase in gene expression for TGF-β, α- SMA, and Col1a1 was observed in mice aﬀected by liver ﬁbrosis and treated with control solution compared to healthy mice (Figure 2), whereas no signiﬁcant variation was shown for GGT expression levels. On the other hand, samples from mice treated with ovothiol A showed a sig- niﬁcant downregulation of mRNA of the TGF-β and α- SMA, whereas the gene expression of Col1a1 and GGT were not aﬀected. 3.3. Eﬀect of Ovothiol on Protein Expression of Key Players in Liver Fibrosis Progression. The protein expression of the hepatic ﬁbrogenic markers, TGF-β, α-SMA, and TIMP1 was evaluated by Western blot analysis (Figure 3(a)). In mice aﬀected by liver ﬁbrosis, the levels of TGF-β, α-SMA, and TIMP1 signiﬁcantly increased compared to healthy mice. After treatment with ovothiol A, the protein expression of TGF-β, α-SMA, and TIMP1 signiﬁcantly decreased com- pared to mice with hepatic ﬁbrosis (Figures 3(a)–3(d)). Previous studies showed that a synthetic ovothiol ana- logue exhibited neuroprotective activity in an in vivo model of brain injury [29], whereas the natural molecule ovothiol A in disulﬁde form was tested only in in vitro models, show- ing anti-inﬂammatory activity in human endothelial cells [30] and antiproliferative eﬀect on human hepatic carcinoma cell lines [31]. 3. Results As appropriate, comparisons among groups were made by Student’s t-test or analysis of variance Oxidative Medicine and Cellular Longevity 5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Tgf-훽 훼-SMA Col1a1 GGT-1 −0.5 −1.0 Expression ratio [2-log] −1.5 −2.0 −2.5 −3.0 Vehicle + CCI4 Ovo + CCI4 ⁎⁎⁎ ⁎⁎⁎ 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Tgf-훽 훼-SMA Col1a1 GGT-1 −0.5 −1.0 Expression ratio [2-log] −1.5 −2.0 −2.5 −3.0 Vehicle + CCI4 Ovo + CCI4 ⁎⁎⁎ ⁎⁎⁎ signiﬁcantly decreased to 36%. Conversely, the treatment with ovothiol A induced an increase in mature protein up to 44% of the total content of GGT (Figures 4(a) and 4(b)). The presence of GGT was also evaluated in mice serum. In all three groups of mice, the serum contained only one band recognized by GGT antibody corresponding to the size of the large subunit of the mature form (Figure 4(c)). 3.4. Ovothiol Aﬀects Membrane-Bound GGT Activity. GGT activity was determined in liver microsomal extracts and nor- malized against the amount of the mature protein, which represents the active form of the enzyme, compared to the inactive precursor polypeptide [11, 12]. GGT activity signiﬁ- cantly increased in ﬁbrotic tissues compared to healthy mice (Figure 5(a)). Treatment with ovothiol A, on the other hand, induced a signiﬁcant reduction in GGT activity. Since GGT activity is closely related to glutathione metabolism, we also evaluated total glutathione levels in the hepatic tissues. As shown in Figure 5(b), total glutathione levels increased in tis- sues from mice aﬀected by liver ﬁbrosis and treated with vehicle solution and decreased in liver tissues from mice treated with ovothiol A. Figure 2: Gene expression analysis of markers of liver ﬁbrosis by real-time qPCR. The levels of gene expression of the ﬁbrotic markers in tissues after treatment with ovothiol A or control solution were compared to tissues from healthy mice (reference baseline). Data were analyzed through the REST software, which considers fold diﬀerences ≥+/−2 to be signiﬁcant. (∗∗∗p < 0 001) represents the signiﬁcance compared to the treated mice with vehicle + CCl4. 3. Results 3.1. Eﬀect of Ovothiol on Liver Histology and Serum Biochemical Parameters. In order to evaluate the eﬀect of ovothiol A on relieving induced liver ﬁbrosis, the histology and functionality of the liver were examined in injured mice treated and not treated with ovothiol compared to control animals. Histological analysis of hepatic tissue of mice treated with CCl4 for 7 weeks compared with healthy mice tissue conﬁrmed that the injection of CCl4 caused the pro- gression of hepatic ﬁbrosis, as demonstrated by the increase of red colored collagen ﬁbers showing established septa linking hepatic veins (see Figure 1(a), C–D compared to A and B). Ovothiol A treatment signiﬁcantly reduced hepatic ﬁbrosis as shown by the reduction of red colored collagen ﬁbers (Figure 1(a), E–F compared to C and D). The amount of collagen was reduced in ﬁbrotic mice treated with ovothiol compared to those treated with vehicle alone (2.7 ± 0.9% vs 5.8 ±1.2%, p < 0 05). 2.7. Glutathione Assay. Total glutathione levels were deter- mined by Glutathione Assay Kit (Sigma). Brieﬂy, frozen liver tissues were ground with a pestle with a mortar in the presence of liquid nitrogen to prepare a ﬁne powder. Then, 100 mg of powder was added to 3 volumes of 5% 5- sulfosalicylic acid and mixed. Then, other 7 volumes of 5% 5-sulfosalicylic acid were added, mixed, left for 5 min at 4°C, and ﬁnally centrifuged at 10,000 × g for 10 min. Diluted samples of the supernatants were used for the assay proce- dure, in which following the incubation with glutathione reductase and NADPH, glutathione was totally recovered in the reduced form and thus determined by monitoring the reduction of 5,5-dithiobis (2-nitrobenzoic acid) to 5-thio-2- nitrobenzoic at 412 nm by a Thermo Scientiﬁc™Multiskan™ FC Microplate Photometer. Serum levels of liver enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phos- phatase (ALP) were assayed to evaluate liver functionality. Levels of AST and ALT increased in mice aﬀected by hepatic ﬁbrosis, whereas the treatment with ovothiol A led to a signif- icant reduction in AST and ALT levels (Figure 1(b)). Con- versely, serum ALP levels in mice with hepatic ﬁbrosis did not signiﬁcantly diﬀer among the treatment groups. 2.8. Statistical Analysis. 4. Discussion (#p < 0 05) and (##p < 0 01) represent signiﬁcance compared to NT; (∗∗p < 0 01) represents signiﬁcance compared to the treated with vehicle + CCl4. GAPDH 35 kDa Vehicle Ovo NT CCI4 TGF훽 48 kDa 훼-SMA 42 kDa TIMP-1 24 kDa (a) Vehicle + CCI4 Ovo + CCI4 NT ⁎⁎ 4 3 TGF-훽 protein expression 2 ## # 1 0 (b) (b) (a) Vehicle + CCI4 Ovo + CCI4 NT 4 # ⁎⁎ 3 훼-SMA protein expression 2 1 0 (c) Vehicle + CCI4 Ovo + CCI4 NT ## ⁎⁎ # 4 5 3 TIMP-1 protein expression 2 1 0 (d) (c) (d) Figure 3: Protein expression of liver ﬁbrosis markers. (a) A representative experiment of Western blot analysis of cytosolic extracts obtained from hepatic tissues of mice treated with ovothiol A or vehicle, after induction of liver ﬁbrosis, compared to samples of healthy mice (NT), using antibodies speciﬁc for TGF-β, α-SMA, and TIMP1. Histograms of the densitometry analysis of protein bands obtained by Western blot for liver markers: (b) TGF-β; (c) α-SMA; and (d) TIMP1. Data were normalized for GAPDH. Data are expressed as mean ± SD, n = 7. The signiﬁcance was determined by ANOVA test. (#p < 0 05) and (##p < 0 01) represent signiﬁcance compared to NT; (∗∗p < 0 01) represents signiﬁcance compared to the treated with vehicle + CCl4. Vehicle Ovo NT GGT 64 kDa 50 kDa GAPDH 35 kDa CCI4 (a) 2 ⁎ ### ### ### # 1 0 GGT protein expression ratio 64 kDa 50 kDa 64 kDa 50 kDa 64 kDa 50 kDa NT Ovo + CCI4 Vehicle + CCI4 (b) GGT 50 kDa Vehicle Ovo NT CCI4 (c) Figure 4: Protein expression of GGT. (a) Representative Western blot performed with GGT-speciﬁc antibody using microsomal extracts of hepatic tissues of mice with liver ﬁbrosis treated with ovothiol or control solution compared to nontreated healthy mice (NT). (b) Histogram of the densitometric analysis of the GGT bands at 64 and 50 kDa. Data were normalized for GAPDH. (c) Representative Western blot performed on mice serum using GGT-speciﬁc antibody. Data are expressed as mean ± SD, n = 6. 4. Discussion These pleiotropic behaviors have been ascribed to diﬀerent mechanisms of action of these molecules in dif- ferent cellular contexts [20]. g The presence of GGT in the liver tissue was also evaluated by immunoblot of microsomal extracts containing membrane- bound GGT. GGT is synthesized as a single 64kDa precursor polypeptide, which undergoes self-proteolysis to form the mature protein composed of two subunits, the largest of which is around 50 kDa (Figure 4(a)). The antibody used in this study is directed against a peptide contained in the major subunit; therefore, it is able to recognize both the large subunit and the precursor. In the liver microsomal extracts of healthy mice, 62% of the total GGT is present in the mature form, while in the ﬁbrotic tissues, the mature form In this study, to deepen the anti-inﬂammatory properties of this class of molecules, we have evaluated the eﬀect of ovothiol A on an in vivo model of liver ﬁbrosis, which is a condition common to many chronic liver diseases, character- ized by the presence of parenchymal damage [4, 5]. The chronic activation of the tissue repair response which leads to liver ﬁbrosis is characterized by a signiﬁcant accumulation of ECM and numerous proﬁbrogenic markers, which is 6 Oxidative Medicine and Cellular Longevity GAPDH 35 kDa Vehicle Ovo NT CCI4 TGF훽 48 kDa 훼-SMA 42 kDa TIMP-1 24 kDa (a) Vehicle + CCI4 Ovo + CCI4 NT ⁎⁎ 4 3 TGF-훽 protein expression 2 ## # 1 0 (b) Vehicle + CCI4 Ovo + CCI4 NT 4 # ⁎⁎ 3 훼-SMA protein expression 2 1 0 (c) Vehicle + CCI4 Ovo + CCI4 NT ## ⁎⁎ # 4 5 3 TIMP-1 protein expression 2 1 0 (d) Figure 3: Protein expression of liver ﬁbrosis markers. (a) A representative experiment of Western blot analysis of cytosolic extracts obtained from hepatic tissues of mice treated with ovothiol A or vehicle, after induction of liver ﬁbrosis, compared to samples of healthy mice (NT), using antibodies speciﬁc for TGF-β, α-SMA, and TIMP1. Histograms of the densitometry analysis of protein bands obtained by Western blot for liver markers: (b) TGF-β; (c) α-SMA; and (d) TIMP1. Data were normalized for GAPDH. Data are expressed as mean ± SD, n = 7. The signiﬁcance was determined by ANOVA test. 4. Discussion Unlike ovothiol, the sulfur of ergothioneine is localized on position 2 of the imidazole ring of histidine, making it stable in its thionic form. In our study, the methyl-5-thiohistidine (ovothiol A) isolated from sea urchin eggs was administered as disulﬁde because the reduced form is unstable and very reactive and can be presumably generated in the cell by recy- cling mechanisms [29]. In the disulﬁde form, ovothiol A inhibited the onset and/or progression of hepatic ﬁbrosis, highlighting the antiﬁbrotic properties for this type of mole- cules. Our ﬁndings clearly indicate that ovothiol A induced a signiﬁcant reduction in the accumulation of collagen ﬁbers in injured hepatic tissues associated with the decrease of the serum levels of the liver enzymes AST and ALT (Figure 1). Indeed, ovothiol A activates ﬁbrinolytic processes on the extracellular ECM matrix through the downregulation of diﬀerent ﬁbrotic markers (Figure 2). In particular, TGF-β, the most important proﬁbrogenic cytokine, was found signif- icantly reduced by ovothiol A treatment also at the protein level (Figure 3). Since the activation of TGF-β is a fundamen- tal step for the activation of HSC and for hepatic ﬁbrogenesis, it is reasonable to infer that a decreased expression of TGF-β, mediated by ovothiol A, is consistent with a reduced amount of activated HSC, remodeling of extracellular matrix, and regeneration of liver function. The reduction of activated HSC after ovothiol A treatment was further conﬁrmed by the reduction of the α-SMA protein isoform, which generally increases in acute hepatopathies, and it is rarely detected in the normal liver. g p The analysis of protein expression and activity of GGT appeared to be more complex. Our ﬁnding indicates that liver ﬁbrosis is characterized by high levels of membrane- bound GGT activity (Figure 5(a)). The total content of GGT protein, consisting of the precursor and the mature form remained unchanged following ovothiol treatment (Figures 4(a) and 4(b)), according to the absence of GGT gene regulation (Figure 2). However, the amount of the membrane-bound mature form decreased in mice aﬀected by liver ﬁbrosis compared to control mice and was restored following ovothiol treatment (Figures 4(a) and 4(b)). The main function of GGT is to regulate the intracellular redox homeostasis by catalyzing the degradation of extracellular GSH and promoting thiols recycling within the cell [11, 12]. 4. Discussion Statistical signiﬁcance was determined by the one-way ANOVA test; (#p < 0 05) and (###p < 0 001) represent the signiﬁcance compared to the corresponding NT band; (∗p < 0 05) represents the signiﬁcance compared to ﬁbrotic mice treated with control solution. (c) (a) (b) Figure 4: Protein expression of GGT. (a) Representative Western blot performed with GGT-speciﬁc antibody using microsomal extracts of hepatic tissues of mice with liver ﬁbrosis treated with ovothiol or control solution compared to nontreated healthy mice (NT). (b) Histogram of the densitometric analysis of the GGT bands at 64 and 50 kDa. Data were normalized for GAPDH. (c) Representative Western blot performed on mice serum using GGT-speciﬁc antibody. Data are expressed as mean ± SD, n = 6. Statistical signiﬁcance was determined by the one-way ANOVA test; (#p < 0 05) and (###p < 0 001) represent the signiﬁcance compared to the corresponding NT band; (∗p < 0 05) represents the signiﬁcance compared to ﬁbrotic mice treated with control solution. responsible for the excessive deposition of collagen ﬁbers [6]. Regeneration from liver ﬁbrosis implies key processes, such as the eradication of pathological agents, apoptosis of HSC, remodeling of ECM, and regeneration of parenchyma and liver function. From the clinical perspective, ﬁbrinolytic therapies reverting advanced ﬁbrosis after the elimination of the causative agent represent a feasible challenge [3]. In fact, in clinical practice, the spontaneous recovery of liver his- tology, after the elimination of the agent causing chronic liver disease, usually occurs only in some patients and takes more 0.12 # 0.11 0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 GGT activity (U/mg protein) NT ⁎⁎ Ovo + CCI4 Vehicle + CCI4 (a) # 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000 nmol of total GSH/mg of tissue ⁎⁎ NT Ovo + CCI4 Vehicle + CCI4 (b) Figure 5: GGT activity and glutathione content. (a) The enzymatic activity of GGT was evaluated on liver tissue microsomal extracts containing membrane-bound GGT. The activity of GGT was normalized compared to the mature protein band (50 kDa) detected by Western blot. (b) The levels of glutathione were determined in hepatic tissue of mice treated with ovothiol A or vehicle, after induction of hepatic ﬁbrosis, compared to samples of healthy tissue mice (NT). Data are expressed as mean ± SD, n = 6. The bars indicated the mean of 7 measures +/−SD (standard deviation). 4. Discussion The signiﬁcance was determined by the ANOVA and post hoc analysis: (#p < 0 05) represents signiﬁcance compared to healthy control; (∗∗p < 0 01) represents signiﬁcance compared to mice treated with vehicle + CCl4. 7 Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity 7 # 0.0045 0.0040 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0.0000 ⁎⁎ NT Ovo + CCI4 Vehicle + CCI4 (b) 0.12 # 0.11 0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 GGT activity (U/mg protein) NT ⁎⁎ Ovo + CCI4 Vehicle + CCI4 (a) (b) (a) Figure 5: GGT activity and glutathione content. (a) The enzymatic activity of GGT was evaluated on liver tissue microsomal extracts containing membrane-bound GGT. The activity of GGT was normalized compared to the mature protein band (50 kDa) detected by Western blot. (b) The levels of glutathione were determined in hepatic tissue of mice treated with ovothiol A or vehicle, after induction of hepatic ﬁbrosis, compared to samples of healthy tissue mice (NT). Data are expressed as mean ± SD, n = 6. The bars indicated the mean of 7 measures +/−SD (standard deviation). The signiﬁcance was determined by the ANOVA and post hoc analysis: (#p < 0 05) represents signiﬁcance compared to healthy control; (∗∗p < 0 01) represents signiﬁcance compared to mice treated with vehicle + CCl4. responsible for the signiﬁcant changes that occur in the composition of the ECM following liver injury and conse- quent alteration of the liver tissue anatomy. The reduction of collagen ﬁbers observed at histological level is therefore likely caused by an indirect action exerted by ovothiol A on the metalloproteinases and on the inhibitors thereof, which are involved in the degradation of ECM and conse- quently in the reversion of the pathological condition. Indeed, we found an increased protein expression of the metalloproteinase inhibitor TIMP1 in ﬁbrotic tissues and a decreased expression after treatment with ovothiol A (Figure 3). Under these conditions, in fact, metalloproteases should be free to degrade collagen ﬁbers in the ECM, thus reducing the ﬁbrotic process. than 3 years. Several sulfur-containing compounds have been shown to induce the reversion of liver ﬁbrosis [10, 13, 14]. Recently, an ergothioneine-rich diet has been shown to ame- liorate liver ﬁbrosis [38]. Ergothioneine is a sulfur-containing histidine produced by some fungi and bacteria [39]. 4. Discussion The cysteinylglycine, resulting from glutathione hydro- lysis, is one of the most reactive thiol compounds able to reduce oxygen by a redox reaction with the iron ion, thus promoting the increase in reactive oxygen species (ROS) and oxidative reactions [40, 41]. Therefore, high levels of GGT activity may induce oxidative stress in the cell, thus contributing to the damage and development of liver ﬁbrosis. It is well known that chronic inﬂammation of the liver can cause damage to the membranes resulting in the release of GGT into the blood [42]. Indeed, GGT and in particular its On the other hand, our results demonstrate that the administration of ovothiol A had no eﬀects on the gene reg- ulation of Col1a1 (Figure 2), a known marker of activation of the ﬁbrogenic process, which encodes the alpha-1 subunit of ﬁbrillar type collagen [6]. The deposition of this protein is Oxidative Medicine and Cellular Longevity 8 Fibrosis GGT ROS Active TGF-훽 Activated HSC CCL4 Ovothiol A CH3 CH3 NH2 NH2 N N S S N N HO2C CO2H Fibrosis GGT ROS Active TGF-훽 Activated HSC TIMP-1 ECM ECM degradation Myofibroblasts 훼-SMA CCL4 Ovothiol A CH3 CH3 NH2 NH2 N N S S N N HO2C CO2H Figure 6: Proposed mechanism of action for ovothiol. During the development of liver ﬁbrosis, membrane-bound GGT activity increases, leading to ROS overproduction. ROS can activate TGF- β, which in turn upregulates α-SMA and TIMP1, favoring ECM deposition. Ovothiol acts as a GGT inhibitor and in turn reduces TGF-β activation, thus inducing a cascade of events leading to downregulation of proﬁbrogenic molecules and induction of ﬁbrolytic enzymes. high serum levels are well-known markers of liver damage pathologies. However, the role of GGT anchored to the outer membrane surface of liver cells is not yet clear. Here, we show that during the process of liver ﬁbrosis, the activity of the mature GGT form anchored to the membrane increases (see Figure 4), probably contributing to oxidative stress and membrane damage, which in turn causes the further release of the mature protein in the serum (Figure 4(c)). Treatment with ovothiol A in mice aﬀected by the ﬁbrotic process causes the reversion of this phenomenon, that is, the reduction of membrane GGT activity at the physiological levels of healthy mice. Abbreviations ECM: Extracellular matrix HSCs: Hepatic stellate cells α-SMA: α-Smooth muscle actin TGF-β: Transforming growth factor TIMP-1: Tissue metalloproteinases inhibitor GGT: γ-Glutamyl transpeptidase MMPs: Metalloproteinases AST: Serum aspartate aminotransferase ALT: Alanine aminotransferase ALP: Alkaline phosphatase GAPDH: Glyceraldehyde 3-phosphate dehydrogenase CCl4: Carbon tetrachloride Col1a1: Fibrillar type collagen 1 NT: Nontreated. Overall, these results suggest that ovothiols can be con- sidered a novel class of sulfur-containing molecules endowed with antiﬁbrotic properties with possible applications as drugs or food supplement for the treatment of chronic inﬂammation of the liver. This study also highlights the key 4. Discussion Indeed, by inhibiting GGT activity, ovothiol A should also reduce the amount of cysteinylglycine in the cell, thus avoiding the accumulation of ROS and oxidative damage. In support of this hypothesis, intracellular thiol levels increased in untreated ﬁbrotic tissues and decreased follow- ing ovothiol A treatment (Figure 5(b)). This may contribute to maintain membrane integrity and reduce the release of the mature membrane-bound GGT into the serum. Indeed, our data suggest that the mature form of GGT is mostly released in the blood during liver ﬁbrosis and that the treat- ment with ovothiol A presumably reduces this release, result- ing in new mature membrane-bound GGT (Figure 4(c)). Another possible explanation could be that the progression of liver ﬁbrosis negatively aﬀects the auto-catalytic cleavage of the GGT precursor, whereas ovothiol A treatment can reinduce the maturation process. The ﬁnding that only the mature form of GGT is released in the serum may depend on the adverse eﬀect of its own overexpression and activity or to a more pronounced anchorage to the membrane of its precursor form. Further studies will be needed to clarify these aspects. Ovothiol A Fibrosis Active TGF-훽 Activated HSC TIMP-1 ECM Myofibroblasts 훼-SMA Myofibroblasts 훼-SMA TIMP-1 ECM TIMP-1 ECM ECM degradation ECM degradation Myofibroblasts 훼-SMA Myofibroblasts 훼-SMA ECM degradation Figure 6: Proposed mechanism of action for ovothiol. During the development of liver ﬁbrosis, membrane-bound GGT activity increases, leading to ROS overproduction. ROS can activate TGF- β, which in turn upregulates α-SMA and TIMP1, favoring ECM deposition. Ovothiol acts as a GGT inhibitor and in turn reduces TGF-β activation, thus inducing a cascade of events leading to downregulation of proﬁbrogenic molecules and induction of ﬁbrolytic enzymes. 5. Conclusions involvement of sulfur groups in the anti-inﬂammatory prop- erties of natural products. Liver ﬁbrosis is known to persist for a long time even after successful pharmacological treatment of hepatitis; therefore, a ﬁbrinolytic therapy to rapidly reverse advanced ﬁbrosis/cir- rhosis would be more advisable. This study indicates that, in the experimental model, repeated cycles of hepatic damage have contributed to a signiﬁcant increase in deposition of col- lagen ﬁbers mediated by proﬁbrogenic cytokines and that the treatment with the disulﬁde form of ovothiol A has led to the reversion of this condition. In our model, ovothiol A inhibits GGT activity and aﬀects GSH metabolism. This could be the speciﬁc mechanism by which ovothiol negatively regulate redox homeostasis and the activation of key ﬁbrotic markers, TGF-β, α-SMA, and TIMP1, which ﬁnally leads to a signiﬁ- cant degradation of the collagen ﬁbers in the ECM (see scheme in Figure 6). To our knowledge, this is the ﬁrst study to highlight the involvement of the membrane-bound GGT form in the evolution of liver ﬁbrosis, thus pointing to this enzyme as a potential target of therapeutic strategies directed to ameliorate liver ﬁbrosis eﬀects. References [1] J. W. Blunt, A. R. Carroll, B. R. Copp, R. A. Davis, R. A. Keyzers, and M. R. Prinsep, “Marine natural products,” Nat- ural Product Reports, vol. 35, no. 1, pp. 8–53, 2018. [18] I. Castellano, O. Migliaccio, S. D’Aniello, A. Merlino, A. Napolitano, and A. Palumbo, “Shedding light on ovothiol biosynthesis in marine metazoans,” Scientiﬁc Reports, vol. 6, no. 1, article 21506, 2016. [2] K. H. Altmann, “Drugs from the oceans: marine natural products as leads for drug discovery,” CHIMIA International Journal for Chemistry, vol. 71, no. 10, pp. 646–652, 2017. [19] A. Palumbo, I. Castellano, and A. 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https://openalex.org/W4220798429	https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/278728/1/irv.12981.pdf	English	null	Analysis of a city‐wide COVID‐19 prevention strategy for aged‐care facilities during third and fifth waves of COVID‐19 in Kyoto City, Kyoto, Japan	Influenza and other respiratory viruses	2,022	cc-by	4,736	O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Received: 28 December 2021 Revised: 28 February 2022 Accepted: 1 March 2022 Received: 28 December 2021 Revised: 28 February 2022 Accepted: 1 March 2022 DOI: 10.1111/irv.12981 Abstract Results: Of the 1,144 facilities subjected to the mass testing, 71.0% participated in the whole program including active PCR testing. The remainder participated in the rest of the programs. The prevalence of ACF-related COVID-19 cases among total COVID-19 cases in Kyoto City decreased from 7.9% in the third wave to 4.1% in the Background: During the third wave of the COVID-19 pandemic at the end of 2020, clusters occurred frequently in aged-care facilities (ACFs), which put pressure on the medical field in Japan. Based on this experience, Kyoto University and Kyoto City collab- orated to promote a citywide COVID-19 prevention strategy to prevent the spread of COVID-19 within ACFs. The aim of this study was to clarify the effect of the prevention strategy among ACFs in Kyoto City during the third and fifth waves of the pandemic. Methods: During the study period, the following measures were adopted as the pre- vention strategy in all ACFs: (1) active polymerase chain reaction (PCR) mass testing and facility-wide testing when a single case was identified, (2) implementation of strat- egies to prevent transmission within a facility, and (3) vaccination program for ACFs. Results: Of the 1,144 facilities subjected to the mass testing, 71.0% participated in the whole program including active PCR testing. The remainder participated in the rest of the programs. The prevalence of ACF-related COVID-19 cases among total COVID-19 cases in Kyoto City decreased from 7.9% in the third wave to 4.1% in the fourth wave and 2.1% in the fifth wave. The incidence of clusters and proportion of severe elderly cases also decreased during the study period. Conclusions: A city-wide multidisciplinary effort including PCR mass testing and a vaccination program in cooperation with a university and local administrative office successfully reduced the clusters and transmission in ACFs in Kyoto City, Japan. 1 \| INTRODUCTION COVID-19 has had a disproportionate impact on vulnerable populations such as the elderly, particularly residents of aged-care facilities (ACFs).1–6 To date, a large amount of evidence has been accumulated on effective strategies to contain and prevent the spread of COVID-19 in ACFs.7–10 Assessing the real-world impact of infec- tion prevention measures in nursing homes is complicated but, at the 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. © 2022 The Authors. Analysis of a city-wide COVID-19 prevention strategy for aged-care facilities during third and fifth waves of COVID-19 in Kyoto City, Kyoto, Japan Miki Nagao1,2 \| Yasufumi Matsumura1,2 \| Masaki Yamamoto1,2 \| Koh Shinohara1,2 \| Satomi Yukawa1,2 \| Taro Noguchi1,2 \| Yasuhiro Tsuchido1,2 \| Takeshi Ikeda3 Miki Nagao1,2 \| Yasufumi Matsumura1,2 \| Masaki Yamamoto1,2 \| Koh Shinohara1,2 \| Satomi Yukawa1,2 \| Taro Noguchi1,2 \| Yasuhiro Tsuchido1,2 \| Takeshi Ikeda3 1Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan 2Department of Clinical Laboratory, Department of Infection Prevention, Kyoto University Hospital, Kyoto, Japan 3Public Health and Welfare Bureau of Kyoto City, Kyoto, Japan 1Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan 2Department of Clinical Laboratory, Department of Infection Prevention, Kyoto University Hospital, Kyoto, Japan 3Public Health and Welfare Bureau of Kyoto City, Kyoto, Japan Abstract Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd. 690 Influenza Other Respi Viruses. 2022;16:690–695. wileyonlinelibrary.com/journal/irv Abstract Abstract Background: During the third wave of the COVID-19 pandemic at the end of 2020, clusters occurred frequently in aged-care facilities (ACFs), which put pressure on the medical field in Japan. Based on this experience, Kyoto University and Kyoto City collab- orated to promote a citywide COVID-19 prevention strategy to prevent the spread of COVID-19 within ACFs. The aim of this study was to clarify the effect of the prevention strategy among ACFs in Kyoto City during the third and fifth waves of the pandemic. Methods: During the study period, the following measures were adopted as the pre- vention strategy in all ACFs: (1) active polymerase chain reaction (PCR) mass testing and facility-wide testing when a single case was identified, (2) implementation of strat- egies to prevent transmission within a facility, and (3) vaccination program for ACFs. Results: Of the 1,144 facilities subjected to the mass testing, 71.0% participated in the whole program including active PCR testing. The remainder participated in the rest of the programs. The prevalence of ACF-related COVID-19 cases among total COVID-19 cases in Kyoto City decreased from 7.9% in the third wave to 4.1% in the fourth wave and 2.1% in the fifth wave. The incidence of clusters and proportion of severe elderly cases also decreased during the study period. Conclusions: A city-wide multidisciplinary effort including PCR mass testing and a vaccination program in cooperation with a university and local administrative office successfully reduced the clusters and transmission in ACFs in Kyoto City, Japan. K E Y W O R D S COVID-19, elderly, infection prevention, mass testing, vaccination Abstract Background: During the third wave of the COVID-19 pandemic at the end of 2020, clusters occurred frequently in aged-care facilities (ACFs), which put pressure on the medical field in Japan. Based on this experience, Kyoto University and Kyoto City collab- orated to promote a citywide COVID-19 prevention strategy to prevent the spread of COVID-19 within ACFs. The aim of this study was to clarify the effect of the prevention strategy among ACFs in Kyoto City during the third and fifth waves of the pandemic. Methods: During the study period, the following measures were adopted as the pre- vention strategy in all ACFs: (1) active polymerase chain reaction (PCR) mass testing and facility-wide testing when a single case was identified, (2) implementation of strat- egies to prevent transmission within a facility, and (3) vaccination program for ACFs. 690 wileyonlinelibrary.com/journal/irv 1 \| INTRODUCTION Influenza Other Respi Viruses. 2022;16:690–695. 690 NAGAO ET AL. 691 same time, critical if the aim is to resume welfare systems and health care delivery systems as they were prior to the pandemic. program for the elderly began. Following the preliminary intervention with a small number of facilities during the third wave, the target facil- ities were expanded to include all ACFs (both residential facilities and day-care centers) in the city, and the following measures were adopted from the beginning of April (fourth wave): (1) mass testing; (2) implementation of code of conduct and infection prevention tech- niques involving staff, elderly, and their families to prevent infection both in the community and within the facilities; and (3) vaccination for the elderly and staff of ACFs, which was of the highest priority along with health care professionals in Japan. In the middle of the COVID-19 pandemic, ACFs accounted for a large proportion of total deaths attributed to the virus in Japan as well.1,11 In Japan, the Ministry of Health, Labour and Welfare has been taking the lead in issuing guidance for ACFs. However, during the third wave of the pandemic, COVID-19 outbreaks occurred fre- quently in medical institutions and ACFs, and there were also high incidences of morbidity and mortality among the elderly, which put pressure on the medical field in Japan. Based on this experience, the local government of Kyoto City (public health) and Kyoto University Hospital (KUH; a university hospital with infectious disease and clini- cal laboratory specialists) collaborated to promote a city-wide COVID- 19 prevention strategy to prevent the spread of COVID-19 within ACFs. The aim of this study was to clarify the effect of the strategy among ACFs during the third and fifth waves of the pandemic in Kyoto City, Kyoto Japan. (1) Mass testing for residents and staff and facility-wide testing strat- egies when a single case was identified Saliva samples for universal serial polymerase chain reaction (PCR) testing were submitted to KUH (in March and April) and a testing company (from April to September) weekly from the end of the third to end of the fifth waves. When a screening PCR test was positive (Step 1), a confirmation test was performed at KUH (Step 2) simulta- neously with facility-wide testing. 2.3.1 \| Prospective, observation study 2.3.1 \| Prospective, observation study Pfizer-BioNTech COVID-19 vaccines were distributed to elderly and staff of ACFs on a priority basis starting in the middle of April from residential-type facilities to day-care centers and including nursing helpers. 1 \| INTRODUCTION The reasons for adopting the two- step policy were as follows: A false-positive result is even more of an issue when the test is performed for asymptomatic individuals and the pre-test probability is low, as it was in this case. In addition, reports of notification must be submitted by a physician in Japan, but the testing company undertaking mass testing did not have such a policy. Eventu- ally, the mass testing ended when the facility finished vaccinating the residents and staff. 2.1 \| The subjects of the study Kyoto is a city of 1.45 million people, with 28.4% of the population aged 65 or over. It is an aging society where the aging rate has dou- bled in the past 5 years. The subjects of this study were users and staff of ACFs including both residential and day-care facilities for the elderly in Kyoto City, Kyoto, Japan. (2) Implementation of strategies to prevent transmission within a facility 2.2 \| Study period As a measure to prevent the introduction of the disease into the facil- ity, educational materials were distributed to facility staff, new users, and their families regarding the request to self-isolate for 2 weeks after using the facility, sick-leave policy, and daily health monitoring. In addition, we provided materials on the appropriate use of personal protective equipment and conducted a training session on infection control using Zoom as an enhanced education program. During the third wave, we realized that there had not been enough guidance to meet the level of infection control at ACFs. As such, in order to fit the actual ACF settings, we modified the manuals and education materials that were used in KUH and distributed them to each facility. The study period was from the third (2020/12/1–2021/2/28) to fifth (2021/6/25–2021/9/30) waves. Because there is no official definition of the third wave, the beginning of the third wave was defined as the day when 30 new cases were identified, and the end of the wave was tentatively defined as February 28, when the number of new infec- tions per day was consistently below 15. The definitions of fourth and fifth waves were based on those of Kyoto Prefecture, from March 1 to June 24, and June 25 to September 30, respectively. 2.5 \| Evaluation of the effect of multifaceted intervention 692 NAGAO ET AL. 692 numbers of participating facilities and tests are shown in the Table 1. Of the 1,144 facilities that were subjected to PCR mass testing, over 70% of them participated in the monitoring program. By the end of August 2021, the two-dose vaccination program involving ACFs was completed. Later, when the fifth wave came to an end, all screening tests were finished. A total of 394,377 mass tests were performed, and 72 (0.018%) were positive, of which 41 were confirmed cases. Ten out of the 41 cases (24.4%) resulted in transmission within facilities. To evaluate the effect of the infection prevention strategy, we ana- lyzed the number of clusters per month, number of COVID-19 cases related to ACFs, and proportion of severe cases between the third wave and fourth and fifth waves. A cluster was defined as an outbreak within a facility involving more than five cases. Major lineages of SARS-CoV-2 in Kyoto City during the study period are also shown in the Table 1. 3.1 \| The results of mass tests and trend of COVID-19 cases The trend in the number of COVID-19 cases during the study period and measures taken are shown in Figure 1. From January 2021, when the third wave peaked, we conducted preliminary interventions for eight ACFs and clarified operational methods and issues. Then, PCR mass testing was launched, targeting the whole facilities. Actual 2.6 \| Ethics approval The Ethics Committee of Kyoto University Graduate School and Fac- ulty of Medicine approved this study (R2379) and waived the need to obtain informed consent from each patient. The proportion of ACF-related COVID-19 cases in total cases decreased from 7.9% in the third wave to 2.9% in the fourth wave and 2.4% in the fifth wave, although the total number of COVID-19 cases spiked in the fifth wave due to the spread of the delta variant. (Figure 1 and Table 1) The number of clusters in ACFs also decreased from 7.3 episodes/month in the third wave to 2.6 episodes/month and 3.7 episodes/month in the fourth and fifth waves, respectively. The proportion of severe cases in the elderly population also decreased during the study period (Figure 2). 2.4 \| Multifaceted infection-prevention measures As such, the infection prevention strategy was expanded from the quarantine measure using a PCR mass testing only to the “shielding measures” by educational programs and vaccination pro- gram to prevent the spread of COVID-19 within in the facilities. This project was originally started as quarantine measure to prevent the introduction of COVID-19 to ACF. During a preliminary interven- tion, an educational project for ACF staff launched and a vaccination 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12981 by Cochrane Japan, Wiley Online Library on [17/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable C 2.5 \| Evaluation of the effect of multifaceted intervention 692 3.3 During the study period, the number of ACF-related COVID-19 cases (both residents/users and staff) was 720, including sporadic and SARS-COV-2 testing and COVID-19 cases in ACFs in Kyoto City, from the third to fifth waves in 2020–2021 T A B L E 1 SARS-COV-2 testing and COVID-19 cases in ACFs in Kyoto City, from the third to fifth waves in 2020–2021 The third wave The fourth wave The fifth wave Without mass testing Serial mass testing 2020/12/1–2021/02/28 2021/03/1–2021/06/24 2021/06/25–2021/09/30 Major lineage of SARS-CoV-2 B.1.1.214 Alpha(B.1.1.7-like) Delta(B.1.617-2-like) Total number of ACF-related COVID-19 cases (sporadic + cluster) 363 147 210 Proportion of cluster-related COVID-19 cases 75.2% 60.5% 42.4% Average number of cluster episodes in a month 7.3 2.6 3.7 Average number of COVID-19 cases in each cluster 12.4 9.9 8.1 Average number of PCR tests in each cluster case 71 141.1 157.3 Number of facilities subject to universal active screening 1,706 1,144 Number of facilities conducting universal active screening (proportion, %) 1,177 (67.2%) 786 (71.0%) Total number of universal active screening tests 182,684 211,693 Number of screening-positive cases 39 33 Positivity rate for screening test (%) 0.020% 0.016% Number of confirmed COVID-19 cases 21 20 False-positive rate (%) 66.7% 70.0% Abbreviations: ACF, aged-care facility; PCR, polymerase chain reaction. y.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12981 by Cochrane Japan, Wiley Online Library on [17 693 NAGAO ET AL. F I G U R E 1 Trend of COVID-19 cases in Kyoto City and the contents of proactive intervention. Preliminary intervention began in the third wave. Targeted facilities were expanded during the fourth and fifth waves (shaded in grey). Mass testing eventually ended when the fifth wave was over 2659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12981 by Cochrane Japan, Wiley Online Library on [17/01/2023]. See the Terms and Conditions (https:// ownloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12981 by Cochrane Japan, Wiley Online Library on [17/01/2023]. See the Terms and Conditions (https://onlinelib F I G U R E 1 Trend of COVID-19 cases in Kyoto City and the contents of proactive intervention. Preliminary intervention began in the third wave. Targeted facilities were expanded during the fourth and fifth waves (shaded in grey). Mass testing eventually ended when the fifth wave was over F I G U R E 1 Trend of COVID-19 cases in Kyoto City and the contents of proactive intervention. 4 \| DISCUSSION This is the first real-world data from Japan showing the effect of com- prehensive efforts to reduce the spread of COVID-19 within ACFs. In addition, collaboration with a national university, local authority, and local ACFs is rare in Japan, and this framework will be of immense value in the event of a future pandemic. As a result of this project, clusters in ACFs, the proportion of ACF-related cases, and severe cases were successfully reduced despite the widespread nature of the delta variant. There is strong observational evidence supporting the merit of serial universal testing of ACF residents and staff to facilitate rapid identification and containment of potential cases.2,12,13 Because there is no test method with 100% sensitivity and specificity, a certain per- centage of false-positives and false-negatives may occur when mass testing is performed. On the other hand, symptom-based screening of residents might fail to identify all COVID-19 cases.4,14 In order to overcome the uncertainty of the testing, it is necessary to perform tests using multiple specimens or different testing methods.15 How- ever, in the case of mass testing, testing only one specimen is practical due to the complexity of specimen collection and cost. When con- ducting tests in situations where the pre-test probability is insuffi- cient, it is always necessary to prepare a safety net similar to that in this study. In addition, considering the increase of cases with previous infection, we need to exercise care when interpreting the results of PCR testing.16 cases identified within clusters decreased over, time and only one quarter of newly identified cases caused intra-facility transmission. Considering the weakened immune system of the elderly, booster vaccination began in December 2021 in Kyoto, starting from ACFs and medical institutions. As COVID-19 cannot be eliminated, it is real- istic to overcome forthcoming waves with both booster vaccinations and evidence-based infection control measures. cluster-related cases. Among them, cluster-related cases comprised 451 cases (staff: 158 cases; seniors: 293 cases) in 42 clusters. Of the 145 staff members whose vaccination history was confirmed, 55 (37.9%) were confirmed to have completed the vaccination pro- gram, and of the 155 seniors, 92 (59.4%) had completed the program. There were 19 clusters in which staff were the index cases, and the average duration of clusters was 25.7 days (ranging from 13 to 44 days). Of the 20 clusters that occurred during the period of mass testing, four were discovered by mass testing. Potential limitations of this study were as follows: Because it was not possible to perform screening and confirmatory tests using the same specimen, we could not specify whether some of the screening positives were truly false-positives. However, in the cases determined to be false-positives, there was no subsequent transmission within the institutions. In addition, we could not evaluate which policy worked to reduce the infection. As is always true with other infec- tions, multifaceted interventions are needed to contain COVID-19 transmission.11,21 We will continue to collect high-quality information regarding ACF-related COVID-19 cases to verify the optimal strategy to deal with COVID-19 in such settings. We also believe that it is nec- essary to verify vaccine coverage at the time of a cluster outbreak and the rate of transmission within a facility, but we were unable to ana- lyze them in this study due to a lack of detailed records. 3.3 Preliminary intervention began in the third wave. Targeted facilities were expanded during the fourth and fifth waves (shaded in grey). Mass testing eventually ended when the fifth wave was over ry.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F I G U R E 2 Proportion of ACF-related COVID-19 cases and severe cases in the elderly during the study period in Kyoto City, Kyoto, Japan. Proportion of ACF-related cases and severe cases in the elderly significantly decreased during the study period F I G U R E 2 Proportion of ACF-related COVID-19 cases and severe cases in the elderly during the study period in Kyoto City, Kyoto, Japan. Proportion of ACF-related cases and severe cases in the elderly significantly decreased during the study period NAGAO ET AL. 694 5 \| CONCLUSION Numbers of COVID-19 clusters and severe cases related to ACFs were markedly reduced by implementing a comprehensive response. Further study is needed to determine a strategy that is both optimal and sustainable, which may be tailored to the level of community transmission in the era of vaccination. ACKNOWLEDGEMENTS We thank Mr. Yoichi Endo and Mrs. Hiromi Inoue of Public Health and Welfare Bureau of Kyoto City, the members of Kyoto City Health Center for COVID-19 public health responses and all staff of ACFs for their proactive efforts to contain the spread of COVID-19 among the facilities. We also thank Kaori Ishizaki, Kazuki Kitamura, Shoichi Nakai, Eiki Kure, Yosuke Kumano, and Mizuki Mori (Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan) for their technical assistance. In addition to the two initiatives of mass testing and improving the quality of infection control, we also promoted a vaccination pro- gram for 40,000 residents and staff at ACFs within 4 months as a shielding measure.17 According to previous reports, implementation of vaccination programs contributed to the decline, and they also stated that vaccination should be a central part of a multifaceted strategy that includes other infection-prevention practices to keep residents in ACFs safe.11, 17–21 Based on our analysis, about 40% of COVID-19 cases involving staff and 60% involving users of ACFs were breakthrough infections, but the rate of serious infections was significantly reduced, which may be attributed to the effectiveness of the vaccine. This work was supported by the COVID-19 Private Fund (to the Shinya Yamanaka Laboratory, CiRA, Kyoto University). REFERENCES 1. Chudasama DY, Milbourn H, Nsonwu O, et al. Penetration and impact of COVID-19 in long term care facilities in England: population surveillance study. Int J Epidemiol. 2021. doi:10.2139/ ssrn.3788914 1. Chudasama DY, Milbourn H, Nsonwu O, et al. Penetration and impact of COVID-19 in long term care facilities in England: population surveillance study. Int J Epidemiol. 2021. doi:10.2139/ ssrn.3788914 16. Whitney SA, Berry SD. Rethinking positive coronavirus results: inter- preting RT-PCR testing in nursing home residents. J am Med Dir Assoc. 2021;22(10):2034–2035. doi:10.1016/j.jamda.2021.07.023 2. Louie JK, Scott HM, DuBois A, et al. Lessons from mass-testing for coronavirus disease 2019 in long-term care facilities for the elderly in San Francisco. Clin Infect Dis. 2021;72(11):2018–2020. doi:10. 1093/cid/ciaa1020 17. Australian Government Department of Health, Operation COVID shield national COVID vaccine campaign plan 2022. https://www. health.gov.au/resources/collections/operation-covid-shield-covid- 19-vaccine-sentiment-summaries 3. Rios P, Radhakrishnan A, Williams C, et al. Preventing the transmis- sion of COVID-19 and other coronaviruses in older adults aged 60 years and above living in long-term care: a rapid review. Syst Rev. 2020;9(1):218. doi:10.1186/s13643-020-01486-4 18. Benin AL, Soe MM, Edwards JR, et al. Ecological analysis of the decline in incidence rates of COVID-19 among nursing home resi- dents associated with vaccination, United States, J Am Med Dir Assoc. 2021;22(10):2009–2015. doi:10.1016/j.jamda.2021.08.004 4. McMichael TM, Clark S, Pogosjans S, et al. COVID-19 in a Long- Term Care Facility - King County, Washington, February 27-March 9, 2020. MMWR Morb Mortal Wkly Rep. 2020;69(12):339–342. doi: 10.15585/mmwr.mm6912e1 4. McMichael TM, Clark S, Pogosjans S, et al. COVID-19 in a Long- Term Care Facility - King County, Washington, February 27-March 9, 2020. MMWR Morb Mortal Wkly Rep. 2020;69(12):339–342. doi: 10.15585/mmwr.mm6912e1 19. Suetens C, Kinross P, Gallego Berciano P, et al. Increasing risk of breakthrough COVID-19 in outbreaks with high attack rates in European long-term care facilities, Euro Surveill. 2021;26(49). doi:10. 2807/1560-7917.ES.2021.26.49.2101070 5. Resciniti NV, Fuller M, Sellner J, Lohman MC. COVID-19 incidence and mortality among long-term care facility residents and staff in South Carolina. J Am Med Dir Assoc. 2021;22(10):2026–2031, e2021. doi:10.1016/j.jamda.2021.08.006 5. Resciniti NV, Fuller M, Sellner J, Lohman MC. COVID-19 incidence and mortality among long-term care facility residents and staff in South Carolina. J Am Med Dir Assoc. 2021;22(10):2026–2031, e2021. doi:10.1016/j.jamda.2021.08.006 20. Giddings R, Krutikov M, Palmer T, et al. Changes in COVID-19 out- break severity and duration in long-term care facilities following vac- cine introduction, England, Euro Surveill. 2021;26(46). doi:10. 2807/1560-7917.ES.2021.26.46.2100995 6. Miki Nagao https://orcid.org/0000-0002-8886-6145 15. Hanson KE, Caliendo AM, Arias CA, et al. Infectious diseases society of america guidelines on the diagnosis of COVID-19. Clin Infect Dis. 2020. NAGAO ET AL. 695 ORCID 14. McMichael TM, Currie DW, Clark S, et al. Epidemiology of Covid-19 in a long-term care facility in King County, Washington. N Engl J Med. 2020;382(21):2005–2011. doi:10.1056/NEJMoa2005412 Miki Nagao https://orcid.org/0000-0002-8886-6145 PEER REVIEW coronavirus 2 in long-term care facilities, Spain. Emerg Infect Dis. 2021;27(10):2595–2603. doi:10.3201/eid2710.211184 The peer review history for this article is available at https://publons. com/publon/10.1111/irv.12981. 12. Dykgraaf SH, Matenge S, Desborough J, et al. Protecting nursing homes and long-term care facilities from COVID-19: a rapid review of international evidence. J Am Med Dir Assoc. 2021;22(10):1969– 1988. doi:10.1016/j.jamda.2021.07.027 DATA AVAILABILITY STATEMENT 13. Neilan AM, Losina E, Bangs AC, et al. Clinical impact, costs, and cost- effectiveness of expanded severe acute respiratory syndrome coro- navirus 2 testing in Massachusetts. Clin Infect Dis. 2021;73(9): e2908-e2917. doi:10.1093/cid/ciaa1418 The data that support the findings of this study are available from the corresponding author upon reasonable request. AUTHOR CONTRIBUTIONS Miki Nagao: Conceptualization; formal analysis; funding acquisition; investigation; methodology; project administration; supervision. Yasufumi Matsumura: Conceptualization; data curation; formal analy- sis; funding acquisition; investigation; methodology; project adminis- tration. Masaki Yamamoto: Conceptualization; funding acquisition; investigation; project administration. Koh Shinohara: Investigation; project administration. Satomi Yukawa: Investigation; project adminis- tration. Taro Noguchi: Investigation; project administration. Yasuhiro Tsuchido: Investigation; project administration. Takeshi Ikeda: Con- ceptualization; project administration; resources; supervision. It has been reported that elderly people lose vaccine antibodies more quickly and that the vaccine is less effective against variant strains.22 Furthermore, Hsu et al. previously reported that the number of transmissions from unvaccinated controls was three times higher than from fully vaccinated patients.22,23 In our cohort, the number of How to cite this article: Nagao M, Matsumura Y, Yamamoto M, et al. Analysis of a city-wide COVID-19 prevention strategy for aged-care facilities during third and fifth waves of COVID-19 in Kyoto City, Kyoto, Japan. Influenza Other Respi Viruses. 2022;16(4):690-695. doi:10. 1111/irv.12981 REFERENCES Shimotsu ST, Johnson ARL, Berke EM, Griffin DO. COVID-19 infec- tion control measures in long-term care facility, Pennsylvania, USA. Emerg Infect Dis. 2021;27(2):644–645. doi:10.3201/eid2702.204265 21. Love J, Keegan LT, Angulo FJ, et al. Continued need for non- pharmaceutical interventions after COVID-19 vaccination in long- term-care facilities. Sci Rep. 2021;11(1):18093. doi:10.1038/ s41598-021-97612-w 7. Tan LF, Tan MF. Pandemic to endemic: New strategies needed to limit the impact of COVID-19 in long-term care facilities (LTCFs). J Am Geriatr Soc. 2021;70(1):72 73. doi:10.1111/jgs.17556 y.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 22. Brockman MA, Mwimanzi F, Lapointe HR, et al. Reduced magnitude and durability of humoral immune responses to COVID-19 mRNA vaccines among older adults. J Infect Dis. 2021. doi:10.1093/infdis/ jiab592 8. Vijh R, Prairie J, Otterstatter MC, et al. Evaluation of a multisectoral intervention to mitigate the risk of severe acute respiratory coronavi- rus virus 2 (SARS-CoV-2) transmission in long-term care facilities. Infect Control Hosp Epidemiol. 2021;42(10):1181–1188. doi:10.1017/ ice.2020.1407 23. Hsu L, Grüne B, Buess M, et al. COVID-19 breakthrough infections and transmission risk: real-world data analyses from Germany’s larg- est public health department (Cologne). Vaccines (Basel). 2021;9(11): 1267. doi:10.3390/vaccines9111267 9. Frazer K, Mitchell L, Stokes D, Lacey E, Crowley E, Kelleher CC. A rapid systematic review of measures to protect older people in long- term care facilities from COVID-19. BMJ Open. 2021;11(10): e047012. doi:10.1136/bmjopen-2020-047012 10. Stratil JM, Biallas RL, Burns J, et al. Non-pharmacological measures implemented in the setting of long-term care facilities to prevent SARS-CoV-2 infections and their consequences: a rapid review. Cochrane Database Syst Rev. 2021;9(9):CD015085. doi:10. 1002/14651858.CD015085 How to cite this article: Nagao M, Matsumura Y, Yamamoto M, et al. Analysis of a city-wide COVID-19 prevention strategy for aged-care facilities during third and fifth waves of COVID-19 in Kyoto City, Kyoto, Japan. Influenza Other Respi Viruses. 2022;16(4):690-695. doi:10. 1111/irv.12981 11. Monge S, Olmedo C, Alejos B, Lapeña MF, Sierra MJ, Limia A, COVID-19 Registries Study Group 2. Direct and indirect effective- ness of mRNA vaccination against severe acute respiratory syndrome
https://openalex.org/W2016765182	https://iiste.org/Journals/index.php/RJFA/article/download/16841/17179	English	null	A Tool for Measuring Organization Performance using Ratio Analysis	Advances in social sciences research journal	2,014	cc-by	5,458	www.iiste.org www.iiste.org Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 www.iiste.org A Tool For Measuring Organization Performance Using Ratio Analysis Elijah Adeyinka Adedeji Introduction The purpose of preparing the financial statements of a company is to convey information on the overall performance and the state of affairs of such an organisation to all interested parties. Besides, users of these financial statements in such a way as to reveal the financial strengths and weaknesses of such an organisation in order to form an opinion as regard her going-concern. However, ratio analysis is one of the ways through which the financial statements could be interpreted. While ratio analysis is also a method used by financial managers and investors alike to compare a company’s financial structure, conditions and performances with standards prevailing in such industry for the purpose of high-lighting improvement or deterioration in the trend of the business performance. Lucey (1988) defined ratio analysis as the systematic products of ratios from both internal and external financial reports so as to summarize key relationships and results in order to appraise financial performance. More so, ratio analysis could serve as a practical means of monitoring and improving performance and it could be enhanced when: i. Ratios are prepared regularly and on a consistent basis so that trends can be highlighted and changes investigated. g g ii. Ratios prepared for and individual firm can be compared with facilitated when the firm has ready access to comparative ratios prepared in a standard manner. g g ii. Ratios prepared for and individual firm can be compared with facilitated when the firm has read access to comparative ratios prepared in a standard manner. p p p iii. Ratios are prepared showing the inter-locking and inter-dependent nature of the factors which contribute to financial success. p p p iii. Ratios are prepared showing the inter-locking and inter-dependent nature of the factors which contribute to financial success. Nevertheless, ratio analysis utilizes figures that routinely appear in the financial statements for a period of several consecutive years, (that is 5years to 10years). One calculated, the ratio may be compare with external industry standards and with internal goals and budgets of the organisation in order to detect trends and estimates, improvement and stability of the measure conditions. Finally, it must be emphasized that ratios must be compared with some prevailing standards, because it cannot in itself convey any useful information. Abstract Ratio analysis has served as a veritable means of monitoring, measuring and improving performance in an organization. Hence, the study examines a tool for measuring organization performance using ratio analysis. It also ascertains the relevance of internal and external financial reports during ratio analysis for the purpose of establishing key relationships and results in order to appraise financial performance. The study confirmed that there is significant relationship between ratio analysis and organizational performances as well as financial ratios highlight the importance of effective management of an organization. Based on the findings of this study, it was recommended that financial ratios should be computed periodically to reveal areas of strengths and weaknesses, as well as, ratio analysis should be used to measure performance in terms of profitability. Keywords: Ratio analysis, Performance, Organization, financial Ratios, Management. Basis of comparison Financial ratio as an index is more useful when it is compared with another index. The basis of comparison includes the following; i. Intra-Firm comparison or previous year basis. ii. Inter-Firm comparison or similar business basis. iii. By basis of ratio established by the management (standard). Statement of the Problem Managerial decision is one of the keys to success in an organisation. And as such, management of a given organisation makes decision based on financial performances prevailing in such establishment. In arriving at such decisions, the management tries to focus their attention on two basics of comparison which are as follows: Current performances are compares with the records of the part years in the organisation at least five (5) years period. Current performances are compared with that attested performances in other similar organizations. As a result of this exercise, in-estimable short comings may arise which could force management to take drastic steps/decisions that could make or mar the organisation. Also, problems may arise when an attempt is made to compare the ratio of one business with those of other organisation, and these could arise as a result of different accounting basis and the aftermath result could not be relied upon. 39 Research Journal of Finance and Accounting www.iis ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Moreover, problem(s) associated with effect of inflation are always being ignored and the resultant ratios are of limited value. As a result of the following, the aforementioned shall be examined in the course of this research work. Literature Review Ratio according to Garbutt (1972) is one number expressed in terms of another. It is defined in the Oxford Dictionary as the relationship between two amounts determined by the number of times one is contained in the other. By the use of ratio analysis techniques, it is possible to facilitate comparison of significant figures, by expressing their relationship in the form of ratios or percentages, thus enabling the accounts of a business to be interpreted by bringing into focus salient features contained in the financial statements. Financial ratios are employed to denote past trends, compare present performances and may given an indication to future trends, performances or operations of a company and thus acts as signposts for plans and policies. It could be deduced from the above that ratios serves as practical means of monitoring and improving performances of a company (Lucey, 1988). Research Hypotheses yp The following hypotheses shall be tested during this research work: yp The following hypotheses shall be tested during this research work: Ho: Financial ratios do not highlight the importance of effective management of an organisation. Hi: Financial ratios highlight the importance of effective management of an organisation Ho: Financial ratios do not highlight the importance of effective management of an organisation. Hi: Financial ratios highlight the importance of effective management of an organisation Hi: Financial ratios highlight the importance of effective management of an organisation. Research Questions Q The following research questions shall be examined during this study: The following research questions shall be examined during this study: g q g y i. Do you use financial ratios as a measurement of management performance? ii. Does the management of this company apply financial ratio in making decisions that affect the company? p y iii. Does ratio analysis help management in taking effective decisions? y p g g iv. Do you agree that financial ratio reveal strengths and weaknesses of an organisation? iv. Do you agree that financial ratio reveal strengths and weaknesses of an organisation? v. Does the interpretation of ratios help to determine whether the activities of the company have been effectively managed? vi. Does interpretation of ratio yield positive results? y g g g v. Does the interpretation of ratios help to determine whether the activities of the company have been effectively managed? vi. Does interpretation of ratio yield positive results? Objective of the Study j y The broad objective of the study is to analysis how ratio analysis can be used to measure performance of an organisation. Also the following specific objectives will be examined in the course of this study: i. To critically analyse the financial statement and evaluate the performance of the company through ratios to ascertain whether resource are optimally and efficiently utilized. i. To critically analyse the financial statement and evaluate the performance of the company through ratios to ascertain whether resource are optimally and efficiently utilized. ii. To evaluate the historical activities of the company such that a projection into the future can be made thereby improving management decision. iii. To analyse the problems associated with the use of financial ratio analysis and proffer possible iv. To identify the importance of financial ratio analysis to every use group. v. To analyse how financial ratio analysis can assist management to detect the various strengths and weaknesses of an organisation. v. To analyse how financial ratio analysis can assist management to detect the various strengths and weaknesses of an organisation. Interested parties to the accounts and financial ratios The fundamental purpose of financial reporting is to communicate economic measurement of information about the resources and performances of an organization useful to those having reasonable right to such information.(SAS 2,1987). Accounting information (and thus ratios calculated) of a business is required by a variety of users. According to the statement of Accounting Standard (SAS2,1987) there are many users, which can be grouped into two, showing clearly why they need such information (Aghoroh, 1999). The interested parties to financial statements and ratios are grouped into two as follows: i. Internal users. ii. External users. ii. External users. Benefits of inter-firm comparison p Garbutt (1972) also noted that the information emerging from comparative surveys may throw new light on points such as follows; p ; i. The actual rate of return on capital being achieved in the industry ii. The industry’s cost structure iii. The main areas of weaknesses and strengths found in a company The areas where there seem be ‘bottleneck’ factors inhibiting economic growth. iv. The areas where there seem be ‘bottleneck’ factors inhibiting economic growth. v. Comparison may also provide realistic quantitative assessments of the scope for increased productivity and efficiency in the industry. Inter statement ratios statement ratios ter statement ratio is calculated by relating items in both the balance sheet and profit and loss account. Inter statement ratios An inter statement ratio is calculated by relating items in both the balance sheet and profit and loss account. An inter statement ratio is calculated by relating items in both the balance sheet and profit and los Garbutt (1972) noted that ratios could be loosely grouped into the following and as a measure of profitability, liquidity or asset use solvency. Another possible and more acceptable method of classifying ratios is according to the financial activity (functions). This method helps to analyse and gives an overview of information required by various parties interested in the company’s financial reports. For example, creditors are interested in the liquidity position of a company; hence, they consider the liquidity ratios. Shareholders are concerned about the net worth and profitability, as a result they monitor the profitability ratios. Ratios used to measure the financial activity of a company can be grouped into four in respect of this research work: Profitability and Efficiency. Profitability and Efficiency. Short term solvency and liquidity. Long term solvency and liquidity/capital structure. Potential and growth investors’ ratio. The basis of ratios established by the management (standard) y g ( ) Financial ratio for the current period may be compared with standard ratios established by the management. This basis follows, budgetary analysis whereby budgeted ratios are compared with actual ratios as calculated from figures in the financial statements and variances indentified. The variances that occur (whether favourable or adverse) will help the management in interpreting the performances of the company which will in turn aid in predicting the company’s future performance. – Lucey, 1988. Classification of financial ratios Three are various way of classifying ratios; this depends on the information need of the analyst of the financial statements. Ratio can be classified in terms of their data source; hence, we have the following classifications: Objective of inter-firm comparison Garbutt (1972) stated that inter-firm comparison is intended to show the management of each firm: i. How its profitability and productivity compare with that of other firms in the same industry. ii. In what respects the firm is weaker or stronger than its competitors. ii. In what respects the firm is weaker or stronger than its competitors. iii. What specific questions of policy or performance should be tackled if the firm’s profitability an productivity are to be raised? iii. What specific questions of policy or performance should be tackled if the firm’s profitability and productivity are to be raised? 40 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 iste.org Balance sheet ratio These are ratios calculated using two related figures from the balance sheet. Profit and loss account ratio These are ratios calculated form related figure in the profit and loss accounts. Operating efficiency This shows how effectively the management utilizes the assets and how the assets are used in generating sales and revenue. Profitability The ratios also help to determine the overall profitability of a company. This helps management to determine whether the company can meet its short and long term debts and still maintain optimum return. Liquidity position According ratios enables various user groups to know or determine the ability of a company to meet its long or short term obligation. Advantages of ratio analysis As stated earlier, there are various techniques which could be employed in the interpretation of the financial statements. These techniques include the straight forward criticism, ratio analysis and movement of funds statements (cash flow statements). The ratio analysis technique has the following advantages over the other techniques. q i. The ratio analysis technique provided a standard through which ratios can be compared at any poin time. ii. The ratios are easy to compute since figures used in computing are picked from the financial statements. iii. Formulates used in calculating ratios are uniform. That is, the formulas are the same all over. ii. The ratios are easy to compute since figures used in computing are picked from the financial statements. iii Formulates used in calculating ratios are uniform That is the formulas are the same all over g iv. Ratios could be combined with other measures, which are also used in evaluating performance of an organization. iv. Ratios could be combined with other measures, which are also used in evaluating performance of an organization. Trend analysis Ratios enable users of financial statement determine whether the financial position of a company is improving or deteriorating over time. The importance of trend analysis is that the analyst knows whether the company is operating on a favourable level or not. Inter-firm comparison p Ratios are often compared among companies in the same industry. This is important because it enables the management of a company to know the position of the company in the industry and among competitors. Importance ratio analysis p y Ratios are effective tool of management in the provision of information and data needed in planning and determining the efficiency of management for a particular period. Ratios are also used to established relationship and trends in the financial statements. Ratios are important and useful to various user groups as viewed from the 41 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 following perspective. Hypothesis One Data Analysis Table 1 2 3 4 5 1 15 15 5 5 - 2 20 15 5 - - 3 15 15 5 2 3 4 10 20 5 - 5 5 60 65 20 7 8 = 160 152 + 152 + 52 + 52 + 202 + 152 SST = + 52 + 152 + 152 + 52 + 22 + 32 - (160)2 + 102 + 202 + 52 + 52 16 2163 – 1600 = 563 SSC = 602 + 652 + 202 + 72 + 82 - (160) 2 5 16 1667.6 - 1600 = 667.6 SSE = SST - SSC = 104.6 Other computations are shown in the table below: Analysis of Variance SOURCE OF VARIATION DF SS MS F Treatment Error 4 12 66.7.6 104.6 166.9 8.72 19.14 Total 16 563 Fcal = 19.14 Ftab @ (0.05, 4, 12) = 5.14 Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. Therefore, there is significant relationship between ratio analysis and organizational performance. Methodology gy The population of study is the staff of PZ CUSSONS PLC, an organization that is reputable for efficiency as a result of management integrity. The population shall be chosen among the staff of finance, marketing and production departments of the organization and they shall constitute the sample for the survey. For the purpose of this research work selected respondents were draw from the total population through simple random sampling. This procedure gives opportunity to all respondents to be selected evenly. Staffs were randomly selected from their departments and given the questionnaires. In the process, only a very few of them collected and filled the questionnaire, while countable number of them allowed to be interviews. Finally, respondents were selected through simple random sampling because it gives the whole population the opportunity of being selected. In selecting the sample size, the total population of the organization was taken into consideration such that a significant part of the staff strength was selected as respondents for the organization. In all, about 40 respondents were selected from the staff in different department. Research instrument refers to the basic tools of the researcher for measuring, evaluating, analyzing and exploring of data – Asika (1991). In the course of this research work, data were collected through the use of well structured questionnaire designed well in accordance with the objectives of the study. The questionnaire made this choice of appreciable language which enables the respondents to understand the questions for appropriate response, while Likert scale and other measuring scales were used for the options. Finally, the questionnaire was divided into two sections, that is section ‘A’ has the demographic characteristics of respondents, and section ‘B’ has questions that relates to the study objectives of the study. The data were grouped using frequency distribution table, and were eventually given percentages in order to ensure further analysis of respondents’ perception. In other words, the percentages gave an insight into 42 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 respondents’ perception in respect of the questions and responses. Hypotheses were tested using analysis of variance and other statistical drawings. respondents’ perception in respect of the questions and responses. Hypotheses were tested using analysis of variance and other statistical drawings. respondents’ perception in respect of the questions and responses. Hypotheses were tested using analysis of variance and other statistical drawings. Hypothesis One Hypothesis One H0: There is no significant relationship between ratio analysis and organisation performance. Hi: There is a significant relationship between ratio analysis and organisation performance. Test of hypothesis Data collected from respondents in questions: 1, 2, 4, and 10 shall be subjected to F-distribution statistical method. Data Analysis Table 1 2 3 4 5 1 15 15 5 5 - 2 20 15 5 - - 3 15 15 5 2 3 4 10 20 5 - 5 5 60 65 20 7 8 = 160 152 + 152 + 52 + 52 + 202 + 152 SST = + 52 + 152 + 152 + 52 + 22 + 32 - (160)2 + 102 + 202 + 52 + 52 16 2163 – 1600 = 563 SSC = 602 + 652 + 202 + 72 + 82 - (160) 2 5 16 1667.6 - 1600 = 667.6 SSE = SST - SSC = 104.6 Other computations are shown in the table below: Analysis of Variance SOURCE OF VARIATION DF SS MS F Treatment Error 4 12 66.7.6 104.6 166.9 8.72 19.14 Total 16 563 Fcal = 19.14 Ftab @ (0.05, 4, 12) = 5.14 Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. Therefore, there is significant relationship between ratio analysis and organizational performance. Hypothesis One H0: There is no significant relationship between ratio analysis and organisation performance. Hi: There is a significant relationship between ratio analysis and organisation performance. yp H0: There is no significant relationship between ratio analysis and organisation performance. Hi: There is a significant relationship between ratio analysis and organisation performance. Test of hypothesis Data collected from respondents in questions: 1, 2, 4, and 10 shall be subjected to F-distribution statistical method. Result and Discussion Result and Discussion The following questions shall be drawn from the questionnaire for the test of hypothesis one (1). Question One: Do you use financial ratios as a measurement of management performance? Question Two: Do the management of this company apply financial ratios in making decisions that affect the company? p y Question Four: Do you agree that financial ratio reveal strengths and weaknesses of an organisation? Question Ten: Ratio Analysis is very effective at various aspect of company performance? Test of hypothesis Data Analysis Table 1 2 3 4 5 1 10 20 5 - 5 2 25 10 - 5 - 3 15 10 5 5 5 4 25 10 5 - - 5 75 50 15 10 10 = 160 Data Analysis Table 1 2 3 4 5 1 10 20 5 - 5 2 25 10 - 5 - 3 15 10 5 5 5 4 25 10 5 - - 5 75 50 15 10 10 = 160 102 + 202 + 52 + 52 + 252 + 102 SST = + 52 + 152 + 102 + 52 + 52 + 52 - (160)2 + 52 + 252 + 102 + 52 15 2450 – 1706.7 = 743.3 SSC = 752 + 502 + 152 + 102 + 102 - (160) 2 4 15 2137.5 - 1706.7 = 430.8 SSE = SST - SSC = 312.5 102 + 202 + 52 + 52 + 252 + 102 SST = + 52 + 152 + 102 + 52 + 52 + 52 - (160)2 + 52 + 252 + 102 + 52 15 2450 – 1706.7 = 743.3 SSC = 752 + 502 + 152 + 102 + 102 - (160) 2 4 15 2137.5 - 1706.7 = 430.8 SSE = SST - SSC = 312.5 Other computations are shown in the table below: Other computations are shown in the table below: Analysis of Variance Source of Variation DF SS MS F Treatment Error 4 11 430.8 312.5 107 28.4 79.29 Total 15 743.3 @ Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. Therefore financial ratios highlight the importance of effective management of an organisation Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. alternative. Therefore, financial ratios highlight the importance of effective management of an organisation. Therefore, financial ratios highlight the importance of effective management of an organisation. Therefore, financial ratios highlight the importance of effective management of an organisation. The following questions shall be drawn from the questionnaire for the test of hypothesis two (2). Question Three: Does ratio analysis help management in taking effective decisions? Question Five: Does the interpretation of rations help to determine whether the activities of the company have been effectively managed? Question Nine: Does the management of this company rely on financial ratios to drawn conclusion on certain issues? Question Twelve: Ratio analysis establishes true picture of company financial status? Hypothesis Two H0: Financial ratios do not highlight the importance of effective management of an organisation. Hi: Financial ratios highlight the importance of effective management of an organisation. Question Three: Does ratio analysis help management in taking effective decisions? Question Five: Does the interpretation of rations help to determine whether the activities of the company have been effectively managed? Question Nine: Does the management of this company rely on financial ratios to drawn conclusion on certain issues? Question Twelve: Ratio analysis establishes true picture of company financial status? Hypothesis Two H0: Financial ratios do not highlight the importance of effective management of an organisation. Hi: Financial ratios highlight the importance of effective management of an organisation. Hypothesis Two H0: Financial ratios do not highlight the importance of effective management of an organisation. Hi: Financial ratios highlight the importance of effective management of an organisation. Test of hypothesis Test of hypothesis Data collected from respondents in questions: 3, 5, 9, and 12 shall be subjected to F-distribution statistical method. Data Analysis Table 1 2 3 4 5 1 10 20 5 - 5 2 25 10 - 5 - 3 15 10 5 5 5 4 25 10 5 - - 5 75 50 15 10 10 = 160 102 + 202 + 52 + 52 + 252 + 102 SST = + 52 + 152 + 102 + 52 + 52 + 52 - (160)2 + 52 + 252 + 102 + 52 15 2450 – 1706.7 = 743.3 SSC = 752 + 502 + 152 + 102 + 102 - (160) 2 4 15 2137.5 - 1706.7 = 430.8 SSE = SST - SSC = 312.5 Other computations are shown in the table below: Analysis of Variance Source of Variation DF SS MS F Treatment Error 4 11 430.8 312.5 107 28.4 79.29 Total 15 743.3 Fcal = 79.29 Ftab @ (0.05, 4, 11) = 5.67 Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. Therefore, financial ratios highlight the importance of effective management of an organisation. Conclusion and Recommendations This research work studied how ratio analysis can be used to measure performance of an organization. Based on the discussions and findings in the course of this study, the following conclusions are made: i. Ratio analysis is a tool of financial analysis, which can be used as a predictive tool for measuring business performance. yp Data collected from respondents in questions: 3, 5, 9, and 12 shall be subjected to F-distribution statistical method. Test of hypothesis Test of hypothesis Data collected from respondents in questions: 1, 2, 4, and 10 shall be subjected to F-distribution statistical method. Data Analysis Table 1 2 3 4 5 1 15 15 5 5 - 2 20 15 5 - - 3 15 15 5 2 3 4 10 20 5 - 5 5 60 65 20 7 8 = 160 152 + 152 + 52 + 52 + 202 + 152 SST = + 52 + 152 + 152 + 52 + 22 + 32 - (160)2 + 102 + 202 + 52 + 52 16 2163 – 1600 = 563 SSC = 602 + 652 + 202 + 72 + 82 - (160) 2 5 16 1667.6 - 1600 = 667.6 SSE = SST - SSC = 104.6 Other computations are shown in the table below: Analysis of Variance SOURCE OF VARIATION DF SS MS F Treatment Error 4 12 66.7.6 104.6 166.9 8.72 19.14 Total 16 563 Fcal = 19.14 Ftab @ (0.05, 4, 12) = 5.14 Other computations are shown in the table below: Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. Therefore, there is significant relationship between ratio analysis and organizational performance. Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. Therefore, there is significant relationship between ratio analysis and organizational performance. Decision: Fcal is greater than (>) Ftab since Fcal is greater than (>) Ftab, then reject null hypotheses and accept the alternative. Therefore, there is significant relationship between ratio analysis and organizational performance. 43 iste.org Conclusion and Recommendations This research work studied how ratio analysis can be used to measure performance of an organization. Based on the discussions and findings in the course of this study, the following conclusions are made: i. Ratio analysis is a tool of financial analysis, which can be used as a predictive tool for measuring business performance. 44 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.5, No.19, 2014 ii. Ratio analysis can be used to show areas of strengths and weaknesses of a company. ii. Ratio analysis can be used to show areas of strengths and weaknesses of a company. Ratio analysis is required for management control decisions, investment decisions and credit contr purposes. iv. Ratio analysis is required to determine whether a company have been improving or is deteriorating financially over a period of time. iv. Ratio analysis is required to determine whether a company have been improving or is deteriorating financially over a period of time. y p Ratio analysis can be used to determine whether a company have met the required standard within the industry. v. Ratio analysis can be used to determine whether a company have met the required standard within the industry. y vi. Profitability ratios are useful to the management of a company. They are used to determine the profitability of a company and the efficiency in the utilization of the resources of a company. vi. Profitability ratios are useful to the management of a company. They are used to determine the profitability of a company and the efficiency in the utilization of the resources of a company. Therefore, the following recommendations are made: i. Ratio analysis should form part of management activities and should be computed periodically reveal areas of strengths and weaknesses of a company. ii. Ratios should be used by the management to measure the profitability of the company and to compare the financial activities of the company with that of other companies within the same industry. This helps to determine whether the company has performed up to the standard required by the industry. y y iii. The investors should use investment ratios to determine how much divided will accrue to them. iv. Conclusion and Recommendations Creditors and loan providers are advised to check the liquidity of a company before granting loans or giving any consideration. Therefore, they should consider ratios such as current ratio and quick assets ratio. iv. Creditors and loan providers are advised to check the liquidity of a company before granting loans or giving any consideration. Therefore, they should consider ratios such as current ratio and quick assets ratio. v. The employees of an organization should be interested in ratios such as the long-term solvency and liquidity ratios. This enables the employees know and measure the security of their jobs. v. The employees of an organization should be interested in ratios such as the long-term solvency and liquidity ratios. This enables the employees know and measure the security of their jobs. References Asika, N. (1999), Research \Methodology in Behavioural Science, Longman Plc, Lagos Garbutt, D. (1972), Carter’s Advanced Accounts, Sir ISAAC Pitman and Sons Ltd, London Lucey, T. (1988), Management Accounting DP Publications Ltd London. SAS 2 (1987), Nigerian Accounting Standard Board, PAT Publications LTD, Lagos. CALL FOR JOURNAL PAPERS There are more than 30 peer-reviewed academic journals hosted under the hosting platform. Prospective authors of journals can find the submission instruction on the following page: http://www.iiste.org/journals/ All the journals articles are available online to the readers all over the world without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself. Paper version of the journals is also available upon request of readers and authors. MORE RESOURCES Book publication information: http://www.iiste.org/book/ References References Asika, N. (1999), Research \Methodology in Behavioural Science, Longman Plc, Lagos Garbutt, D. (1972), Carter’s Advanced Accounts, Sir ISAAC Pitman and Sons Ltd, London Lucey, T. (1988), Management Accounting DP Publications Ltd London. SAS 2 (1987), Nigerian Accounting Standard Board, PAT Publications LTD, Lagos. 45 The IISTE is a pioneer in the Open-Access hosting service and academic event management. The aim of the firm is Accelerating Global Knowledge Sharing. The IISTE is a pioneer in the Open-Access hosting service and academic event management. The aim of the firm is Accelerating Global Knowledge Sharing. More information about the firm can be found on the homepage: http://www.iiste.org IISTE Knowledge Sharing Partners EBSCO, Index Copernicus, Ulrich's Periodicals Directory, JournalTOCS, PKP Open Archives Harvester, Bielefeld Academic Search Engine, Elektronische Zeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe Digtial Library , NewJour, Google Scholar
https://openalex.org/W3179678468	https://jyx.jyu.fi/bitstream/123456789/77681/1/1-s2.0-S014362282100117X-main.pdf	English	null	Land-use and climate related drivers of change in the reindeer management system in Finland: Geography of perceptions	Applied geography	2,021	cc-by	17,587	This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Title: Land-use and climate related drivers of change in the reindeer management system in Finland : Geography of perceptions Title: Land-use and climate related drivers of change in the reindeer management system in Finland : Geography of perceptions Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ © 2021 The Authors. Published by Elsevier Ltd. Published version Rasmus, S., Wallen, H., Turunen, M., Landauer, M., Tahkola, J., Jokinen, M., & Laaksonen, S. (2021). Land-use and climate related drivers of change in the reindeer management system in Finland : Geography of perceptions. Applied Geography, 134, Article 102501. https://doi.org/10.1016/j.apgeog.2021.102501 2021 Sirpa Rasmus a,e,, Henri Wallen a,f, Minna Turunen a, Mia Landauer a,g, Juho Tahkola b, Mikko Jokinen c, Sauli Laaksonen d Sirpa Rasmus a,e,, Henri Wallen a,f, Minna Turunen a, Mia Landauer a,g, Juho Tahk Mikko Jokinen c, Sauli Laaksonen d a Arctic Centre, University of Lapland, P.O. Box 122, FI-96101, Rovaniemi, Finland b Reindeer Herders’ Association, P.O. Box 8168, FI-96101, Rovaniemi, Finland c Natural Resources Institute, Ounasjoentie 6, FI-96200, Rovaniemi, Finland d Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, FI-00014, Helsinki, Finland e Department of Biological and Environmental Science, University of Jyv¨askyl¨a, University of Jyv¨askyl¨a, P.O.Box 35, FI-40014, Finland f Department of Archaeology, Faculty of Humanities, University of Oulu, P.O Box 8000, FI-90014, Oulu, Finland g Risk and Resilience Program, International Institute for Applied Systems Analysis (IIASA), Schlossplatz 1, A-2361, Austria 1. Introduction (Eide et al., 2017; Soppela & Turunen, 2017). Reindeer husbandry is considered regionally important as it employs people, keeps remote areas inhabited, and provides economic benefits. It also represents cul tural continuity and a way of life connected to traditions, indigenous rights, and trans-generational values (Helle & Jaakkola, 2008; Kumpula & Siitari, 2020; Turunen & Vuojala-Magga, 2013, 2014). A R T I C L E I N F O Keywords: Climate change Cumulative effects Land use Northern fennoscandia Practitioner knowledge Reindeer husbandry Drivers of change in the reindeer management system are rather well-known. But when developing the gover nance to support the traditional livelihoods, it is crucial to understand also practitioner perceptions. Systematic research on these is lacking. We analyzed the land-use and climate related drivers within the reindeer man agement area (RMA) in Finland, and, using a perception geography approach, studied the herder perceptions towards these. We conducted an on-site questionnaire survey with herders from 51 herding districts. Factors directly affecting the welfare of reindeer were perceived as crucial by herders, for example basal icing affecting the forage availability, and land-use related factors limiting the seasonal pasture access. Perceptions of herders on biophysical factors were rather homogeneous. The regional heterogeneities in perceptions towards land-use related factors could be explained by spatial differences in land-use and varying herding traditions. Cumulative land-use impacts raised particular concerns. Our approach can be utilized in the co-planning of the northern land-use and more widely in the co-management of natural resources. Please cite the original version: Rasmus, S., Wallen, H., Turunen, M., Landauer, M., Tahkola, J., Jokinen, M., & Laaksonen, S. (2021). Land-use and climate related drivers of change in the reindeer management system in Finland : Geography of perceptions. Applied Geography, 134, Article 102501. https://doi.org/10.1016/j.apgeog.2021.102501 Applied Geography 134 (2021) 102501 Available online 8 July 2021 0143-6228/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.apgeog.2021.102501 Received 10 August 2020; Received in revised form 23 June 2021; Accepted 23 June 2021 * Corresponding author. Arctic Centre, University of Lapland, P.O. Box 122, FI-96101, Rovaniemi, Finland. E-mail address: sirpa.rasmus@ulapland.fi (S. Rasmus). 1 When referring to reindeer as a livelihood, we use the term “reindeer husbandry”. “Herding” refers to day-to-day practices (and it also appears in the term “reindeer herding district”), whereas husbandry considers reindeer as a resource and is related for example to the profits, breeding, and social mechanisms. “Reindeer management” is related to all of the practices pertaining to the keeping of reindeer, including governance (Forbes, 2006). Available online 8 July 2021 0143-6228/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). g ; ; p y 0143-6228/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. Arctic Centre, University of Lapland, P.O. Box 122, FI-96101, Rovaniemi, Finland. E-mail address: sirpa.rasmus@ulapland.fi (S. Rasmus). 1.1. Reindeer management in Finland 1.1. Reindeer management in Finland Reindeer husbandry1 is one of the traditional livelihoods in northern Finland (Itkonen, 1948; Kortesalmi, 2007). The Reindeer management area (RMA) covers 36% of Finland’s total area. In this area, semi-domesticated reindeer (Rangifer tarandus tarandus) have a free grazing right which is not dependent on land ownership (Reindeer Herding Act 1990/848, Heikkinen et al., 2012). There is a vast cultural, Reindeer husbandry1 is one of the traditional livelihoods in northern Finland (Itkonen, 1948; Kortesalmi, 2007). The Reindeer management area (RMA) covers 36% of Finland’s total area. In this area, semi-domesticated reindeer (Rangifer tarandus tarandus) have a free grazing right which is not dependent on land ownership (Reindeer Herding Act 1990/848, Heikkinen et al., 2012). There is a vast cultural, historical and geographical diversity within the RMA. Both Indigenous Saami and Finnish people practice reindeer husbandry in Finland, unlike in Sweden and Norway where it is mainly an exclusive right of the Saami In a globalizing north, reindeer husbandry shares the same opera tional space with several other land-use forms. Economic development, such as industrial infrastructure projects and tourism activities, is growing in the Arctic (AMAP, 2017; Finger & Heininen, 2019; Forbes, 2006). In Fennoscandia, reindeer husbandry has conflicting interests with other land-use forms, mainly forestry (timber harvest), agriculture, exploration and extraction of natural resources, outdoor recreation and historical and geographical diversity within the RMA. Both Indigenous Saami and Finnish people practice reindeer husbandry in Finland, unlike in Sweden and Norway where it is mainly an exclusive right of the Saami i 1 When referring to reindeer as a livelihood, we use the term “reindeer husbandry”. “Herding” refers to day-to-day practices (and it also appears in the term “reindeer herding district”), whereas husbandry considers reindeer as a resource and is related for example to the profits, breeding, and social mechanisms. “Reindeer management” is related to all of the practices pertaining to the keeping of reindeer, including governance (Forbes, 2006). Applied Geography 134 (2021) 102501 S. Rasmus et al. area subsidy policy since reindeer husbandry in Finland belongs to the EU’s unified food and agriculture sector (Rees et al., 2008; Turunen & Vuojala-Magga, 2014). Other land use in the RMA is governed first and foremost by the National Land Use Guidelines (2017) which steer regional and municipal zoning in Finland. 1.1. Reindeer management in Finland Herders are consulted during planning of land-use projects affecting their livelihood (for example during the Environmental Impact Assessment procedures), but they do not necessarily have enough power to affect decision making (Landauer & Komendantova, 2018). In the Saami homeland, legislation on the rights of the Saami as an Indigenous people plays a central role in land-use planning (Markkula et al., 2019). i tourism, and energy production (Pape & L¨offler, 2012; Pettersson et al., 2017; Sarkki et al., 2018). Intensifying land use has led to fragmentation and deterioration of reindeer pastures, causing the available pasture area to shrink (Anttonen et al., 2011; Jaakkola et al., 2013; Kumpula et al., 2014). The warming climate poses additional challenges, among them increased risk of ice formation on the winter pastures (Rasmus et al., 2018; 2020a) and lengthening of the heat periods and increased insect harassment during the summer (Soppela & Turunen, 2017; Tur unen et al., 2016). , Reindeer husbandry is dependent on management decisions of external public authorities (Heikkil¨a, 2006). It is steered by the Ministry of Agriculture and Forestry of Finland. Furthermore, the European Union steers the livelihood by means of national livestock and arable Economic profitability of reindeer husbandry depends on several factors such as legislation, markets and historical legacies (Bernes et al., Table 1 Table 1 The drivers of change in the reindeer management system in Finland and examples of their impacts on reindeer husbandry, based on national reviews and reports (1919–2020). The shading marks the drivers considered in our study (Grotenfelt, 1919; J¨a¨ask¨o, 2001; Pakkanen & Valkonen, 2012; Sonnenfeld, 1972; Working committee, 1992). The drivers of change in the reindeer management system in Finland and examples of their impacts on reindeer husbandry, based on national reviews and reports (1919–2020). The shading marks the drivers considered in our study (Grotenfelt, 1919; J¨a¨ask¨o, 2001; Pakkanen & Valkonen, 2012; Sonnenfeld, 1972; Working committee, 1992). Applied Geography 134 (2021) 102501 S. Rasmus et al. 2015). These are continuously evolving and diversifying. Still, the basis for successful reindeer husbandry is the productive herd of healthy an imals (Kynk¨a¨anniemi, 2020). The welfare of reindeer is largely deter mined by the quantity and quality of available forage (Helander-Renvall, 2014; Kitti et al., 2006). Forage adequacy ensures productivity of dams, which, in turn, affects the calving success and calf weight, both being pivotal to the profitability of the livelihood (Muut toranta et al., 2014). In addition to the area and state of pastures, limited access to pasture resources reduces forage availability: “It is more often a question of access to the forage than the amount of forage as such” (Kitti et al., 2006, p. 149). Factors affecting the accessibility of forage are mostly biophysical (Forbes, 2006; Rasmus et al., 2018); some are related to the management choices or governance of land use (e.g., Eira et al., 2018; Riseth et al., 2016). conditions, various snow types, and predator behavior (Porsanger & Guttorm, 2011; Turunen & Vuojala-Magga, 2014). Perception geography approach has been applied for example in studies on risk management (Ren et al., 2016) and urban planning (Bergeron et al., 2014). Using local perceptions together with the quantitative data from monitoring surveys has been as a tool to get to more holistic understanding of the changes in ecosystems and the nature-based livelihoods such as reindeer husbandry (Rasmus, Turunen, Luomaranta, et al., 2020), fishing (Coll et al., 2014) or forest use (Meijaard et al., 2013). Geographical distribution of perceptions has been studied using map-based methods, which enable the collection and presentation of information about local people’s views of their environment and important places, and can make their incorporation to decision-making easier (Nikula et al., 2020; Ren et al., 2016). Table 1 Public Participation Geographic Information Systems (PPGIS) can be used to locate percep tions on a map and are designed to involve wide audience. During recent years these have been applied in land use studies both in urban and rural areas (Brown & Kytt¨a, 2014; Kahila-Tani et al., 2016; Kantola et al., 2018; Nikula et al., 2020). Understanding people’s perceptions towards for example certain forms of land use may facilitate the management of conflicts between land users with different interests in the same resource (Brown et al., 2020). Understanding the spatial differences both in the drivers of change and in the perceptions towards them will help develop jurisdictional and institutional strategies to support governance and future of the northern livelihoods such as reindeer husbandry. Reindeer management represents an example of a social-ecological system (SES) (cf. Ostrom, 2009; McGinnis & Ostrom, 2014) in which biophysical and socio-economic factors are interacting as drivers of change (K¨ayhk¨o & Horstkotte, 2017). For reindeer management in Finland, these drivers and related impacts on the livelihood are rather well-known (Table 1). Already about a hundred years ago deterioration of pastures and conflicts between reindeer husbandry and other land-use forms were listed as factors causing pressure on reindeer husbandry (Table 1; Kortesalmi, 2007). A recent national report (Kumpula & Siitari, 2020) presented recommendations on how to support and develop “ecologically, economically, culturally and socially sustainable use of reindeer pastures, as well as the vitality of reindeer husbandry”. The fact that reindeer husbandry faces varying challenges in different areas within the RMA in Finland was strongly emphasized in the report. Still, local and regional differences are currently not sufficiently considered in the governance of the livelihood. Systematic research on herders’ per ceptions on factors affecting their livelihood is lacking. In addition, more knowledge is needed of the cumulative effects of various factors – ones resulting from the combined effects of multiple activities affecting reindeer management over space or time. Our work, for its part, ad dresses this gap. This work is based on a questionnaire survey conducted during a project “Reindeer health in the changing environment” (2016–2018, Laaksonen, 2016). The survey focused on herders’ perceptions on factors affecting reindeer welfare. This means factors related to drivers of change such as climate and weather, pasture conditions and land use (shaded in Table 1). Table 1 Consequently, this paper also focuses on these concrete factors herders experience in their daily herding work; factors affecting the success of the livelihood through reindeer condition and available forage. In reindeer management, some drivers, such as governance institutions, are of socio-economic or cultural nature (Table 1). In this study we do not focus on these drivers. Our research questions are: 1.2. Perception geography approach We use a perception geography approach (Downs, 1970, Sonnenfeld, 1972), basing our analysis on herder perceptions on and observations of their operational environment. The concept ‘Perception geography’ or ‘Perceptual geography’ emerged in the 1960s from the wider conceptual framework of behavioral geography (see Bunting & Guelke 1979; Tuan, 2003) and it provides tools and methods to discuss and quantify land scape research and is applicable also in natural resources management. 1) Which factors of the climatic, ecological and land-use related drivers of change are perceived by herders as important for reindeer welfare and why? 2) What kinds of regional differences are there in these perceptions within the RMA? 3) Can the regional differences be explained by regional variation in land-use patterns, climatic and ecological conditions, or varying herding traditions and practices? Perceptual geography is characterized by a common idea that experience affects perception, which leads to the conclusion that per ceptions vary because individuals’ life experiences differ (Tuan, 2003). Perceptions are understood as points of contact between people and their environment and as a basis for spatial reasoning and decision making. Perception is the process that encodes the objective environ ment as a subjective one (Golledge & Stimson, 1997), with the subjective environment and past experiences influencing our behaviour and ac tions (Sonnenfeld, 1972; Guelke, 2003). 2.1. The study area The study area covers the RMA in Finland, situated between 64.5◦N and 70.1◦N (Fig. 1a, Fig. S1). The sparsely populated region is charac terized by boreal coniferous forests, mires, subarctic mountain birch woodlands and fells. For the time period from 2000 to 2019, the maximum number of reindeer over one year old allowed within the region has been set at 203,700. The numbers are set by the Ministry of Agriculture and Forestry every ten years. This is the size of the winter stock; furthermore, ca. 100,000 calves are born in spring (RHA 2018). Of this total number, ca. 80,000 calves and 20,000 over one-year-old reindeer are slaughtered in autumn. Finland has 54 herding districts. A reindeer herder can practice herding within one herding district at a time, and every district is responsible for reindeer herding within its area. The districts are regulated by the Reindeer Husbandry Act (1990/848). During the study period, there were approximately 4500 Herder perceptions are relevant when studying the reindeer man agement SES. Herders are central actors in the system: they experience and deal with drivers of change such as land use and climate change in their daily professional life (Landauer et al., 2021). Their perceptions affect the functioning of the system for example through decision making and risk preparedness. Perceptions also carry culture, and local and traditional knowledge (Forbes et al., 2020; Jaakkola et al., 2018). This means knowledge and practices, developed during centuries and handed down from generation to generation (Berkes 2008). Knowledge also accumates and develops through context-situated learning in new situations. Knowledge can consider for example reindeer behavior, forage and diseases, suitable herding practices during adverse weather 3 Applied Geography 134 (2021) 102501 S. Rasmus et al. State and privately owned land used for forestry and the present and planned land-use projects in the reindeer management area (RMA; region ensity in the northern area and close to the eastern border, or agricultural regions in the southern area not shown). b) The change in the annual ) in the number of warm weeks in the winter season in 1981–2010; locations with significant trends (at 5% significance level) are marked with b ta: Finnish Environment Institute (2020) and Finnish Meteorological Institute. Fig. 1. Table 2 Table 2 Factors related to climate and weather, usability of pasture resources, and other land use and disturbances, considered in the survey. Driver of change Factor considered Factor explained Climate and weather1,2,3,4 Timing of spring Cold and snow are harmful for newborn calves, and lactating reindeer benefit from natural fresh forage. Summer temperature Multiple impacts on reindeer welfare: affecting forage and water supply and insect harassment, causing heat stress. Cold spells in winter Long periods of very low temperatures deplete energy storages of reindeer Deep snow Makes reindeer difficult to access ground lichens; risk of predator attacks increase; herding work gets difficult. Icing events: -icy snow structure -basal ice on pastures Makes reindeer difficult to access ground lichens; decreased usability of pastures. Insect harassment in summer11 Increases stress, energy consumption and vector borne diseases of reindeer. Wild mushroom yield in autumn Important for reindeer to gain fat and energy storage for winter. Usability of pasture resources5,6,7 Summer pasture condition and availability Diverse and peaceful summer pastures needed for reindeer to recover from winter and improve the body condition, and for calves to grow. Winter pasture condition and availability Lichen pastures with adequate quantity and quality needed for winter survival, welfare, calving success and calf weight Fragmentation of pastures Fragmented pastures are difficult to utilize, managing the herds and controlling the grazing becomes difficult for herders. Availability of nature conservation areas Less human disturbance and other land use in conservation areas; diverse and peaceful seasonal pastures and increased winter forage availability because of old-growth forests. Other land use1,7,8,9,10 Forestry Forestry decrease the amount of old-growth forests important for reindeer as winter pastures; increases fragmentation of pastures; harvesting of dense forests can improve lichen growth. Mining Mining districts occupy pasture land and increase fragmentation of pastures; operations and transportation increase the risk of accidents, noise and dust impacts. Peat extraction Peat extraction destroys summer pastures and calving areas, reindeer may drown in deep dikes; areas can be utilized by reindeer to avoid insect harassment. Hunting/dogs Free-running dogs can kill or injure reindeer or cause extra work for herders by scattering the herd. The impacts are most common during rut, and can affect calf production. Disturbances by human activities Reduced forage availability through decreased access or usability of pastures; may disturb calving and grazing. 2.1. The study area a) State and privately owned land used for forestry and the present and planned land-use projects in the reindeer management area (RMA; regions with high predator density in the northern area and close to the eastern border, or agricultural regions in the southern area not shown). b) The change in the annual degree day sum and c) in the number of warm weeks in the winter season in 1981–2010; locations with significant trends (at 5% significance level) are marked with black check marks. Data: Finnish Environment Institute (2020) and Finnish Meteorological Institute. Fig. 1. a) State and privately owned land used for forestry and the present and planned land-use projects in the reindeer management area (RMA; regions with high predator density in the northern area and close to the eastern border, or agricultural regions in the southern area not shown). b) The change in the annual degree day sum and c) in the number of warm weeks in the winter season in 1981–2010; locations with significant trends (at 5% significance level) are marked with black check marks. Data: Finnish Environment Institute (2020) and Finnish Meteorological Institute. Applied Geography 134 (2021) 102501 S. Rasmus et al. 2.2. Data collection method According to regulation on land-use, the land in this area should not be used in a way detrimental to herding. The 13 northernmost districts belong to the Saami Homeland area (SHA) in Finland (Reindeer Husbandry Act 848/1990, N¨akk¨al¨aj¨arvi & Jaakkola, 2017). In the ASR, and especially in the SHA, the herds are generally larger and reindeer husbandry is more commonly the main source of livelihood, whereas in the southern districts, reindeer husbandry is traditionally more often combined with other livelihoods, particularly small-scale agriculture and forestry and reindeer-based tourism (Jaak kola et al., 2018; Soppela & Turunen, 2017). Supplementary winter feeding in enclosures is a more common practice in central and southern parts of the RMA. In the north, especially in the SHA, the livelihood is more based on herding the reindeer on natural pastures (Helle & Jaak kola, 2008; Turunen & Vuojala-Magga, 2014). For these reasons, we present some of the results separately for the northern area (ASR including the SHA) and the southern area (the rest of the RMA). The survey respondents were asked to estimate their perceptions towards 32 factors on a scale from −4 to 4, whereby −4 denotes a considerable negative effect, 0 denotes no effect and 4 denotes a considerable positive effect. They were asked to consider the latest ten- year period in their answers. Our study covers 94% of herding districts within the RMA (51 out of total of 54). The survey was conducted mainly on-site in the reindeer herding districts the herders represent by one of the co-authors (J.T.) between May 2016 and January 2017. In three cases herders were interviewed by phone. All respondents were male and full-time herders. Chiefs of districts (porois¨ant¨a) were selected to represent each district as key informants because they have the most holistic knowledge of the district they represent (purposive sampling; Bernard, 1995). i Topography, vegetation and climate vary within the RMA as well. Clear warming trends have been observed during the past decades (Fig. 1b and c). In reindeer management, impacts of changing climate are experienced through changing seasonal weather conditions. These impacts are already being observed across the area but in varying degree (Rasmus, Turunen, Luomaranta, et al., 2020). The key findings are presented in the form of maps, while all answers The key findings are presented in the form of maps, while all answers 2.2. Data collection method reindeer owners in Finland of which an estimated 900 are full-time herders (RHA 2018). Herding cultures, seasonal herding practices, reindeer numbers per district, as well as the intensity and type of other land use vary greatly within the RMA (Table S1, Fig. 1a). The 20 northernmost districts belong to the area specially intended for reindeer husbandry (ASR; “northern area”). According to regulation on land-use, the land in this area should not be used in a way detrimental to herding. The 13 northernmost districts belong to the Saami Homeland area (SHA) in Finland (Reindeer Husbandry Act 848/1990, N¨akk¨al¨aj¨arvi & Jaakkola, 2017). In the ASR, and especially in the SHA, the herds are generally larger and reindeer husbandry is more commonly the main source of livelihood, whereas in the southern districts, reindeer husbandry is traditionally more often combined with other livelihoods, particularly small-scale agriculture and forestry and reindeer-based tourism (Jaak kola et al., 2018; Soppela & Turunen, 2017). Supplementary winter feeding in enclosures is a more common practice in central and southern parts of the RMA. In the north, especially in the SHA, the livelihood is more based on herding the reindeer on natural pastures (Helle & Jaak kola, 2008; Turunen & Vuojala-Magga, 2014). For these reasons, we present some of the results separately for the northern area (ASR including the SHA) and the southern area (the rest of the RMA). Data for our analysis were gathered using an on-site questionnaire survey which was part of semi-structured interview conducted with 51 chiefs of herding districts in the RMA. In the survey factors affecting reindeer welfare were considered. The factors were selected by re searchers based on existing knowledge of external drivers of change affecting reindeer (Tables 1 and 2 and S1). Herding practices as internal drivers of change were discussed as well (Table 3). More information about the factors considered and their impacts on reindeer welfare and reindeer husbandry can be found in references in the tables. Reindeer management SES and the relationships between the key factors are thoroughly explained and graphically presented e.g. in K¨ayhk¨o & Horstkotte (2017) and Landauer et al. (2021). Herding cultures, seasonal herding practices, reindeer numbers per district, as well as the intensity and type of other land use vary greatly within the RMA (Table S1, Fig. 1a). The 20 northernmost districts belong to the area specially intended for reindeer husbandry (ASR; “northern area”). Table 2 Other land use (wind farms, hydropower, infrastructure like roads etc.) Land use occupies pasture land and increases fragmentation of pastures; reduced forage availability through decreased access or usability of pastures; grazing pressure on remaining pastures increases. Predation Wolverine (Gulo gulo), wolf (Canis lupus), brown bear (Ursus arctos), lynx (Lynx lynx) and golden eagle (Aquila chrysaetos) hunt and kill reindeer, which can cause severe damage to herds and extra work for herders though mitigating the damage and looking for carcasses (needed to get compensations). Decreases the usability of certain pastures. 1Pape & L¨offler 2012, 2Turunen et al.,2016, 3Jaakkola et al.,2018, 4Rasmus et al.,2020a, 5Kitti et al.,2006, 6Kivinen et al.,2012, 7Kumpula et al.,2014, 8Anttonen et al. Cold and snow are harmful for newborn calves, and lactating reindeer benefit from natural fresh forage. Multiple impacts on reindeer welfare: affecting forage and water supply and insect harassment, causing heat stress. Makes reindeer difficult to access ground lichens; risk of predator attacks increase; herding work gets difficult. i Makes reindeer difficult to access ground lichens; risk of predator attacks increase; herding work gets difficult. i i Makes reindeer difficult to access ground lichens; decreased usability of pastures. i Makes reindeer difficult to access ground lichens; decreased usability of pastures. Increases stress, energy consumption and vector borne diseases of reindeer. I f i d i f d f i Diverse and peaceful summer pastures needed for reindeer to recover from winter and improve the body condition, and for calves to grow. Diverse and peaceful summer pastures needed for reindeer to recover from winter and improve the body condition, and for calves to grow. Lichen pastures with adequate quantity and quality needed for winter survival, welfare, calving success and calf weight i Lichen pastures with adequate quantity and quality needed for winter survival, welfare, calving success and calf weight i Fragmented pastures are difficult to utilize, managing the herds and controlling the grazing becomes difficult for herders. i Less human disturbance and other land use in conservation areas; diverse and peaceful seasonal pastures and increased winter forage availability because of old-growth forests. i Less human disturbance and other land use in conservation areas; diverse and peaceful seasonal pastures and increased winter forage availability because of old-growth forests. Forestry decrease the amount of old-growth forests important for reindeer as winter pastures; increases fragmentation of pastures; harvesting of dense forests can improve lichen growth. 11not a climatic factor, but related to for example seasonal temperature, precipitation and wind conditions. Table 3 Due to poor winter pasture resources or limited access to forage, for example, due to icy snow, reindeer are fed with supplementary feeds in their natural pastures (often in forests); helps also controlling the herds and protecting reindeer from predators. Feeding in the pasture area to support active herding. On-the-spot management of the movement and foraging of herds; moving herds with or without the aid of hay from one pasture area to another, shepherding. Practice where certain pastures are reserved for certain seasons and natural seasonal behaviour of reindeer is supported by fences separating the pastures, and by active herding. are annually treated with antiparasitic medication, to improve the condition. In enclosure calving, reindeer give birth within a fenced pasture area. The calves are ear-marked immediately after their birth with the owner’s earmark i In free-ranging type of calving reindeer give birth in their natural calving regions and specific spots such as forested areas or the southern slopes of fells. Reindeer are gathered from pastures to summer round-ups, in which the calves are ear-marked with the owner’s reindeer earmark from midsummer onwards. Timing of slaughtering3,4,5 Impacts on amount of meat to sell and meat quality. The later the slaughter, the poorer the condition of reindeer generally is; they start loosing weight after the snow cover forms. Impacts on amount of meat to sell and meat quality. The later the slaughter, the poorer the condition of reindeer generally is; they start loosing weight after the snow cover forms. Pekkarinen et al., 2015, 2Horstkotte et al., 2020, 3Forbes 2006, 4Helle & Jaakkola 2008, 5K¨ayhk¨o & Horstkotte, 2017, 6Laaksonen et al., 2017. brmss (Bürkner, 2017) was used for fitting the GLMs and bayesplot (Gabry & Mahr, 2021) for creating visualizations from the models. i of the on-site questionnaire survey are shown in Tables S2-S5 – in the Supplementary material. The survey respondents had the possibility to comment on their answers (15 comments received), provide additional information on calving success of reindeer (37 answers received) and give suggestions on how to increase the welfare of reindeer (50 sug gestions received). Some excerpts of this free-form material are pre sented as part of the results to illustrate the themes of this article. Table 3 Table 3 Factors related to herding practices in use (internal drivers), considered in the survey. Driver of change Factor considered Factor explained Herding practices Supplementary winter feeding in enclosures1,2 Due to poor winter pasture resources or limited access to forage, for example, due to icy snow, reindeer are taken into enclosures and given supplementary feeds daily for several months; also protects reindeer from predators and keeps them off the roads and settlements. Supplementary winter feeding in the field1,2 Due to poor winter pasture resources or limited access to forage, for example, due to icy snow, reindeer are fed with supplementary feeds in their natural pastures (often in forests); helps also controlling the herds and protecting reindeer from predators. “Herding feeding”1,2 Feeding in the pasture area to support active herding. Active herding3,4,5 On-the-spot management of the movement and foraging of herds; moving herds with or without the aid of hay from one pasture area to another, shepherding. Pasture rotation3,4,5 Practice where certain pastures are reserved for certain seasons and natural seasonal behaviour of reindeer is supported by fences separating the pastures, and by active herding. Antiparasitic medication6 Reindeer are annually treated with antiparasitic medication, to improve the condition. Managing calving in enclosures3,4,5 In enclosure calving, reindeer give birth within a fenced pasture area. The calves are ear-marked immediately after their birth with the owner’s earmark Earmarking of calves during summer3,4,5 In free-ranging type of calving reindeer give birth in their natural calving regions and specific spots such as forested areas or the southern slopes of fells. Reindeer are gathered from pastures to summer round-ups, in which the calves are ear-marked with the owner’s reindeer earmark from midsummer onwards. Timing of slaughtering3,4,5 Impacts on amount of meat to sell and meat quality. The later the slaughter, the poorer the condition of reindeer generally is; they start loosing weight after the snow cover forms. 1Pekkarinen et al., 2015, 2Horstkotte et al., 2020, 3Forbes 2006, 4Helle & Jaakkola 2008, 5K¨ayhk¨o & Horstkotte, 2017, 6Laaksonen et al., 2017. Due to poor winter pasture resources or limited access to forage, for example, due to icy snow, reindeer are taken into enclosures and given supplementary feeds daily for several months; also protects reindeer from predators and keeps them off the roads and settlements. Table 3 To show the significance of cumulative effects of different land-use activities we calculated how many of the following factors were considered as harmful per district: forestry, mining, peat extraction, hunting/dogs, other disturbances by human activities (such as outdoor recreation), other land use (such as wind farms, hydropower, infra structure; see Table 2). We interpreted answers −3 or −4 as a harmful effect and 3 or 4 as a beneficial effect. The dataset was rescaled from range [-4,4] to [-1,1] for the regression models. Essentially all negative values were considered as negative were considered as negative effect (−1), zero values as neutral (0) and positive values as positive (1) effect. Perceptions with only negative and neutral (icing, predation, human disturbance, mining, hunting) or neutral and positive (herding, conser vation areas, mushroom yield) values were omitted from the analysis. Detailed background information on the herding practices of each district was also collected. Due to the quality and, in some cases, the confidentiality of the material, they are not shared in full as part of the original data set for this study, but referred to under the Results section to provide further insight into the commonness of and regional differ ences in certain herding practices. Our study also draws on articles published in the professional journal Poromies [Reindeer herder] in order to describe the changes herders have experienced in the pasture environment and the subsequent adoption of the supplementary winter feeding (Supplementary text S1). The journal has been published since 1931 by the Reindeer Herders’ Association in Finland, and it is a commonly used source of information in research on reindeer management (Kortesalmi, 2007; Helle & Jaak kola, 2008, Vuojala-Magga et al., 2011; Turunen et al., 2017). We used absence/presence data for analyzing the perceptions regarding peat production and mining. In these models the A/P variable was used as a predictor for the corresponding perception. Perceptions on forestry were analyzed using log-transformed ratio of private and state owned forests within each reindeer herding district as a predictor. Regression models were fitted using tight priors as constraints as sug gested by Gelman et al. (2020). Details are found in Table S9 in the Supplementary material. Table 2 transportation increase the risk of accidents, noise and dust impacts. eat extraction destroys summer pastures and calving areas, reindeer may drown in deep dikes; areas can e utilized by reindeer to avoid insect harassment. Peat extraction destroys summer pastures and calving areas, reindeer may drown in deep dikes; areas can be utilized by reindeer to avoid insect harassment. Free-running dogs can kill or injure reindeer or cause extra work for herders by scattering the herd. The impacts are most common during rut, and can affect calf production. Reduced forage availability through decreased access or usability of pastures; may disturb calving and grazing. Land use occupies pasture land and increases fragmentation of pastures; reduced forage availability through decreased access or usability of pastures; grazing pressure on remaining pastures increases. Wolverine (Gulo gulo), wolf (Canis lupus), brown bear (Ursus arctos), lynx (Lynx lynx) and golden eagle (Aquila chrysaetos) hunt and kill reindeer, which can cause severe damage to herds and extra work for herders though mitigating the damage and looking for carcasses (needed to get compensations). Decreases the usability of certain pastures. 1Pape & L¨offler 2012, 2Turunen et al.,2016, 3Jaakkola et al.,2018, 4Rasmus et al.,2020a, 5Kitti et al.,2006, 6Kivinen et a 2011, 9Rasmus et al.,2020b, 10Landauer et al.,2021 11not a climatic factor, but related to for example seasonal temperature, precipitation and wind conditions. 5 Applied Geography 134 (2021) 102501 S. Rasmus et al. S. Rasmus et al. 3. Results Depending on the factor studied, the distribution of answers shows agreement, disagreement and even polarization of perceptions. The general agreement on certain biophysical factors is clear (Fig. 3a and b). Perceptions on deep snow cover (Fig. 3c) give a good example of disagreement among the survey respondents. Generally, deep snow makes reindeer foraging more difficult, but in some districts with ample forest pastures it may ease grazing on arboreal lichen. Moreover, snow depth is not a significant factor in districts where most of the reindeer are fed in enclosures. Also, some land-use related factors such as forestry were considered as neutral or even beneficial by some respondents, although forestry was generally seen as harmful for reindeer husbandry (Fig. 3d). All respondents perceived hunting negatively, although the importance of this factor varied (Fig. 3e). Three herders from the southern area explain the effects on reindeer: “Hunting dogs disturb rutting [reindeer], which has an impact on the calf percentage … Sometimes the [presence of]hunting dogs delays the rut so that calves are lighter when slaughtered … We should put an end to the barking of the elkhounds early in the autumn because it breaks up the herds, and the calves may become separated from the dams.” 2.3. Analyses We divided the observations into two groups according to borders presented in Fig. 1a. The first group (n = 17) consists of herding districts north of the ASR -border, “northern area”, including the Saami Home land area (SHA). For the second group (n = 34) we combined the dis tricts within the rest of the RMA (“southern area”). Differences in herding cultures and practices between these groups, as well as the in tensity of other land use and the land-use regulations, justify this divi sion. Considering the SHA separately was not considered possible because of small number of survey respondents from that area (n = 10). We analyzed the group differences on perceptions using Bayesian ordinal regression framework as presented by Bürkner and Vuorre (2018). Cumulative models with probit -link were fitted using each perception as response variable and group as a predictor. The perceptions of the herders were analyzed by using exploratory data analysis methods and the differences of perceptions between the groups were analyzed using Bayesian generalized linear regression models (GLM). The survey data were combined with existing spatial data- sets on land use of the study region (RHA 2018; Mets¨ahallitus, 2019, Finnish Environmental Institute, 2020) to explain and discuss the results. Regional comparisons of the data were conducted by mapping the data into choropleth maps and compared visually. Data preparation, visual isations and analyses were done in R (R Core Team, 2020). The Tidyverse package (Wickham et al., 2019) was used for data preparation and visu alisations, the tmap package (Tennekes, 2018) for plotting the maps and the sf package (Pebesma, 2018) for spatial data operations. R-package S. Rasmus et al. S. Rasmus et al. Applied Geography 134 (2021) 102501 3. Results 3.2. Regional heterogeneity in perceptions Perceptions on the importance of factors related to climate and weather were rather homogeneous within the whole RMA (Table S2). Cold and rainy summers were mainly perceived as a harmful factor (Fig. 4a), but in some southern districts as a beneficial one. Few districts from the central region viewed also hot summers positively, although this factor is generally considered harmful to reindeer (Rasmus, Tur unen, Luomaranta, et al., 2020). Survey respondents explain how climate-related factors directly affect the welfare of the animals and, thus, the future calving success: “After a hot summer, calf production was very weak. The dams were not in heat” (Southern area); “Warm autumns led to a weak rutting period, the stags got lazy … Rutting is delayed in warm autumns. The dams need frosts to be in heat” (two herders from the southern area); “A cold winter may also cause dams to abort their calf” (Northern area). Despite the fact that forestry is practiced in most of the reindeer herding districts (Fig. 1a) and it is considered as a disturbing factor to reindeer husbandry by other studies (Table S1), perceptions of our survey respondents on forestry were diverse (Fig. 5c) and, overall, less negative than expected. Interestingly, perceptions on forestry were more often negative in regions where forests are mostly state owned (Fig. 5f), compared to regions where forest ownership is mostly private. When reading the results of a 30-year-old comparable survey together with ours (Supplementary text S1), the intensification and diversification of land use within the RMA during the past decades is clearly visible. Approximately 30 years ago, forestry stood out very clearly as the most harmful form of land use affecting reindeer hus bandry. Only a few other factors were mentioned by the respondents then, tourism and agriculture being the most common ones. Several other land-use related factors – hunting, human disturbance, peat extraction, other land use and mining – are nowadays seen as equally or more harmful than forestry in the districts in which forestry was considered as the most harmful factor in the 1980s. (Supplementary text S1, Table 4). Perceptions on factors related to pasture resources were heteroge neous (Table S4). Summer pastures were nearly unanimously viewed as beneficial for reindeer welfare. Less agreement was seen in the percep tions on winter pastures (Fig. 4b). 3.1. Importance of the drivers There was a strong agreement among the survey respondents that certain biophysical factors are harmful. For example, 75% of the herders perceived the impact of predators and 80% considered icy foraging conditions as harmful (Fig. 2a; See also Tables S2–S5). The respondents also agreed on benefits of certain biophysical factors such as abundance of mushroom (92% considered beneficial; Fig. 2b) and early arrival of spring (69% considered beneficial). As one herder from the northern area put it: “Dams’ success for becoming pregnant depends on mushroom yield”. Disturbance-related factors such as hunting were generally perceived as harmful. Perceptions on certain herding practices diverged the most. Feeding in enclosures was seen as an important, positive factor in the southern part of the RMA, but in the north it was mostly seen as a method which should not be preferred. Instead, herders in the northern part of the RMA preferred practices such as active herding (moving herds, shepherding) and feeding to support this (“herding feeding”). 7 Fig. 2. Factors perceived as harmful (a) for the welfare of reindeer; the percentage of respondents sharing the view (answer −3 or −4; N = 51); Factors perceived as beneficial (b) for the welfare of reindeer, the percentage of respondents sharing the view (answer 3 or 4; N = 51). Fig. 2. Factors perceived as harmful (a) for the welfare of reindeer; the percentage of respondents sharing the view (answer −3 or −4; N = 51); Factors perceived as beneficial (b) for the welfare of reindeer, the percentage of respondents sharing the view (answer 3 or 4; N = 51). 7 Applied Geography 134 (2021) 102501 S. Rasmus et al. Applied Geography 134 (2021) 1025 ig. 3. Distribution of herder perceptions on selected factors: a) basal ice on the pastures, b) abundance of mushrooms, c) deep snow cover, d) forestry, e) huntin ogs, f) peat extraction areas, g) pasture rotation, h) active herding (−4 denotes a considerable negative effect, 0 no effect and 4 a considerable positive effect Rasmus et al. Fig. 3. Distribution of herder perceptions on selected factors: a) basal ice on the pastures, b) abundance of mushrooms, c) deep snow cover, d) forestry, e) hunting/ dogs, f) peat extraction areas, g) pasture rotation, h) active herding (−4 denotes a considerable negative effect, 0 no effect and 4 a considerable positive effect). S. Rasmus et al. 3.1. Importance of the drivers Applied Geography 134 (2021) 102501 areas were considered beneficial for reindeer husbandry, also by herders from districts lacking such areas. Perceptions on peat extraction (Fig. 3f) give an example of localized views. Most of the survey respondents considered peat extraction as a neutral factor, but some perceived it as very harmful. Perceptions on some herding practices were also localized, pasture rotation being an example (Fig. 3g). It was perceived mostly as a neutral factor, except by districts benefiting from it in their own work. Active herding was most often perceived either neutrally or very positively (Fig. 3h). The importance of natural pastures was seen in many of the free-form comments of the respondents, as well as the need for actions to foster the pasture quality: “Grazing peace and diverse pastureland are needed” (Northern area); “Old-growth forests should not be cut. In late winter [they are] very important for reindeer” (Northern area); “Pasture regeneration would be important” (Southern area). Land-use related factors were generally perceived either as neutral or negative (Table S3). For example, perceptions on peat extraction (Fig. 5a) and mining (Fig. 5b) were localized. These forms of land use were considered harmful especially in areas where such activities had existed in the past, currently existed or were under planning (Fig. 5d and e, see also Fig. 1a and Table S1). Several herders from the southern area wanted to see the peat extraction areas restored: “Reindeer husbandry should be considered in the re-use of peatlands” (Southern area). Fig. 4. a) Perceptions of herders on factors “Cold and rainy summer” and b) “Winter pastures” (−4 denotes a considerable negative effect, 0 no effect and 4 de notes a considerable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 4. a) Perceptions of herders on factors “Cold and rainy summer” and b) “Winter pastures” (−4 denotes a considerable negative effect, 0 no effect and 4 de notes a considerable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3.2. Regional heterogeneity in perceptions We assume that the perceptions are linked to the particular situation of each district: quality and usability of seasonal pastures and pasture accessibility. Good summer pastures are most often available, but in some regions lichen pastures for winter foraging are scarce and the quality of the remaining winter pastures is low. Especially interesting is a region in the middle of the RMA, where winter pastures were seen as a factor affecting reindeer welfare nega tively, most probably due to the impact of forestry. Nature conservation 9 Fig. 4. a) Perceptions of herders on factors “Cold and rainy summer” and b) “Winter pastures” (−4 denotes a considerable negative effect, 0 no effect and 4 de notes a considerable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Applied Geography 134 (2021) 102501 S. Rasmus et al. Perceptions of herders on the factor “Peat extraction”, the black dots show the peat extraction areas; b) “Mining”, the black dots show the locations of stricts; c) “Forestry” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect), d-e) Marginal effects of absenc of peat extraction/mining districts on the perceptions of herders on factors “Peat extraction and “Mining” (posterior mean with 89% credible intervals effect of forestry ownership ratio (private or state) on the perceptions of herders on factor “Forestry” (posterior mean with 89% credible intervals). See r the land used for forestry. Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red l order of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web versio e.) Fig. 5. 3.4. Perceptions on herding practices Antiparasitic medication as well as early slaughtering were generally stated as beneficial factors by the survey respondents (Table S5). Of the districts studied, 92% aimed at early slaughter annually during the study period and 94% gave antiparasitic medication to the majority of their animals. Then again, some herders expressed somewhat critical views of medication: “Medication should be developed as the reindeer will become immune or new diseases will appear” (Southern area); “Medication should be given only to [the reindeer] in poor condition” (Southern area). According to our data, in 90% of the districts studied majority of reindeer got some supplementary feed during a typical winter during the study period. Several forms of feeding (feeding in the forest/pastures, feeding in the enclosures, “herding feeding” to support moving the herds) were used depending on the need and situation in an individual district. At least some feeding in the forest/pastures was practiced in 55% of the districts (the main form of feeding in four of the studied districts). At least some “herding feeding” was practiced in 29% the districts (the main form of feeding in seven districts, six of them belonged to the Saami Homeland area). At least some reindeer were fed in enclosures in 88% of the districts (the main form of feeding in 34 districts). Interestingly, perceptions on some herding practices varied a great deal either locally or regionally (Table S5). Pasture rotation is a practice where certain grazing lands are reserved for certain seasons. Natural seasonal behaviour of reindeer is supported by fences separating the pastures, and by active herding of animals from one area to another. Pasture rotation may not be possible if grazing lands are fragmented due to competing land-use forms and the related infrastructure (Anttonen et al., 2011), or if some seasonal pasture types are missing from the area of the district. Also, low lichen biomass on winter pastures may hinder the use of these (Kumpula et al., 2014). Overall, 39% of the total number of the districts studied – and all districts situated in the northern part of the RMA, including the Saami Homeland area – use pasture rotation. Most respondents considered pasture rotation as beneficial (Fig. 7a), although the importance of this factor varied. Similarly, as feeding reindeer in enclosures seems to divide the RMA in distinct regions (Fig. 9a), also perceptions on it were rather polarized (Fig. 9b and c). 3.3. Cumulative effect of land use enclosures in 49% of the districts studied. Half or more of the calves were born in enclosures only in three districts; these were located in the northern area. Earmarking of calves during summer was common, with 82% of the studied districts practicing this. Herders may also mark calves earlier in the spring in the case of enclosure calving, or marking may be postponed until autumn if there are problems with collecting the animals in the summer or if there is a risk of heat stress during hot pe riods (Rasmus, Turunen, Luomaranta, et al., 2020). At least one land-use related factor was considered harmful by 86% of the survey respondents. Many districts listed several factors as harmful. When the sum of these views (number of these factors per district) was mapped, some hot-spot areas of land use could be seen (Fig. 6a). Characteristic of these hot-spot areas is their location within the forestry region of the RMA, and close to the southern border of the RMA and/or roads with heavy traffic. The pasture lands of districts host active or planned mines and wind farms, hydropower reservoirs and peat extraction areas (Fig. 1b, Table S1). The districts perceiving several land-use related factors as harmful often also considered fragmentation of pastures as a problem (Fig. 6b). Some overlap is also seen with the region where the winter pasture situation was perceived negatively (Fig. 4b). Particularly heterogeneous perceptions were related to supplemen tary winter feeding. Some feeding of reindeer has been practiced in northern Fennoscandia for centuries (Helle & Jaakkola, 2008; Salmi et al., 2020). In the past, during difficult foraging conditions in winter lichen has been pulled off the trees, trees rich in lichen have been cut down, and hard snow cover has been broken to make digging easier for reindeer (Turunen & Vuojala-Magga, 2014). Annual feeding was adop ted especially in the southern districts in the 1970s. Due to the scarcity of forest pastures rich with ground and arboreal lichen, reindeer are nowadays provided with supplementary feed in the forest, or they are kept and fed in enclosures for some winter months or even throughout the winter (Helle & Jaakkola, 2008; Turunen & Vuojala-Magga, 2014; see also Supplementary text S1.) Table 4 Table 4 Percentage of districts where certain land-use related factors were seen as harmful as or more harmful than forestry in our study (number of districts = 34; only those districts analyzed, which had seen forestry as the single most negatively affecting activity within their district during 1986–1987, see Supplementary text S1 for details). Factor Factor as harmful as or more harmful than forestry (% of districts) Hunting/dogs 79 Disturbance (tourism, traffic, etc.) 59 Peat extraction 29 Other land-usea 29 Mining 18 a including wind farms, hydropower, and related infrastructure (power transmission lines, roads etc.). Table 4 Percentage of districts where certain land-use related factors were seen as harmful as or more harmful than forestry in our study (number of districts = 34; only those districts analyzed, which had seen forestry as the single most negatively affecting activity within their district during 1986–1987, see Supplementary text S1 for details). Factor Factor as harmful as or more harmful than forestry (% of districts) Hunting/dogs 79 Disturbance (tourism, traffic, etc.) 59 Peat extraction 29 Other land-usea 29 Mining 18 a including wind farms, hydropower, and related infrastructure (power transmission lines, roads etc.). 3.2. Regional heterogeneity in perceptions a) Perceptions of herders on the factor “Peat extraction”, the black dots show the peat extraction areas; b) “Mining”, the black dots show the locations of the mining districts; c) “Forestry” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect), d-e) Marginal effects of absence or presence of peat extraction/mining districts on the perceptions of herders on factors “Peat extraction and “Mining” (posterior mean with 89% credible intervals), f) Marginal effect of forestry ownership ratio (private or state) on the perceptions of herders on factor “Forestry” (posterior mean with 89% credible intervals). See also Fig. 1a, for the land used for forestry. Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 10 Applied Geography 134 (2021) 102501 S. Rasmus et al. 3.4. Perceptions on herding practices According to the free-form answers of the survey re spondents, the northern districts emphasized the harmfulness of the intensive winter feeding: “Keeping reindeer in enclosures is harmful for them. Field feeding [of reindeer] should be controlled to avoid spoiling of the soil.” Also, several southern districts saw negative sides in enclosure feeding: “Reindeer will become lazy when they are kept in enclosures.” “In some regions the reindeer stag population is weak due to feeding in enclosures, because it keeps the animals in a restricted area.” However, enclosure feeding was considered necessary in the southern area: “There should be a shift from enclosure feeding into forest feeding, but predator pressure is too strong”; “We would feed [the reindeer]in the forests, if we had some [forests], but on private land it is not possible”. Several development needs and ideas about enclosure feeding were given by herders from the southern area: “If [reindeer] must be fed in enclosures, the reindeer should have enough space and clean area”. Active herding was a common practice within most of the RMA until the 1960s (Helle & Jaakkola, 2008). Now it is especially considered as a relevant part of the Saami herding tradition (Jaakkola et al., 2018). On the other hand, growing predator populations and increasing predation pressure on reindeer has increased the need for monitoring and con trolling of herds also in some of the southern districts (Turunen et al., 2017). In our study, active herding was locally seen as a beneficial factor (Fig. 7b). Enclosure calving (Fig. 8a) and summertime earmarking of calves (Fig. 8b) were perceived either as beneficial or harmful locally. Ac cording to one respondent from the northern area: “[The animals] should be handled only when necessary. Bringing reindeer to enclosures for calving is not good for reindeer health.” At least some calvings was managed in 11 Applied Geography 134 (2021) 102501 S. Rasmus et al. Applied Geography 134 (2021) 102501 4 Discussion and conclusions harmful were mainly climatic or related to land use factors Cumulative Fig. 6. a) Hot-spot areas of land-use related factors: the number of land-use forms perceived as harmful per district (forestry, mining, peat extraction, hunt ing, human disturbance, other land-use), b) Percep tions of herders on the factor “Fragmentation of pastures” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive ef fect). 3.4. Perceptions on herding practices Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 7. a) Perceptions of herders on the factors “Pasture rotation” and b) “Herding” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) S. Rasmus et al. Fig. 6. a) Hot-spot areas of land-use related factors: the number of land-use forms perceived as harmful per district (forestry, mining, peat extraction, hunt ing, human disturbance, other land-use), b) Percep tions of herders on the factor “Fragmentation of pastures” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive ef fect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 7. a) Perceptions of herders on the factors “Pasture rotation” and b) “Herding” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of hi i l ) Fig. 7. a) Perceptions of herders on the factors “Pasture rotation” and b) “Herding” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). 3.4. Perceptions on herding practices (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4. Discussion and conclusions harmful were mainly climatic or related to land-use factors. Cumulative land-use impacts raised particular concerns. 4.1. Herder perceptions on factors affecting reindeer welfare and reindeer husbandry 4.1. Herder perceptions on factors affecting reindeer welfare and reindeer husbandry (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) S. Rasmus et al. S. Rasmus et al. Fig. 8. a) Perceptions of herders on the “Managing calving in enclosures” and b) “Earm of calves during summer” (−4 denotes a consi negative effect, 0 no effect and 4 denotes a erable positive effect). Purple line: southern b the area specially intended for reindeer hu (ASR; “northern area” in this study). Red line: border of the Saami Homeland area (SHA interpretation of the references to colour in th legend, the reader is referred to the Web ve this article.) Fig. 8. a) Perceptions of herd “Managing calving in enclosures” of calves during summer” (−4 de negative effect, 0 no effect and erable positive effect). Purple line the area specially intended for (ASR; “northern area” in this stud border of the Saami Homeland interpretation of the references to legend, the reader is referred to this article.) Fig. 8. a) Perceptions of herders on the factors “Managing calving in enclosures” and b) “Earmarking of calves during summer” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a consid erable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 9. a) Percentage of reindeer in enclosure feeding during a typical winter, b) Perceptions of herders on factors “Feeding in enclosures” and c) “Herding feeding (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect). Purple line: southern border of the area specially intended fo reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 9. a) Percentage of reindeer in enclosure feeding during a typical winter, b) Perceptions of herders on factors “Feeding in enclosures” and c) “Herding feeding” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect). 4.1. Herder perceptions on factors affecting reindeer welfare and reindeer husbandry According to the survey respondents, factors directly affecting the welfare of reindeer and the calving success are crucial. These are climate-related factors directly affecting the forage availability and grazing such as basal icing, and land-use related factors limiting the seasonal pasture access. For example, ample mushroom yield was considered as highly beneficial as it is associated with increasing the body condition of the reindeer by the onset of winter. Furthermore, in autumn, the presence of hunting dogs may disperse reindeer herds during the rutting period and thus disturb the calving success next spring. Nature conservation areas were perceived as beneficial, as they secure pasturelands from development activities. Indeed, nature con servation has prevented industrial land use on important grazing lands such as old-growth forests. However, the disadvantage for reindeer We studied perceptions of reindeer herders from Finland on factors affecting the welfare of reindeer and the consequent success of reindeer husbandry. These factors could be divided into three categories: 1) factors which were perceived unanimously positively (for example early spring) or unanimously negatively (for example predation), 2) factors on which perceptions were heterogeneous or even polarized (for example supplementary winter feeding), and 3) factors on which perceptions were localized (for example peat extraction). Factors that were most often considered as beneficial were mainly related to pasture resources or certain herding practices. Factors that were most often seen as 12 Applied Geography 134 (2021) 102501 Applied Geography 134 (2021) 102501 Fig. 8. a) Perceptions of herders on the factors “Managing calving in enclosures” and b) “Earmarking of calves during summer” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a consid erable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 9. a) Percentage of reindeer in enclosure feeding during a typical winter, b) Perceptions of herders on factors “Feeding in enclosures” and c) “Herding feeding” (−4 denotes a considerable negative effect, 0 no effect and 4 denotes a considerable positive effect). Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). 4.1. Herder perceptions on factors affecting reindeer welfare and reindeer husbandry Purple line: southern border of the area specially intended for reindeer husbandry (ASR; “northern area” in this study). Red line: souther border of the Saami Homeland area (SHA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) husbandry is that these areas are also habitats for predators (Turunen et al., 2017). Some land-use types raise concerns among the herders even if they do not yet exist but are planned in a particular area. Cumulative long-term effects of other land-use forms on reindeer husbandry have been studied earlier for example by looking at the ef fects of forestry actions (Kivinen, 2015) or infrastructure development (Nellemann et al., 2003; Vistnes et al., 2001). Combined local effects of individual tourist resorts (Nellemann et al., 2000) and extractive in dustries (Fohringer et al., unpublished) have also been studied. Tools to assess the cumulative effects of different drivers have been developed (AMAP, 2017) but they have not been empirically tested for reindeer management in northern Fennoscandia. Our analysis of cumulative land use showed the existence of land-use related hotspots as illustrated by herders’ negative perceptions. Some land-use activities are harmful during certain seasons (for example, land use in the proximity of calving grounds or on or along the route from one seasonal pasture to another, Anttonen et al., 2011) or in certain locations. The disturbing effects of some industrial land use, for example open-pit mines, can cover large areas, considerably larger than the spot where the activity takes place. We compared our results with those presented in a recent national report about the sustainability of reindeer husbandry in Finland (Table S1; Kumpula & Siitari, 2020). Also according to that report at least one land-use related factor was considered as “a moderate or considerable problem” to reindeer husbandry in nearly all herding dis tricts. Multiple land-use types were considered problematic in 63% of the districts. 4.4. Limits of the study We are aware of the fact that our analysis is lacking some important factors. Economic, socio-cultural, and governance aspects were not part of the questionnaire survey although these affect herder perceptions and give preconditions for decision-making and herding practices adopted. As perceptions cannot be objectively measured or observed, mis understandings and misinterpretations are possible both by those taking part and by those conducting the survey and further analyses. In our case, there is actually also an interesting latent level of interpretation in the study setting. We asked the survey respondents to consider either positive or negative effects of various factors on reindeer welfare and reindeer husbandry as a whole. What herders actually provided was not only their experiential knowledge of the subject, but also their inter pretation of “the preferences” of those herded – the reindeer. Thus, when providing their answers they also, whether consciously or uncon sciously, came to share their insights on what is good for the reindeer survival and reproduction from the animal point of view. How a certain factor is perceived among the herders seems to be connected also to historical land-use developments of the district. Forestry serves as a good example. Forestry measures are known to have unfavorable impacts on reindeer husbandry, beginning from the loss and fragmentation of pastures and ending with complicated work conditions (Berg et al., 2008; Helle & Jaakkola, 2008; Jaakkola et al., 2013; Kivinen et al., 2012, 2010; Moen & Keskitalo, 2010; Turunen et al., 2020). On the other hand, forestry as a form of land use has been present in the RMA for over 100 years and reindeer husbandry must have been adapting to it (Helle & Jaakkola, 2008; Turunen et al., 2020). Comparing our results to the results of the 30-year-old survey (Supple mentary text S1; Nieminen, 1988; Nieminen & Autto, 1989) provided interesting insights into the diversification and intensification of land use within the RMA. The majority of the survey respondents listed several other land-use pressures that nowadays override the effect of historical and present-day forestry. One might ask whether herders have, for example, got used to practicing herding in managed forests. Also, some framing effect cannot be ruled out due to the selection and phrasing of the factors considered in the questionnaire survey. 4.5. Challenges for land-use planning In nature-based livelihood SESs (K¨ayhk¨o & Horstkotte, 2017), one biophysical or socio-economic driver can affect another. The impact experienced by reindeer herders over a certain period of time is both the sum of impacts and their accumulation over time. This makes the governance of SESs difficult. Competing forms of land use, predation, degradation and fragmentation of pasture resources pose challenges to reindeer husbandry and give rise to conflicts with other land users (Hukkinen et al., 2003; K¨ayhk¨o & Horstkotte, 2017; Merist¨o et al., 2004; Pohjola & Valkonen, 2012; Soppela & Turunen, 2017). Industrial land use such as mining, wind farms and forest clear-cuts causes local but long-lasting impacts on reindeer husbandry. 4.4. Limits of the study The human memory tends to emphasize the most recent and unordinary conditions (Gray, 1955), so it is also possible that recent weather events or topical land-use projects affected some of the responses presented in this study. Only one person (although the key informant) was inter viewed per district in our study. This means that perception of one in dividual has been used to represent rather large land areas. This has limited also further data analyses. Developing detailed models to explain the regional differences in perceptions or studying spatial correlations between the actual land-use activities and perceptions in detail did not seem possible, based on our data. It would be very interesting to deepen the analysis by gathering more data per district. Also, it would be valuable to broaden the analysis to encompass other Nordic countries or even those parts of Russia where reindeer husbandry is practiced. p g p g g g Interestingly, also forest ownership within herding districts seem to affect perceptions towards forestry (Fig. 5f). Perceptions were negative in districts where state is the main owner of forest lands. Also, in Saami Homeland area forestry was seen as a harmful factor. This can be referred to environmental and political struggles between indigenous Saami people and state forestry (Jokinen 2014). Meanwhile, in the south-east part of the RMA in Finland forestry was often seen as a beneficial factor by herders. Most of the forest land there is privately owned. Many herders are also forest owners, and historically reindeer husbandry must have adapted to operate on private lands and with private forest owners. In social terms it is probably easier to express critics towards state-based forestry than local private forestry. Our re sults hint that herder perceptions towards forestry are not determined only by the ecological impacts on pastures, described above. They seem to be based on social, cultural and economic aspects as well. 4.2. Reasons for heterogeneity in herder perceptions Places and landscapes have various meanings which affect the per ceptions. What is perceived as important means that it has value for an individual; for biological survival, and for providing cultural good (Tuan, 1990). For herders, places and landscapes are both working en vironments and grazing lands for their herds. They also carry socio-cultural meanings and heritage. In the language of ecosystem 13 Applied Geography 134 (2021) 102501 S. Rasmus et al. have also shown that herder perceptions influence their actions (AMAP, 2017; Landauer et al., 2021). Local herding tradition and culture may carry traditional knowledge essential in coping during adverse condi tions. They may also limit the willingness to adapt certain new practices, as they may be perceived as harmful to reindeer or the livelihood. services: in addition to providing provisional and supporting services for them and for their herds, landscapes and places provide non-material benefits in the form of cultural ecosystem services (CAFF, 2015; Kettu nen et al., 2012; Markkula et al., 2019). Working as a herder means communality, social ties and identity (Heikkinen et al., 2012; Helle & Jaakkola, 2008; Kumpula & Siitari, 2020). Herding maintains important features of rural landscape, such as pastures and built structures such as reindeer fences and huts, as well as intangible assets of cultural heritage and tradition (Kumpula & Siitari, 2020). In the Saami Homeland area, reindeer livelihood is linked to vitality of the Saami languages and indigenous rights (Jaakkola et al., 2018; Markkula et al., 2019). Regional heterogeneity in perceptions, especially towards herding practices, can party be explained by varying herding traditions and culture in the northern study area (including the Saami Homeland area) and more southern herding districts. 4.3. From perceptions to action? One long-term question in perception geography is how perceptions translate into action (Bunting & Guelke 1979). Generally, our survey respondents considered herding practices used by them as beneficial. It may be that practices are valued because choises to use them have already been made and actions taken. On the other hand, developing and adopting herding practices are considered as ways to cope with changing conditions (Armitage et al., 2011; Turunen & Vuojala-Magga, 2014). Herding practices were in this study mainly considered as in ternal drivers (Table 3), affecting reindeer welfare from their part. On the other hand, they can be considered as coping strategies to mitigate conditions considered as harmful or to utilize the opportunities during conditions perceived beneficial (Table 2). This way the perception (what is harmful/beneficial to reindeer) indeed translates into action (which herding practices to use) and as potential to affect the welfare of rein deer and the success of the livelihood. Furthermore, climate-related risks affect the livelihood (Kumpula & Siitari, 2020; Peltonen-Sainio et al., 2017; Rasmus, Turunen, Luomar anta, et al., 2020; Turunen et al., 2016). Effects of climate change become visible through seasonal weather events which are stochastic and rather short-lived. The probability of extreme weather events such as hot summer periods, icing events and deep snow covers increases within the RMA in the warming climate (Abram et al., 2019; Jylh¨a et al., 2008; Rasmus, Turunen, Luomaranta, et al., 2020). During an extreme weather event, welfare of reindeer can be negatively affected. It all comes down to the sufficiency and diversity of pastures as well as pasture accessibility (Kitti et al., 2006) – or if needed, supplementary forage (L´epy et al., 2018; Pekkarinen et al., 2015). What is particularly detrimental to reindeer husbandry is the Earlier studies on climate change adaptation of reindeer husbandry 14 Applied Geography 134 (2021) 102501 S. Rasmus et al. combination of a harmful weather event and intensive land-use. Similar conclusions were also made about the situation in Norway in a recent review by Tyler et al. (2021). Herders need new strategies to adapt to the changes (Peltonen-Sainio et al., 2017; Rasmus, Turunen, Luomaranta, et al., 2020). 4.3. From perceptions to action? Their capacity to cope with extreme weather events is limited and climate vulnerability is increasing if there is no flexibility in the use of pasture resources, such as seasonal pasture rotation (Anttonen et al., 2011; Degteva et al., 2017; Eira et al., 2018; Pape & L¨offler, 2012). There is a need for more holistic regional land-use planning, which would take several overlapping and neighboring livelihoods into ac count. One solution would be to acknowledge the needs of reindeer husbandry by allocating space to ensure flexibility in pasture use (Kumpula & Siitari, 2020). However, planning should not be targeted at the mean conditions or even at the most probable event since extreme events tend to cause the most harm. just been completed. The number was set by the Ministry of Agriculture and Forestry of Finland, but it was negotiated within a stakeholder working group. The negotiations resulted also in a new process: putting together herding management plans for the pasture areas of every herding district. What this plan will contain in practice is not yet clear, and new biannual negotiations within the stakeholder group and with herding districts will soon begin. In this process, methods to bridge different knowledge sources will be needed. The approach and data presented in this work could be of use in this process, and also in other land-use planning processes aiming at genuine co-management. Perception is not only subjectively interprating the environment, but also acting accordingly. As Thomas and Thomas (1928) formulated: “If men define situations as real, they are real in their consequences”. Herders are central actors in the reindeer management SES. Their per ceptions translate into decision making, planning, and risk prepared ness. Interpretation of a situation, or in our case, perception on drivers of change, leads to actions and shapes the future of reindeer husbandry. In land-use planning, the needs of all land users should be under stood in order to be able to generate synergies, negotiate difficult trade- offs and manage conflicts. Environmental conflicts are mostly consid ered to be caused by differences in knowledge and irreconcilable values (Pettersson et al., 2017). Environmental conflicts can emerge and continue to persist because of a clash of diverging cultural models and frames that stakeholders carry in their individual and collective minds (Jokinen, 2019). These concepts come close to the concept of perception used in our study. Funding Financial support was provided by the Finnish Cultural Foundation (project “Gradual changes and abrupt crises - changing operational environment of Finnish reindeer herding”), Nordforsk (NCoE “Reindeer Husbandry in a Globalizing North – Resilience, Adaptations and Path ways for Actions”, project number 76915) and the Finnish Ministry of Agriculture and Forestry (MAKERA/2016 “Reindeer health in the changing environment” and “Sustainable bioeconomy on reindeer pas tures” projects). Declaration of competing interest None. 4.3. From perceptions to action? Improved understanding of local people’s perceptions could inform and shape political agendas regarding land use, sustain ability and people’s rights, and could lead to more equitable societal processes (Meijaard et al., 2013; Raymond et al., 2009). Author statement Individual contributions to the paper: Sirpa Rasmus: Conceptualization; Methodology; Writing - original draft; Writing - review & editing, Project administration. Henri Wallen: Formal analysis; Methodology; Software; Visualiza tion; Writing - review & editing. Minna Turunen: Conceptualization; Methodology; Writing - original draft; Writing - review & editing. Practitioner knowledge of herders is about local observations but also about interpretations and preferences. Presently, incorporating these types of facts in the environmental assessments and planning procedures is not easy (Chapman & Schott, 2020) and not adequately recognized by decision-makers and land use planners, but would be urgently needed. Local perceptions carry relevant information about the relationships between people and their environments. These subjective and intangible aspects are part of the knowledge of the experienced environment and cannot be excluded even from practical approaches. This is one argument for bringing the perspectives of local communities and livelihoods to the joint planning table. Mia Landauer: Methodology; Writing - original draft; Writing - re view & editing. Juho Tahkola: Investigation; Data curation; Validation; Writing - review & editing. Mikko Jokinen: Methodology; Software; Visualization; Writing - re view & editing. Sauli Laaksonen: Conceptualization, Supervision, Funding acquisi tion, Resources, Writing - review & editing. 4.6. Conclusions - contribution of this study to the participatory environmental governance We revisit the rather old approach of perception geography, where individual values towards, perceptions on, and observations of the environment are studied. Why study perceptions instead of just concentrating on proven land-use pressures or detrimental weather events? Some of these pressures and climate indicators are scarcely studied and poorly known. Understanding the perceptions is needed in managing the present-day and future environmental conflicts (Brown et al., 2020). Participatory environmental governance and public participation in environmental management are increasingly adopted (Adenskog, 2018; Huntington et al., 2019; J¨aske, 2018). Participatory decision making is believed to lead to more deliberate, inclusive and sustainable solutions. These processes have also been criticized for poor stakeholder involvement (e.g., Komendantova et al., 2015). Reindeer herders have experienced power imbalances in the negotiations with the governance of the livelihood, feeling that their herding practices do not get enough recognition and support and their voice is not heard (Lan dauer & Komendantova, 2018; Markkula et al., 2019). Our approach presents one tool which can be used to facilitate these processes. Local perceptions are needed as a relevant part of balanced discussion. They also carry valid local and traditional knowledge that can be bridged with scientific knowledge of the issues studied (Abu et al., 2019; Chapman & Schott, 2020). 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Participatory environmental governance of infrastructure projects affecting reindeer husbandry in the Arctic. Journal of Environmental Management, 223, 385–395. Raymond, C. M., Bryan, B. A., MacDonald, D. H., Cast, A., Strathearn, S., et al. (2009). Mapping community values for natural capital and ecosystem services. Ecological Economics, 68, 1301–1315. Landauer, M., Rasmus, S., & Forbes, B. C. (2021). What drives reindeer management towards social and ecological tipping points? Regional Environmental Change. https:// doi.org/10.1007/s10113-021-01757-3 Rees, W. G., Stammler, F. M., Danks, F. S., & Vitebsky, P. (2008). Vulnerability of European reindeer husbandry to global change. Climatic Change, 87(1–2), 199. Ren, X., Che, Y., Yang, K., & Tao, Y. (2016). Risk perception and public acceptance toward a highly protested Waste to Energy facility Waste Management 48 528 539 Rees, W. G., Stammler, F. M., Danks, F. S., & Vitebsky, P. (2008). Vulnerability of European reindeer husbandry to global change. Climatic Change, 87(1–2), 199. R X Ch Y Y K & T Y (2016) Ri k ti d bli t Rees, W. G., Stammler, F. M., Danks, F. S., & Vitebsky, P. (2008). Vulnerability of European reindeer husbandry to global change. Climatic Change, 87(1–2), 199. Ren, X., Che, Y., Yang, K., & Tao, Y. (2016). Risk perception and public acceptance toward a highly protested Waste-to-Energy facility. Waste Management, 48, 528–539. RHA (Reindeer Herders’ Association). (2018). References Muuttoranta, K., Holand, Ø., Røed, K. H., Tapio, M., Nieminen, M., & M¨aki-Tanila, A. (2014). Genetic variation in meat production related traits in reindeer (Rangifer tarandus). Rangifer, 34(1), 21–36. y j j j Tennekes, M. (2018). tmap: Thematic maps in R. Journal of Statistical Software, 84(6), 1–39. https://doi.org/10.18637/jss.v084.i06 N¨akk¨al¨aj¨arvi, K., & Jaakkola, J. (2017). Saamelaiset ja muutos. In M. Tennberg, J. Haapala, A. Hannukkala, J. P. Jaakkola, T. Jouttij¨arvi, K. Jylh¨a, S. Kauppi, et al. (Eds.), Barentsin alue muuttuu – Miten Suomi sopeutuu? Valtioneuvoston selvitys- ja tutkimustoiminnan julkaisusarja 31. Thomas, W. I., & Thomas, D. S. (1928). The child in America: Behavior problems and programs (pp. 571–572). New York: Knopf. Thomas, W. I., & Thomas, D. S. (1928). The child in America: Behavior problems and programs (pp. 571–572). New York: Knopf. Tuan, Y.-F. (1990). Topohilia (Morningside Edition). Columbia University Press. l d l l h l f h National Land Use Guidelines. (2017). Land-use guidelines in Finland. https://www.ym paristo.fi/en-US/Living_environment_and_planning/Land_use_planning_system/Na tional_land_use_guidelines. Tuan, Y.-F. (2003). Perceptual and cultural geography: A commentary. Annals of the Association of American Geographers, 93(4), 878–881. Turunen, M., Rasmus, S., Bavay, M., Ruosteenoja, K., & Heiskanen, J. (2016). Coping with increasingly difficult weather and snow conditions: Reindeer herders’ views on climate change impacts and coping strategies. Climate Risk Management, 11(2016), 15–36. https://doi.org/10.1016/j.crm.2016.01.002 Nellemann, C., Jordhøy, P., Støen, O.-G., & Strand, O. (2000). Cumulative impacts of tourist resorts on wild reindeer (Rangifer tarandus tarandus) during winter. Arctic, 53 (1), 9–17. Nellemann, C., Vistnes, I., Jordhøy, P., Strand, O., & Newton, A. (2003). Progressive impact of piecemeal infrastructure development on wild reindeer. Biological Conservation, 113, 307–317. Turunen, M., Rasmus, S., J¨arvenp¨a¨a, J., & Kivinen, S. (2020). Relations between forestry and reindeer husbandry in northern Finland: Perspectives of science and practice. Forest Ecology and Management, 117677. https://doi.org/10.1016/j. foreco.2019.117677 Nieminen, M. (1988). Porojen laitumet ja ruokinta poronhoitovuonna 1986-87. I Laitumet ja niiden kunto. Poromies, 6. Turunen, M., Rasmus, S., Norberg, H., Kumpula, J., Kojola, I., & Ollila, T. (2017). Porot ja pedot – kuinka poronhoidon sopeutuminen petoihin on muuttunut 90 vuodessa? Suomen Riista, 63, 19–42. Nieminen, M., & Autto, P. (1989). Porojen laitumet ja ruokinta poronhoitovuonna 1986- 87. II Rehut ja ruokinta. Poromies, 2. 17 S. Rasmus et al. Turunen, M., & Vuojala-Magga, T. (2014). Past and present winter feeding of reindeer in Finland: Herders adaptive learning of the practices. Arctic, 67(2), 173–188. https:// doi.org/10.14430/arctic4385 Turunen, M., & Vuojala-Magga, T. (2013). Porojen talviruokinta: Luppopuiden hakkuusta tarharuokintaan. [With English summary: Reindeer winter feeding: From lichen tree cuttings to pen feeding]. Suomen Riista, 59, 86–99. Turunen, M., & Vuojala-Magga, T. (2013). Porojen talviruokinta: Luppopuiden hakkuusta tarharuokintaan. [With English summary: Reindeer winter feeding: From lichen tree cuttings to pen feeding]. Suomen Riista, 59, 86–99. Turunen, M., & Vuojala-Magga, T. (2014). Past and present winter feeding of reindeer in Finland: Herders adaptive learning of the practices. Arctic, 67(2), 173–188. https:// doi.org/10.14430/arctic4385 Wickham, et al. (2019). Welcome to the tidyverse. Journal of Open Source Software, 4(43), 1686. https://doi.org/10.21105/joss.01686 Applied Geography 134 (2021) 102501 Vistnes, I., Nellemann, C., Jordhøy, P., & Strand, O. (2001). Wild reindeer: Impacts of progressive infrastructure development on distribution and range use. Polar Biology, 24, 531–537. Wickham, et al. (2019). Welcome to the tidyverse. Journal of Open Source Software, 4(43), 1686. https://doi.org/10.21105/joss.01686 Working committee. (1992). Poronhoitolain seurantaty¨oryhm¨an muistio. Rovaniemi: Poronhoitolain seurantaty¨oryhm¨a [Working committee for the follow-up of the herding legislation]. 18
https://openalex.org/W2409269922	https://actamedica.lfhk.cuni.cz/media/pdf/am_2010053010035.pdf	English	null	TIPS Creation in Patients with Persistent Left Superior Vena Cava	Acta Medica	2,010	cc-by	2,948	TIPS CREATION IN PATIENTS WITH PERSISTENT LEFT SUPERIOR VENA CAVA Vendelín Chovanec1, Antonín Krajina1, Petr Hůlek2, Miroslav Měšťan3, Ondřej Renc1 Charles University in Prague, Faculty of Medicine and University Hospital Hradec Králové, Czech Republic: Department of Radiology1, 2nd Department of Internal Medicine, Division of Gastroenterology2, 1st Department of Internal Medicine3 Summary: Transjugular intrahepatic portosystemic shunt is a minimally invasive endovascular procedure that has played an important role in the treatment of acute or repeated variceal bleeding or refractory ascites. The standard venous access route for this procedure is the right jugular vein. Sometimes it is better to use the left jugular vein because of lower pro- bability of life threatening complication or technical failure. In this case reports the authors have described their experi- ence with TIPS creation in two patients with persistent left and absent right superior vena cava and recommend using the left jugular vein as an access route in this rare anatomical variant. umbilical hernia, and large portosystemic collaterals via the coronary vein and short gastric veins. The protrombine time was elevated at 18.2 sec, total bilirubin 25 μmol/L, al- bumin 27.8 g/L. An attempt to introduce a Swan-Ganz catheter, in order to perform hemodynamic measurement, via the left subclavian vein failed. The catheter was left in the left subclavian vein and the patient was transferred to the angiographic suite, because of the suspicion of the SVC anomaly – the left sided SVC above all. Phlebography per- formed through the catheter confirmed the LSVC draining into the right atrium via hypertrophic coronary sinus. After this, the right femoral vein was punctured and the catheter was passed through the dilated coronary sinus into the left IVJ (Fig. 1A) and straightened by a very stiff guidewire (J Back-up Meier, Boston Scientific/Meditech, Watertown, USA). The patient tolerated this manoeuvre without dys- rhythmia, and this confirmed the possibility of TIPS crea- tion using the left IJV approach. TIPS procedure was performed 5 days after the diagnostic angiogram with of a surgeon and anesthesiologist present in view of the poten- tial risk of cardiac or large vessel injury. After gaining left IJV access, a soft guidewire (Bentson, Cook Europe, Bjaeverskov, Denmark) and 5F catheter was advanced via the LSVC and coronary sinus into the inferior vena cava. The soft wire was replaced by an extra stiff guidewire over which the 10F sheath of TIPS set (TIPSI-100, Cook Euro- pe, Bjaeverskov, Denmark) was inserted. CASE REPORT CASE REPORT Introduction Transjugular intrahepatic portosystemic shunt (TIPS) is an accepted method for treatment of symptomatic portal hypertension especially in the management of uncontrolled or repeated variceal bleeding in spite of endoscopic therapy or in the management of refractory ascites. TIPS is a mini- mally invasive procedure and the right internal jugular vein (IJV) is recommended as a standard venous access, for there is lower risk of mediastinal injury than using the left IJV (5). The left jugular approach is used in patients in whom the right IJV is thrombosed and can not be cannu- lated, or portal vein access is unsatisfactory or unsuccessful via the right IJV due to horizontal orientation of the hepa- tic vein (5). We report successful TIPS creation via the left IJV in two patients with the congenital central venous ano- maly such as persistent left superior vena cava (LSVC) with absent right superior vena cava. TIPS CREATION IN PATIENTS WITH PERSISTENT LEFT SUPERIOR VENA CAVA The rigid steel cannula was advanced into the right hepatic vein. After se- veral unsuccessful needle passes, with one of them compli- cated by the puncture of the liver capsule, the guidewire ACTA MEDICA (Hradec Králové) 2010;53(1):35–38 Case 1. A 64-year-old female with cryptogenic liver cirrhosis Child-Pugh class C and refractory ascites was admitted to our hospital for TIPS creation in April 1994. She had no previous episode of gastroesophageal variceal bleeding. The physical examination revealed large ascites, umbilical her- nia, Caput Medusae, and bilateral perimaleolar edema. Com- puted tomography confirmed ultrasonographic findings of the shrunken liver, splenomegaly, massive ascites with an 35 ACTA MEDICA (Hradec Králové) 2010;53(1):35–38 Fig. 1: A) Venogram through catheter in the left jugu vein introduced from the right femoral approach. LSV (black arrow) and pulmonary artery (white arrow) are op cified by iodinated contrast media. B) Initial transjugu portography shows large portosystemic collaterals and p tent portal vein. C) Final portogram after TIPS creati with selfexpandable stent. A B C A Fig. 2: A) Carbon dioxide wedged hepatic venogram right branch of portal vein is marked with black arrow the middle hepatic vein with white arrow. B) Final p venogram after coronary vein embolization (black ar and shunt creation. C) Phlebography through the retra sheath with extra stiff wire left in the cranial end of (white arrow). Black arrow marks persistent LSVC w enters the right atrium via dilated coronary sinus. Blac rowhead shows central venous catheter. A B C A A B B B Fig. 2: A) Carbon dioxide wedged hepatic venogram. The right branch of portal vein is marked with black arrow and the middle hepatic vein with white arrow. B) Final portal venogram after coronary vein embolization (black arrow) and shunt creation. C) Phlebography through the retracted sheath with extra stiff wire left in the cranial end of TIPS (white arrow). Black arrow marks persistent LSVC which enters the right atrium via dilated coronary sinus. Black ar- rowhead shows central venous catheter. Fig. 2: A) Carbon dioxide wedged hepatic venogram. The right branch of portal vein is marked with black arrow and the middle hepatic vein with white arrow. B) Final portal venogram after coronary vein embolization (black arrow) and shunt creation. C) Phlebography through the retracted sheath with extra stiff wire left in the cranial end of TIPS (white arrow). Black arrow marks persistent LSVC which enters the right atrium via dilated coronary sinus. Black ar- rowhead shows central venous catheter. Fig. 1: A) Venogram through catheter in the left jugular vein introduced from the right femoral approach. Case 2. A 66-year-old man with portal hypertension and repeated variceal hemorrhage in spite of endoscopic sclerotherapy was admitted to another hospital for TIPS creation in March 2009. The last episode of variceal bleeding led to hemorr- hagic shock. Portal hypertension was secondary to alcoho- lic cirrhosis. The patient was class C according to Child-Pugh classification and he suffered from chronic he- art failure with 30 % ejection fraction. The radiologist can- nulated the right IJV and he performed phlebography because he could not introduce guidewire into SVC. Phlebography revealed the LSVC drained into the right at- rium through a hypertrophic coronary sinus. The right SVC was absent. The procedure was abandoned because of the high risk of mediastinal injury during introducing stiff me- tallic cannula of TIPS set in the hepatic vein. The patient was transferred to our institution. The second procedure was performed 2 days after the first attempt. In our experi- ence of this anomaly, the course of guidewire from left IJV to the hepatic vein through this anomaly is less curved than from the right IJV and so interventional radiologist selected the left IJV as an access site. Curved catheter was introdu- ced into the middle hepatic vein and 10 F sheath was pus- hed in the hepatic vein over the extra stiff guidewire (Amplatz super stiff, Boston Scientific Corporation, Natick, USA). Carbon dioxide wedged portography was performed (Fig. 2A) and the rigid metallic cannula of TIPS set (TIP- SI-100, Cook Europe, Bjaeverskov, Denmark) was inserted in the hepatic vein over extra stiff guidewire. The right branch of portal vein was punctured at the fourth attempt and carbon dioxide portography through the pigtail cathe- ter was performed. Direct portography depicted porto- systemic collaterals rising from the coronary vein. The coronary vein was embolized with mixture of n-butyl 2-cya- noacrylate (Histoacryl, B Braun Aesculap, Tuttlingen, Germany) and oil contrast media (Lipiodol Ultrafluide, Guerbert, Ceres, France). The shunt was created using 12 mm x 90 mm selfexpandable stent (Wallstent-Uni, Boston Scientific Corporation, Natick, USA) which was dilated with 8mm balloon (Fig. 2B). Portosystemic gradient decre- ased from 19 mmHg to 10 mmHg. At the end of the proce- dure the sheath was retracted to the left IJV and Congenital anomalies of the SVC are rare with an inci- dence in general population 0.3–0.5 % (2). The most com- mon of them is the LSVC. Case 1. LSVC (black arrow) and pulmonary artery (white arrow) are opa- cified by iodinated contrast media. B) Initial transjugular portography shows large portosystemic collaterals and pa- tent portal vein. C) Final portogram after TIPS creation with selfexpandable stent. Fig. 1: A) Venogram through catheter in the left jugular vein introduced from the right femoral approach. LSVC (black arrow) and pulmonary artery (white arrow) are opa- cified by iodinated contrast media. B) Initial transjugular portography shows large portosystemic collaterals and pa- tent portal vein. C) Final portogram after TIPS creation with selfexpandable stent. 36 entered into the branch of the right portal vein. Direct por- tography revealed varices filled via coronary and short ga- stric veins (Fig. 1B). The TIPS was created using 10 mm x 70 mm selfexpandable stent (Wallstent-Uni, Boston Scien- tific Corporation, Natick, USA) (Fig. 1C). Portal pressure fell down from 38 mmHg to 27 mmHg and portosystemic gradient decreased from 21 mmHg to 10 mmHg. Portosyste- mic collaterals were not opacified at the digital portogram after the stent placement. Postprocedural course was with- out complication. Doppler sonography 6 days after the pro- cedure demonstrated a patent shunt with good function and the patient was discharged. Unfortunately she died of an unrelated myocardial infarction one month later. phlebography showed no extravasation (Fig. 2C). Ultra- sound on the first postoperative day a revealed patent shunt and the patient was transferred back to the referring hospi- tal. Five months after procedure he lives with patent shunt and no symptoms of portal hypertension. Discussion The right IJV is the standard access route for TIPS pro- cedure because the IJV, brachiocephalic vein, SVC on the right side and right atrium form a straight channel which is more suitable for manipulation with a stiff cannula during procedure than the left sided approach. Any congenital anomaly of these veins can cause technical problems and TIPS creation may be difficult or impossible via the right IJV. References The primary use of the left IJV approach was chosen in these cases, because this allowed easier cannulation of the hepatic vein. There is a higher risk of dysrhythmia, retro- sternal pain, cardiac arrest with persistent LSVC because devices are introduced into the hepatic vein through heart causing distortion of the coronary sinus and right atrium (8). 1. Bahramipour P, Abu-Judeh HH, Miller J. Transjugular intrahepatic portosystemic shunt placement in a patient with absent right superior vena cava and persistent left superior vena cava. AJR 1999;173:631–2. 2. Campbell M, Deuchar DC. The left-sided superior vena cava. Br Heart J 1954; 16:423–439. 3. Chovanec V, Krajina A, Lojík M, Hůlek P, Vaňásek T. TIPS creation in a patient with situs inversus totalis. Cardiovasc Intervent Radiol 2002;25:447–9. 4. Druart F, Cosse P, Aboul-Hosn H, Carre Jl, Guyot G. Left superior vena cava: a cause of failure of transjugular liver biopsy and difficulty inserting a pacemaker catheter. Nouv Presse Med 1981;10:36–45. The right IJV vein approach is preferred for the follow- ing reasons: the right IJV continues in a straight line with the SVC down to the inferior vena cava facilitating easy ma- nipulation. The thoracic duct is on the left side, the apex of the right lung is lower in most cases than the left one re- ducing the risk of procedural comlications. The course from the left side through the left brachiocephalic vein to the SVC is angled. This may cause the thoracic pain from stretching of the mediastinal vessels with the stiff metallic cannula. Hausegger at al. (5) found the left side approach helpful in patients with horizontal course of the right hepa- tic vein and in the patients with cranially located the right portal vein. The stiff cannula takes a diagonal course through the mediastinum and reaches the right hepatic vein at a more obtuse angle which can facilitate positioning and stabilisation of the metallic cannula in this vein. 5. Hausegger KA, Tauss J, Karaic K, Klein GE, Uggowitzer M. Use of the left in- ternal jugular vein approach for transjugular portosystemic shunt. AJR 1998;171:1637–9. 6. Higgs AG, Paris S, Potter F. Discovery of left-sided superior vena cava during central venous catheterization. Br J Anaesth 1998;81:260–1. 7. Köcher K, Černá M, Hutyra M, Novotný J, Kozák J, Buřval S. Persistent left su- perior vena cava- possible source of the paradoxical embolisation. Ces Radiol 2009;63:129–32. 8. Krajina A, Lojík M. Case 2. Two types of the LSVC have been described – the first one is associated with normal location of visceral organs and the LSVC usually enters the heart via dilated coronary sinus. The second one is associated with complete situs viscerum inversus and enters directly in the morphologic right atrium lying on the left side. Successful TIPS placement has been described in both type of the LSVC (1, 3). Between September 1992 and August 2009 we found 3 cases of the SVC anomalies in our group of 815 pa- tients (0.4 %) who underwent TIPS placement. Two patiens had first type of LSVC and one had situs viscerum inversus. The incidence of persistent LSVC is about 0.3 % in ge- neral population and 4.4 % in patients with congenital car- diac disease (1). The LSVC is the most common anomaly of systemic vein circulation. It is associated with patent right SVC in 82–90 % (double superior vena cava) and ab- sence of the left brachiocephalic vein in 65 % of patients. Nearly all the LSVC (in 92 % cases) enter the right atrium via the coronary sinus. The rest drains into the left atrium, hepatic vein or inferior vena cava. Drainage to the left atri- um cause right to left shunting, which can lead to para- doxical embolisation (7). The anomaly is due to failed obliteration of the left anterior cardinal vein. The first ex- planation for embryonic development of the persistent left SVC was described by Marshall in 1850 (10). Increasing number of multidetector row computed tomo- graphy (MDCT) and magnetic resonance (MR) imaging including angiography, central venous cannulation, endo- vascular venous procedures such as pacemaker, defibrilator leads implantation, vena cava filter implantation, TIPS cre- ation or transjugular biopsy has contributed to more fre- quent detection of LSVC during the life of a patient. This anomaly is rare, but an operator must be able to modify a procedure when it is discovered. The diagnosis can be anticipated by signs on chest X-ray, such as paramediastinal strip, paramediastinal bulging below the aortic arch, wide- ning of aortic shadow, crescent along the left heart border in persistent LSVC (6, 14) or revealed by noninvasive methods 37 like MDCT or MR angiography (15). Vendelín Chovanec, MD, Ph.D., University Hospital, Department of Radiology, Sokolská 581, 500 05 Hradec Králové, Czech Republic; e-mail: chovanec.v@seznam.cz Case 2. There are two reports describing successful TIPS placement and inferior vena cava filter placement through the jugular access (2, 11) and one article of unsuccessful transjugular biopsy due to per- sistent LSVC (4). The authors used the right IJV for im- plantation of vena cava filter. From the right side there is a longer distance between the infrarenal part of vena cava and jugular vein. This condition allows a gentle curvature of a long introducer sheath, hence it is possible to use either the right or left jugular vein in this case (11). approaches should be considered much more complicated in cases with the persistent left SVC and absent right SVC (9, 12, 13). Based on our experience we conclude that the access via left jugular vein should be considered as the method of choice in liver interventions including transju- gular liver biopsy and TIPS in the presence of a congenital central venous anomaly such as persistent LSVC with ab- sent right superior vena cava. References TIPS – Technique. In Hůlek P, Krajina A: Current practice of TIPS. Olga Štambergová Publishing House, Hradec Králové 2001, 51–119. 9. LaBerge JM, Ring EJ, Gordon RL. Percutaneous intrahepatic portosystemic shunt created via a femoral vein approach. Radiology 1991;181:679–81. 10. Marshall J. On the development of the great anterior thoracic veins in man and mammalia: including an account of certain remnants of fetal structure found in the adult, a comparative view of these great veins in the different mammalia, an analysis of their occasional peculiarities in the human subject. Philos Trans R Soc Lond 1850;140:133–69. 11. Nair S, Ettles D, Robinson G, Scott P. Inferior vena cava filter from left sided su- perior vena cava. CardioVasc Intervent Radiol 2008;31:S171–3. 12. Raza SA, Walser E, Hernandez A, Chen K, Marroquin S. Transhepatic puncture of portal and hepatic veins for TIPS using a single-needle pass under sonographic guidance. AJR 2006;187:W87–92. We have described two cases of a rare congenital cent- ral venous anomaly in patients who underwent successful TIPS procedure using a simple modification of the routine technique. Others have described different techniques of TIPS creation such as the femoral, transmesenteric or transhepatic approach. According to our experience these 13. Rozenblit G, Del Guercio LR, Savino JA, et al. Transmesenteric-transfemoral method of intrahepatic portosystemic shunt placement with minilapparotomy. J Vasc Interv Radiol 1996;7:499–506. 14. Spearman P, Leier CV. Persistent left superior vena cava, unusual wave countour of left jugular vein as the presenting feature. Am Heart J 1990;120:999–1002. 15. White CS, Baffa JM, Haney PJ, Pace ME, Campbell AB. MR imaging of conge- nital anomalies of the thoracic veins. RadioGraphics 1997;17:595–608. Received: 02/11/2009. Accepted in revised form: 17/12/2009. Corresponding author: 38
https://openalex.org/W4393087707	https://www.mdpi.com/2073-445X/13/4/405/pdf?version=1711097586	English	null	Integrating Health and Smartness—New Sustainable Paradigms for the Urban Environment: A Case Study in Lianshi Town (China)	Land	2,024	cc-by	12,893	Citation: Pietra, C.; Venco, E.M. Integrating Health and Smartness—New Sustainable Paradigms for the Urban Environment: A Case Study in Lianshi Town (China). Land 2024, 13, 405. https://doi.org/10.3390/ land13040405 Keywords: smart cities; healthy cities; environmental sustainability; industrial regeneration; urban renewal Article Integrating Health and Smartness—New Sustainable Paradigms for the Urban Environment: A Case Study in Lianshi Town (China) Caterina Pietra and Elisabetta Maria Venco * Department of Civil Engineering and Architecture, University of Pavia, 27100 Pavia, Italy; caterina.pietra@unipv.it * Correspondence: elisabettamaria.venco@unipv.it; Tel.: +39-0382-98-5409 Abstract: The concepts of healthy cities and smart cities are popular in emerging research in the 21st century. This study focuses on the existing interrelations between the two notions in terms of socio-spatial quality, technology, and innovation, particularly regarding industrial sites that no longer have a role and constitute ‘urban voids’ with high volumetric concentrations. The fast expansion of cities and the de-industrialization phenomena have resulted in such void-producing blights that compromise public health; environmental quality; and social, economic, and living conditions. Therefore, the authors intend to emphasize the relevance of citizens’ and communities’ engagement in shaping new healthy and smart urban environments. The present method relies on a literature review to describe the current theoretical and practical dimensions of such topics, identifying synergies and trade-offs. After this, a case study in China is presented to support the discussion. The site, a former granary, is located in Lianshi, a traditional water-edge town in the central area of the Yangtze River Delta. The proposed example enhances solutions that meet healthy and smart requirements, transforming the former industrial area into a social catalyst, acting as an effective motivator for urban development. One such theoretical approach is exemplified by a project that won a national architecture competition in 2018, the ‘Taihu Cultural Heritage Rehabilitation Competition’. The latter is then validated through practical solutions in a real-world context by analyzing the ‘Cuckoo’ project developed in 2021 for the same area. Overall, the integration of healthy and smart elements is proposed by the authors as an effective method to achieve more holistic and sustainable city development from both theoretical and practical points of view. land land land land 1. Introduction Nowadays, contemporary cities serve as the epicenter of human experience, technolog- ical advancement, and public health. The rapid pace of urbanization significantly impacts the emergence and progression of health and well-being issues, developing challenges that experts must confront and resolve [1–5]. While certain temporary, immediate, or short-term measures [6] might be identified, these actions are incapable of guaranteeing sustainable and enduring solutions. The latter typically require more profound alterations in the composition and behavior of a system. Received: 1 February 2024 Revised: 19 March 2024 Accepted: 20 March 2024 Published: 22 March 2024 It may also be vacant, derelict, or contaminated. Therefore, a brownfield site is not necessarily available for immediate use without intervention”.iii y In China, the term ‘brownfield’ was first mentioned by Niu [15]. During the first industrialization period, the decline in traditional industries and the relocation of many factories led to a large number of unused and abandoned sites. Officially, in 2010, the World Bank’s Waste Management in China: Problems and Suggestions stated that there were “at least 5000 brownfield sites”; the real estimation is undoubtedly higher [16]. Moreover, at present, there are no specific policies or regulations for brownfield management and redevelopment, but there are numerous national-level documents related to soil pollution control [13]. Europe, where approximately 70% of the population lives in urban or suburban areas [17], is challenged by urban sprawl, scattered development, urban dispersion, soil sealing, and air, soil, and water pollution. Investments in green space and brownfield restoration are seen as new development opportunities [18]. In this context, for over 150 years, an extensive and continually growing body of research has shown that the planning and administration of cities significantly influence the health and well-being of urban residents [5,19]. Urban health is the result of interactions within the environmental, social, and economic domains [20–22]. Consequently, since challenges to urban health emerge as integral components of various cause-and-effect relationships across urban sectors, potential solutions must consider the complexity of the issue [23]. In this sense, urban regeneration constitutes an integrated approach involving vision and action, favoring the overall improvement of disadvantaged places (ranging from buildings, neighborhoods, cities, and regions) to increase their sustainability level [24]. In the 2016 Shanghai Declaration, the World Health Organization (WHO) stated that health acts as a highly significant indicator of a city’s sustainable development [25], reinforcing its position in advocating the necessity to promote ‘healthy cities’ [26]. The urban system derives health benefits if the interconnectivity layer facilitates access to diverse data and resource flows. In particular, the interconnectivity among urban areas must enhance the availability of spaces that can improve health and well-being, contingent on the level of infrastructure development in each sector [5]. The imperative for cities to actively engage in the ongoing ‘data revolution’ implies the need for expanded discussions and debates concerning the utilization of technology, big data, and citizen science for the promotion of smart cities and the advancement of urban planning [27–29]. p y Regarding urban issues, the widespread presence of ‘urban voids’ due to the rapid growth of cities and the de-industrialization phenomenon significantly affects citizens’ quality of life and their environmental impact. Indeed, industrial sites commonly present a multifaceted pollution profile involving an array of pollutants dispersed in the air, water, and soil [7]. Copyright: © 2024 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/). The widespread economic restructuring and long recession of the 1970s led many industrial cities into an era of de-industrialization, characterized by severe economic de- cline and the loss of employment in manufacturing industries. Furthermore, suburban https://www.mdpi.com/journal/land Land 2024, 13, 405. https://doi.org/10.3390/land13040405 Land 2024, 13, 405 2 of 19 2 of 19 growth caused the decentralization of production, trade, and population away from the central urban areas. Additional reasons that led to such consequences are related to the outsourcing of production in developing economies, the obsolescence of certain public in- frastructure, more general modifications of the new economy, many changes in production techniques and processes, and the cessation of activities that were no longer competitive in the local market or no longer met environmental hygiene standards [8–11]. Thus, the highly accessible and intensely infrastructure-industrialized areas have turned into brownfields. growth caused the decentralization of production, trade, and population away from the central urban areas. Additional reasons that led to such consequences are related to the outsourcing of production in developing economies, the obsolescence of certain public in- frastructure, more general modifications of the new economy, many changes in production techniques and processes, and the cessation of activities that were no longer competitive in the local market or no longer met environmental hygiene standards [8–11]. Thus, the highly accessible and intensely infrastructure-industrialized areas have turned into brownfields. Starting from the 1980s, several definitions have arisen [12,13]. In the United States, the Environmental Protection Agency (US EPA) defines brownfields as abandoned, idling, or underutilized industrial and commercial facilities where expansion or redevelopment is complicated by real or perceived contamination. The definition proposed by Alker et al. [14] is quite broad and widely accepted: “any land or premises which has previously been used or developed and is not currently fully in use, although it may be partially occupied or utilized. In this case, the primary implementation tool is represented by technology and innovation. Overall, smart cities positively and actively support strategies exploiting innovation to strengthen the global competitiveness and sustainability of the urban environment [30,31]. g p y Healthy and smart cities notions are both centered on the achievements related to the level of socio-spatial quality, which is derived from applying solutions enhancing the value of virtual and material services and urban infrastructures (the systems providing water, energy, food, shelter, transportation, communication, health, waste management, public areas, etc.). Land 2024, 13, 405 3 of 19 3 of 19 Additionally, insights drawn from the complexity theory of cities point out the es- sential need to integrate both scientific and societal elements in actively addressing urban challenges by providing access and enhancing people’s capabilities [5,32–34]. Therefore, social infrastructure plays a fundamental role in the approach to urban regeneration, in- volving science in producing knowledge and supporting a broader collective process that considers multiple spatial scales, sectors, stakeholders, and research disciplines [35]. Particularly, relationships with citizens, living close to the neglected areas, and sharing specific objectives has to be enhanced to build positive cultural networks and support social cohesion for the healthy and smart regeneration process. The current work presented by the authors supports the urban regeneration process towards the creation of healthier and smarter cities. Indeed, it demonstrates the potential of brownfields for redevelopment and interdisciplinary experimentation to enhance com- munity health and innovation. More specifically, attention is placed on a former industrial area in Lianshi, a traditional water-edge town located in the central area of the Yangtze River Delta. The case study emphasizes urban strategies that can facilitate the area’s pos- itive, sustainable transformation. Moreover, the authors stress the importance of citizen engagement in shaping new urban environments. The case study is verified through both a theoretical and practical framework, converging into a holistic approach linking healthy and smart elements. 2. Healthy and Smart Cities: A Comparison The concept of healthy cities emerged in the 1980s as part of a World Health Organi- zation (WHO) initiative driven by the goal of promoting health on a global scale through local actions [36]. The concept of smart cities, on the other hand, emerged more recently and originates from technology companies in the private sector, evolving with scientific methods and computational analysis in urban planning. Smart cities refer to the utilization of innovations and technological resources in urban infrastructure and services. The Eu- ropean Commission defines a smart city as a place where digital and telecommunication technologies enhance traditional networks and services for the benefit of inhabitants and businesses [37,38]. Consequently, healthy cities emphasizes enhancing individuals’ environmental, eco- nomic, and living conditions to promote overall health [39]. In smart cities, the focus shifts towards fostering the concentration of human capital, attracting businesses, and catalyzing activities that transform cities into hubs of global competitiveness [40].i A healthy city is defined as follows: “it is one that is continually creating and improv- ing those physical and social environments and strengthening those community resources which enable people to mutually support each other in performing all the functions of life and achieving their maximum potential” [41]. In particular, the fostering of healthy cities includes notions deriving from: 1. Primary health care: implementing the WHO Health for All [42] strategy to prioritize essential healthcare services accessible to all community members; 2. Health promotion: encouraging proactive measures to improve health and prevent illnesses; 3. Ottawa Charter (1986): embracing the idea of health as a continuous process and recognizing the need for new skills, processes, styles, and governance structures; 4. Health beyond the absence of disease: viewing health holistically, considering physi- cal, mental, and social dimensions; 5. Agenda 21: aligning with the principles of sustainable development and addressing environmental and social factors affecting health. The health policies implemented over time were founded on robust partnerships, health determinants, community development, and monitoring initiatives [43]. Efforts were directed towards clarifying the community’s responsibility throughout and beyond the process, encompassing both the initial framing of the problem and the final evaluation concerning work outcomes. Land 2024, 13, 405 4 of 19 The healthy cities (HCs) movement underscores the significance of the procedural aspect, dividing it into three primary components. 2. Healthy and Smart Cities: A Comparison The first component mandates that the involved cities establish a substantial political commitment connected to a shared vision at the local level. The second component addresses the engagement of a diverse range of stakeholders, including local communities [5]. This action determines a shift from an exclusively expert-led health agenda to one that emphasizes the role of the community, fostering empowerment and participation [44–46]. The last part necessitates the implemen- tation of the strategy within the local government, requiring a city health plan derived from intersectoral partnerships and stakeholder engagement. When evaluating city transformations, the healthy cities concept presupposes that people possess knowledge about the territory, which enables them to act in their favor. Consequently, strategies are expected to arise from the community, playing a pivotal role in shaping public policies with governments, emphasizing the bottom-up approach in this concept. When considering the development of a smart city, both bottom-up approaches from citizens and top-down approaches from planners and managers are taken into account. In this context, Dameri [47] put in evidence a top-down approach involving the application of government rules and policies, as well as a bottom-up approach grounded in applying technology to urban challenges. This approach recognizes that integrating technology into urban activities can broaden citizens’ potential access to the city, with citizens contributing data to collaboratively address shared issues. y In terms of achieving outcomes, the requirements for being labeled as ‘smart’ or ‘healthy’ and the directions for action also vary. In the field of smart cities, particularly when considering the perspective of private initiatives, interventions are often geared towards yielding returns within short-term horizons [5]. In contrast, within the context of healthy cities, the focus is on an ongoing improvement process and long-term perspectives to overcome the risk of ‘projectism’ [48] and to promote sustainability and non-exclusivity. p j p y y Political commitment denotes a crucial objective linked to the correct utilization of resources aimed at mitigating inequalities. Securing engagement and dedication from the political sphere, in conjunction with valuable stakeholder support, form critical prerequi- sites for implementing enduring change. Additionally, the healthy city vision provides local governments with a leadership role, empowering them to safeguard and enhance citizens’ health and well-being [5]. The robustness of the approach is evident when utilizing process indicators, repre- senting a pivotal element in measuring actions and engaging directly with the community. 2. Healthy and Smart Cities: A Comparison These indicators can also assess the sustainability aspect of healthy city projects [49]. Finally, another aspect where the distinctions between the concepts of a Healthy City and a smart city are noteworthy is in their treatment and understanding of the environment. While both concepts express a commitment to preserving natural resources, there is an im- plicit difference. In healthy cities, nature is regarded as an essential natural asset for health and quality of life, considered a universal right that should not be monetized. On the other hand, the concept of a smart city emphasizes nature as a natural resource serving the needs of society’s reproduction. In this case, the focus is on rationality, efficiency, and occasionally the valorization of specific areas in urban spaces distinguished by environmental amenities, with secondary consideration given to concerns about social relations and overall social and environmental issues. Overall, a smart city contributes to maintaining a stable ecosystem, a crucial aspect for a healthy city. Consequently, initiatives for a smart city have the potential to contribute to the development of a healthy city, and vice versa. This synergy arises from the understanding that individuals with high levels of health, education, quality of life, etc., are more likely to actively participate in the transformation of cities. The authors embraced the healthy and smart perspective to allow planners and pol- icymakers to consider a broad range of factors that can contribute to the overall urban quality of life. It also helps uncover potential trade-offs that may arise between different urban development goals. Moreover, understanding the overlaps and differences between Land 2024, 13, 405 5 of 19 e betwe diﬀ 5 of 19 e betwe d ﬀ healthy and smart city initiatives enables more informed decision-making processes that consider multiple dimensions of urban well-being. Lastly, comparing smart and healthy cities provides insights into how these challenges can be addressed through integrated approaches that leverage technological innovation, public health strategies, and community engagement, plus ensuring long-term environmental, social, and economic sustainabil- ity. Figure 1 effectively summarizes the main components characterizing both concepts and highlights common elements to be taken into consideration and implemented in the proposed case study. y y g p consider multiple dimensions of urban well-being. 2. Healthy and Smart Cities: A Comparison Lastly, comparing smart a cities provides insights into how these challenges can be addressed through approaches that leverage technological innovation, public health strategies, a nity engagement, plus ensuring long-term environmental, social, and econom bility. Figure 1 eﬀectively summarizes the main components characterizing bo and highlights common elements to be taken into consideration and impleme proposed case study. Figure 1. Main components of healthy and smart city concepts. Figure 1. Main components of healthy and smart city concepts. Figure 1 Main components of healthy and smart Figure 1. Main components of healthy and smart city concepts. 3 M i l 3. Materials and Methods 3. Materials and Methods The work presented by the authors analyses the case study of a form which is part of Lianshi Town’s built heritage, by taking into consideration h The work presented by the authors analyses the case study of a former granary, which is part of Lianshi Town’s built heritage, by taking into consideration healthy and smart elements to demonstrate both theoretical and practical urban regeneration approaches. smart elements to demonstrate both theoretical and practical urban regen proaches. In particular, the materials and methods encompass a multidisciplinary approach, including three main steps. The process started in 2018, when the site was selected to participate in the ‘Taihu Cultural Heritage Rehabilitation Competition’. The goal was to investigate the preservation and rehabilitation of this built heritage within the framework of the on-going transformation of urban development models. Indeed, the architectural contest was part of a bigger event held on 27 November 2018, in the Nanxun Ancient Town titled ‘Nanxun Summit Forum on Cultural Heritage Around Taihu Lake’. The latter was co- sponsored by the College of Architecture and Urban Planning (CAUP) of Tongji University, the Huzhou Municipal Bureau of Culture, the Nanxun District People’s Government, and the Huzhou Municipal Planning Bureau. p g The ‘Community Condenser’ is one of the ten projects that participated in the compe- tition. One of the authors developed the proposal in collaboration with other three team members from different international institutions and won the third prize. Land 2024, 13, 405 6 of 19 In the context of national development and urban planning, architectural competitions emerge as effective catalysts for innovation and excellence [50–53]. Moreover, they can reflect the nation’s identity and values: through the design process, architects often draw inspiration from a country’s history, culture, and heritage. This opportunity facilitated the development of a cohesive vision tackling the key challenges and opportunities for the selected area and the urban context in which it is located. The selected designs, therefore, go beyond mere structures, representing symbols of national pride and cultural expression. By investing in the selection of remarkable designs, governments send a strong message regarding their dedication to create aesthetically pleasing, functional, smart, and sustainable spaces that contribute to the overall well-being of their citizens. p g The Lianshi Town case study is a valuable example of how these principles can be applied and validated in a real-world context. 3.1. Lianshi Case Study 3.1. Lianshi Case Study Lianshi Town, included in Nanxun District of Huzhou City, Zhejiang Province, is situated at the geographical center of the Hangjiahu Plain, the most extensive plain of the province [54]. Moreover, Lianshi is located in the southern part of Lake Tai, China’s third-largest freshwater lake, in the central region of the Yangtze River Delta, approximately 3 h away from Shanghai (Figure 3). The Beijing–Hangzhou Grand Canal traverses the entire Lianshi territory from south to north. W 8 of 21 Figure 3. Lianshi Town localization. Figure 3. Lianshi Town localization. Figure 3. Lianshi Town localization. Figure 3. Lianshi Town localization. Notably, the focus is placed on the regeneration project developed for the former granary in the southern Lianshi area. The site, occupying an area of 15,000 sq.m, is recog- nized as an integral part of the built heritage. Originating around the 1950s as a granary for rice collection and trade, the complex was strategically constructed along one of the a al b a hi oﬀthe Beiji Ha hou G a d Ca al It de elo e t a ub e A traditional water-edge town setting and a rich natural environment surround Lianshi Town, making this area among the most developed in Chinese history. Additionally, it is renowned as the primary grain and silk production hub in the Yangtze River Delta. Therefore, the region is pivotal in contributing to the agricultural and textile industries. canals branching oﬀ the Beijing–Hangzhou Grand Canal. Its development was subse- quently inﬂuenced by the various events that marked China’s history, particularly follow- ing the establishment of the People’s Republic of China. Indeed, the granary was aban- doned in the 2000s, evolving into an integral architectural element linked to the cultural heritage of that era. Therefore, standing as a symbol of traditional local culture, it under- scores the necessity for preservation and rehabilitation. 3.2. Brief Historical Background For over a thousand years, Lianshi has stood as a riverside market town along the Jiangnan Canal, witnessing the passage of numerous travelers moving between the North Notably, the focus is placed on the regeneration project developed for the former granary in the southern Lianshi area. The site, occupying an area of 15,000 sq.m, is recognized as an integral part of the built heritage. 3.1. Lianshi Case Study Originating around the 1950s as a granary for rice collection and trade, the complex was strategically constructed along one of the canals branching off the Beijing–Hangzhou Grand Canal. Its development was subsequently influenced by the various events that marked China’s history, particularly following the establishment of the People’s Republic of China. Indeed, the granary was abandoned in the 2000s, evolving into an integral architectural element linked to the cultural heritage of that era. Therefore, standing as a symbol of traditional local culture, it underscores the necessity for preservation and rehabilitation. 3 M i l 3. Materials and Methods Indeed, the second phase of the process refers to what occurred in 2021, when the project was concretely implemented by the Office Canopy of Architecture in collaboration with Xiangban Cultural Tourism to renovate the granary station area in Lianshi Town, translating the conceptual designs and programs into tangible urban interventions. The Office Canopy of Architecture represents an architectural firm in Hangzhou that proactively intervenes to implement a new local identity to the site, named ‘Cuckoo’, emphasizing sustainable development and urban renewal. p g p The third and last step of the methodology consists of the effective synthesis and discussion of the healthy city (HC) and smart city (SM) solutions that have been integrated in both projects, demonstrating how the competition results enabled the actual urban development in Lianshi Town. Figure 2 displays a diagram explaining the components of the methodology. 7 of 21 Figure 2 Methodological diagram of the study Figure 2. Methodological diagram of the study. Figure 2. Methodological diagram of the study. Land 2024, 13, 405 7 of 19 7 of 19 and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background yp g tural town catering to the needs of the surrounding villages and serving as a hub for the distribution of agricultural products, including silk and rice [55]. As the 21st century unfolded, together with the decline in traditional waterway trans- portation, the town underwent an accelerated pace of urban industrialization, with Lianshi establishing itself as a stronghold in key industries such as equipment manufac- turing, new metal materials, and textile apparel. Therefore, Lianshi achieved its status as For over a thousand years, Lianshi has stood as a riverside market town along the Jiangnan Canal, witnessing the passage of numerous travelers moving between the North and South. The Jiangnan Canal is one of the six sections into which the Beijing–Hangzhou Grand Canal is divided. Indeed, before the 1980s, Lianshi functioned as a typical agricul- tural town catering to the needs of the surrounding villages and serving as a hub for the distribution of agricultural products, including silk and rice [55]. Land 2024, 13, 405 8 of 19 8 of 19 As the 21st century unfolded, together with the decline in traditional waterway trans- portation, the town underwent an accelerated pace of urban industrialization, with Lianshi establishing itself as a stronghold in key industries such as equipment manufacturing, new metal materials, and textile apparel. Therefore, Lianshi achieved its status as an industrial strength in Huzhou City through diligent and pragmatic efforts by the government. Today, nearly half of the town’s area is now dedicated to planned industrial land (Figure 4). W 9 of 21 Figure 4. Industrial land distribution in Lianshi Town (picture: © Oﬃce Canopy of Architecture). Figure 4. Industrial land distribution in Lianshi Town (picture: © Office Canopy of Architecture). Figure 4. Industrial land distribution in Lianshi Town (picture: © Oﬃce Canopy of Architecture). Figure 4. Industrial land distribution in Lianshi Town (picture: © Office Canopy of Architecture). In this speciﬁc urban context, the abandoned granary, situated at the heart of the town, represents a signiﬁcant testimony of the initial Lianshi industrialization and the evolving times. Established in the 1950s, the grain depot includes a large open-air granary with a 15 m diameter, ten conical silos arranged in a single row, and over ten rectangular factory buildings (Figure 5). The overall industrial complex is well-preserved and stands as the area’s most architecturally valuable vernacular site. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background The Optimal Strategy for Regeneration: Health and Smartness Combined In 1956, several private rice businesses in Lianshi transitioned to p 3.3. The Optimal Strategy for Regeneration: Health and Smartness Combined nerships, and the building was subsequently renamed as the state-owned Lianshi Ri Factory. It marked the earliest industrial enterprise in the history of Lianshi and emerge as the sole state-owned enterprise at that time. In 1969, the ﬁrst export of Chinese gra originated from this production plant. In November 1970, subsequently merging with th Grain Management Oﬃce, the factory changed its name to Lianshi People’s Grain Statio [55]. In the 1980s, a period of reforms occurred, during which the construction of a ne road was approved, i.e., Zhongji Road, with the granary cluster located on its east an west sides. The latter was later enlarged in the 2000s through the demolishing of som Currently, Lianshi Town has a stable population of around 106,000 inhabitants, com- prising roughly 85,000 residents and approximately 21,000 migrant residents [54]. As evident from previous descriptions, the actual numbers derive from the area’s historical evolution, which is strictly linked to its industrial history. Indeed, statistical reports from different years show that the global industrial output value of Lianshi Town was CNY 2.1 billion in 2000, further reaching CNY 26 billion in 2022, marking a remarkable tenfold increase over a span of more than 20 years. This growth highly affected the population rate, shifting from 18.6% in 2000 to 78.9% in 2020 [55]. west sides. The latter was later enlarged in the 2000s through the demolishing of som ndustrial buildings that were part of the cluster. After various additional transformation t ultimately ceased production and went out of business in the second half of 2009. 3.3. The Optimal Strategy for Regeneration: Health and Smartness Combined g [ ] Data demonstrate that a significant portion of the local population has migrated to major cities over the past two decades. Nevertheless, at the same time, the rural and immigrant populations have adapted to the urbanization and industrial development occurring in Lianshi from the beginning of the 21st century, thus filling the void left by the outflow. Currently, Lianshi Town has a stable population of around 106,000 inhabitants, com prising roughly 85,000 residents and approximately 21,000 migrant residents [54]. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background Moreover, it has been listed among the municipal-level cultural heritage buildings in Huzhou City [55]. In this specific urban context, the abandoned granary, situated at the heart of the town, represents a significant testimony of the initial Lianshi industrialization and the evolving times. Established in the 1950s, the grain depot includes a large open-air granary with a 15 m diameter, ten conical silos arranged in a single row, and over ten rectangular factory buildings (Figure 5). The overall industrial complex is well-preserved and stands as the area’s most architecturally valuable vernacular site. Moreover, it has been listed among the municipal-level cultural heritage buildings in Huzhou City [55]. In 1956, several private rice businesses in Lianshi transitioned to public–private part- nerships, and the building was subsequently renamed as the state-owned Lianshi Rice Factory. It marked the earliest industrial enterprise in the history of Lianshi and emerged as the sole state-owned enterprise at that time. In 1969, the first export of Chinese grain originated from this production plant. In November 1970, subsequently merging with the Grain Management Office, the factory changed its name to Lianshi People’s Grain Station [55]. In the 1980s, a period of reforms occurred, during which the construction of a new road was approved, i.e., Zhongji Road, with the granary cluster located on its east and west sides. The latter was later enlarged in the 2000s through the demolishing of some Land 2024, 13, 405 9 of 19 industrial buildings that were part of the cluster. After various additional transformations, it ultimately ceased production and went out of business in the second half of 2009. 10 of 2 Figure 5. Bird’s-eye view of the old granary station (picture: © Oﬃce Canopy of Architecture) Figure 5 Bird’s-eye view of the old granary station (picture: © Office Canopy of Architecture) igure 5. Bird’s-eye view of the old granary station (picture: © Oﬃce Canopy of Architecture Figure 5. Bird’s-eye view of the old granary station (picture: © Office Canopy of Architecture). Figure 5. Bird’s-eye view of the old granary station (picture: © Oﬃce Canopy of Architec Figure 5. Bird’s-eye view of the old granary station (picture: © Office Canopy of Architectur In 1956, several private rice businesses in Lianshi transitioned to pu 3.3. The Optimal Strategy for Regeneration: Health and Smartness Combined In 1956, several private rice businesses in Lianshi transitioned to p 3.3. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background g q y g y p The initial phase of the competition proposal started considering the possible evolution of such neglected industrial areas, and the opportunities included [62]: • Reactivation by renovating the existing facilities (like maintaining the initial on-site activity with possible technological upgrades towards the so-called green economy); • Abandonment with or without protection measures; • Reactivation by renovating the existing facilities (like maintaining the initial on-site activity with possible technological upgrades towards the so-called green economy); • Abandonment, with or without protection measures; activity with possible technological upgrades towards the so called green economy); • Abandonment, with or without protection measures; • Abandonment, with or without protection measures;i • Structural reconversion (keeping an industrial profile with a rearrangement of the buildings); • Functional and/or structural reconversion (change in function, with or without physi- cal and historic preservation, and reuse linked to typically urban functions but pro- viding urban systems with a high incidence of public services and equipment to significantly improve urban quality). To this end, the engagement with residents has been decisive in defining the correct functional program: through a direct survey, it was possible to collect some key points. To this end, the engagement with residents has been decisive in defining the correct functional program: through a direct survey, it was possible to collect some key points. Over 50 interviews were conducted by the authors among residents ranging in age from 13 to 91 years old. The interviewed people recalled the site not just as an industrial space but also as a gathering spot for enjoyment, reminiscing about meeting friends, and playing games like chasing each other around the granaries. In fact, a significant number of them expressed the desire to witness the transformation and enhancement of the area into a new big public space, accounting for 33% of all the answers. Others expressed the desire for new spaces for cultural activities (14%), commercial functions (14%), or new housing (5%). A good number of people expressed the will to preserve the site (24%), while others preferred not to express an opinion (10%) [63]. Most residents envisioned it as a place where people of all ages, including those from neighboring communities, could interact and participate in various activities catering to diverse needs. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background As ev ident from previous descriptions, the actual numbers derive from the area’s historical evo lution, which is strictly linked to its industrial history. Indeed, statistical reports from di ferent years show that the global industrial output value of Lianshi Town was CNY 2 billion in 2000, further reaching CNY 26 billion in 2022, marking a remarkable tenfol increase over a span of more than 20 years. This growth highly aﬀected the populatio rate, shifting from 18.6% in 2000 to 78.9% in 2020 [55]. Data demonstrate that a signiﬁcant portion of the local population has migrated t major cities over the past two decades Nevertheless at the same time the rural and im A careful survey conducted by Tang and Jiang [54] subdivided Lianshi into three main demographic categories: elderly, middle-aged individuals, and teenagers. Notably, a con- siderable segment of the population comprises the elderly and teenagers, constituting one fundamental element of the overall regeneration strategy. As previously discussed, healthy and smart paradigms recognize the pivotal role of citizens and emphasize the importance of social participation. Additionally, the synergy between technological innovation, citizen engagement, and a focus on well-being contributes to smartness and promotes the residents’ health and quality of life. Anttiroiko et al. and Hollands [56,57] assert that placing the citizen at the core of urban innovations is essential, given that the primary challenges in urban areas derive from sociological rather than technological foundations.i major cities over the past two decades. Nevertheless, at the same time, the rural and im migrant populations have adapted to the urbanization and industrial development occu ring in Lianshi from the beginning of the 21st century, thus ﬁlling the void left by th outﬂow Therefore, integrating healthy and smart elements within the specific urban context of the disused granary leads to some considerations since industrial area requalification may represent an extremely complicated process requiring the significant mobilization Land 2024, 13, 405 10 of 19 10 of 19 of resources. However, current urban policies strongly support preserving and reusing these residual buildings, open unbuilt areas, and urban public spaces to develop more high-quality, mixed-use, and high-density spaces [11,58–61]. of resources. However, current urban policies strongly support preserving and reusing these residual buildings, open unbuilt areas, and urban public spaces to develop more high-quality, mixed-use, and high-density spaces [11,58–61]. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background Concerning smart features, the following principles were selected and implemented: • Integrated mobility: intelligent transportation systems for traffic management and improved public transit; • Integrated mobility: intelligent transportation systems for traffic management and improved public transit; Land 2024, 13, 405 11 of 19 11 of 19 • Sustainable energy: integration of renewable energy sources and smart energy management • Sustainable energy: integration of renewable energy sources and smart energy management; d f d d d f ffi Sustainable energy: integration of renewable energy sources and smart energy management; • Connected infrastructure: interconnected devices and systems for efficient resource utilization;i • Connected infrastructure: interconnected devices and systems for efficient resource utilization; • Smart buildings: intelligent construction and management systems for energy effi- ciency and occupant comfort; • Digital inclusion: ensuring access to technology and digital services for all residents; • Digital inclusion: ensuring access to technology and digital services for all residents; • Innovation ecosystem: support for technology-driven innovation and entrepreneurship • Innovation ecosystem: support for technology-driven innovation and entrepreneurship Globally, as emphasized by Anthopoulos [65], smart cities comprise different cases in urban spaces where technology is applied for different purposes, from virtualizing cities to attracting business relocations. In the case of the renovation of the granary cluster, the main goal is to create urban spaces that are not exclusively technologically advanced but mostly adaptable, responsive, and capable of fostering economic growth and a high quality of life for the residents of Lianshi Town. The final project output, which has been submitted to the competition, can be syn- thesized into three main actions that have been implemented in the design of the new masterplan, encapsulating healthy and smart conditions. The latter are preservation, con- nectivity, and activation, which are able to function simultaneously. These elements defined the comprehensive revitalization of the industrial site and incorporated this specific project into a broader plan that addresses and connects various parts of the city [66]. Therefore, the entire settlement’s infrastructure system is preserved and converted in-to a novel public pedestrian system. The vacant spaces between the massive build- ings are transformed into various public recreation areas to bolster community connec- tions. Activities and features along internal paths encourage social aggregation and evoke collective memories. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background p p g Therefore, ‘Community Condenser’ proposes an overall intervention, enhancing healthy and smart solutions based on the existing social, economic, and environmental context characterizing the granary cluster. Qualitative techniques such as case studies, exploration, observation, and description contribute to a comprehensive comprehension of the evolving trends that characterize both smart and healthy cities. Most of the local requests were in line with the healthy guidelines promoted by WHO. Indeed, the new grain station masterplan integrates: • Green and leisure spaces: ample parks and recreational areas promoting physical activity and mental well-being; • Clean environment: effective waste management, air quality control, and pollution reduction measures; • Safe infrastructure: secure public spaces, well-lit streets, and efficient emergency services; • Healthy lifestyle promotion: community programs promoting healthy habits, nutrition, • Safe infrastructure: secure public spaces, well-lit streets, and efficient emergency services; • Healthy lifestyle promotion: community programs promoting healthy habits, nutrition, and fitness; i • Social inclusion: places and facilities to ensure the well-being of diverse populations; • Active transportation: infrastructure network supporting walking, cycling, and other forms of eco-friendly transportation. • Active transportation: infrastructure network supporting walking, cycling, and other forms of eco-friendly transportation. It is worth noting that from the healthy city point of view, the focus is on ongoing improvement processes and long-term perspectives that must be evaluated through perfor- mance indicators, which contribute to planning and management [64]. On the other hand, concerning smart cities, private initiatives are much more involved, generating interven- tions geared towards achieving short-term returns. Thus, the emphasis lies on leveraging technology to optimize urban operations and resource management. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background New granary station masterplan submitted to the competition [62]. 3.4. From Architectural Competition to Execution: Enhancing Lianshi’s Regeneration 3.4. From Architectural Competition to Execution: Enhancing Lianshi’s Regeneration 3.4. From Architectural Competition to Execution: Enhancing Lianshi’s Regeneration 3.4. From Architectural Competition to Execution: Enhancing Lianshi’s Regeneration The national competition opportunity to redesign the granary cluster of Lianshi rep- resented more than just a design contest; it is recognized as a transformative exercise that will shape the stakeholders’ physical, cultural, and social landscape. Governments’ atten- tion to these competitions demonstrates a commitment to fostering innovation, celebrat- ing cultural identity, and building healthy environments. In the presented case study, the competition became a driving force for progressive and forward thinking regarding urban development in Lianshi Town. The national competition opportunity to redesign the granary cluster of Lianshi repre- sented more than just a design contest; it is recognized as a transformative exercise that will shape the stakeholders’ physical, cultural, and social landscape. Governments’ attention to these competitions demonstrates a commitment to fostering innovation, celebrating cultural identity, and building healthy environments. In the presented case study, the competition became a driving force for progressive and forward thinking regarding urban development in Lianshi Town. p The social urban context previously outlined was also the same after the participation in the competition, as conﬁrmed by Tang and Jiang [54]. Indeed, they obtained interesting insights from 120 questionnaires that were randomly distributed within each of the three age groups mentioned before (elderly individuals, middle-aged individuals, and teenag- ers). Of the total number of questionnaires distributed, 112 were successfully retrieved, corresponding to a response rate of 93.3%, including 89 locals, 18 migrants, and 5 tourists, with a relatively balanced gender ratio. The social urban context previously outlined was also the same after the participation in the competition, as confirmed by Tang and Jiang [54]. Indeed, they obtained interesting insights from 120 questionnaires that were randomly distributed within each of the three age groups mentioned before (elderly individuals, middle-aged individuals, and teenagers). Of the total number of questionnaires distributed, 112 were successfully retrieved, corre- sponding to a response rate of 93.3%, including 89 locals, 18 migrants, and 5 tourists, with a relatively balanced gender ratio. y g Even in this case, the residents, especially elderly individuals, expressed an urgent need for large-area spaces suitable for physical exercise activities such as ﬁtness, dancing, badminton, etc. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background The ‘Community Condenser’ has been designed to shape social behavior and serve as a social catalyst for Lianshi, providing the historical granary with the opportunity to serve as a focal point and act as a cultural motivator for the area’s urban development. Operating at a neighborhood scale, the project aims to demonstrate how heritage can be revitalized in contemporary cities while preserving the unique characteristics of communities, ultimately creating a vibrant and smart urban environment. More specifically, regarding the action of preservation, the approach involves trans- forming the entire urban area along the canal, where the granary was built from the beginning, into a historic district, highlighting the distinctive features of the existing struc- tures. This includes creating new public spaces along the canal to reinstate social relations and stimulate economic growth through incentives aimed at renewing the architecture in the area. Regarding connectivity, the plan entails developing new pathways through surround- ing neighborhoods to connect residents to the granary and historic district along the canal. Improved circulation is promoted through specific signage and open green spaces. Regarding connectivity, the plan entails developing new pathways through surround- ing neighborhoods to connect residents to the granary and historic district along the canal.i g g g y g Improved circulation is promoted through specific signage and open green spaces. Finally, the activation element focuses on designing open and landscaped areas for community gatherings, establishing ICT hubs in the granaries aiming for efficiency and improved services, converting the south and west areas of the granary into a modern residential block, and developing commercial spaces around the southeast area of the site. The proposed intervention is well expressed through the detailed design of the new site (Figure 6), which represents both a healthy and smart approach to the redevelopment of an urban area. Overall, putting effort into addressing healthy dimensions can contribute to establishing a smart dimension and vice versa. In conclusion, the proposed intervention harmoniously combines healthy and in- telligent aspects, creating an urban environment that promotes the physical and mental well-being of the community while innovatively leveraging history and technology for sustainable urban development. Land 2024, 13, 405 12 of 19 al well- sustain- Figure 6. New granary station masterplan submitted to the competition [62]. Figure 6. New granary station masterplan submitted to the competition [62]. Figure 6. New granary station masterplan submitted to the competition [62]. Figure 6. Figure 7. New granary station masterplan designed by Oﬃce Canopy of Architecture (picture: © Oﬃce Canopy of Architecture). Figure 7. New granary station masterplan designed by Office Canopy of Architecture (picture: © Office Canopy of Architecture). Also, in this case study, eﬀorts were made to open walls, activate street corners, an readjust the scale of the empty squares among the abandoned buildings. As previousl discussed, similar interventions allow citizens to engage with and utilize cultural relic Considering Lianshi’s recent substantial transformation into the most populous indus- trial town in the Nanxun district, the area must face challenges competing with renowned ancient towns. Therefore, the focus shifted to meeting daily residents’ needs. eﬀectively. Moreover, the design studio promoted the concept of ‘creating local future scenarios This approach involves addressing the needs of speciﬁc customer groups and developin g y The planning phase proposed ‘community commerce’ as the main function to be implemented, aiming to attract residents during holidays and position Lianshi as a sought- after destination. pp g p g p p innovative, community-oriented solutions through non-standard commercial projec centered around the concept of ‘locality’. Considering Lianshi’s recent substantial transformation into the most populous in dustrial town in the Nanxun district, the area must face challenges competing with re nowned ancient towns. Therefore, the focus shifted to meeting daily residents’ needs. The planning phase proposed ‘community commerce’ as the main function to be im plemented, aiming to attract residents during holidays and position Lianshi as a sough after destination. ‘C k ’ th f th b t th it hit t l d l d ‘Cuckoo’, the name of the new urban space, strengthens its architectural and landscape design by maintaining the granary styles and principal features. Through a strategic combination of demolition, restoration, and renovation, the goal is to eliminate congestion and scattered elements, creating a continuous space flowing from the riverbank to the south. Accordingly, the preservation action is enhanced to give value to structures with historical significance (such as the 15 m diameter open-air granary and the ten conical silos) [55]. Consequently, the site’s inherent identity or character is either maintained or altered to meet the new demands. This approach enhances sustainability’s significance, which is crucial for innovation-driven urban development and heritage conservation (Figure 8). ‘Cuckoo’, the name of the new urban space, strengthens its architectural and land scape design by maintaining the granary styles and principal features. and South. The Jiangnan Canal Grand Canal is divided. Indeed 3.2. Brief Historical Background Additionally, they desired leisure areas for napping, soaking up the sun, and conversing. Adults were interested in outdoor spaces for dining and socializing dur- ing their spare time. Meanwhile, teenagers’ preferences revolved around recreational ac- tivities and more entertainment venues Even in this case, the residents, especially elderly individuals, expressed an urgent need for large-area spaces suitable for physical exercise activities such as fitness, dancing, badminton, etc. Additionally, they desired leisure areas for napping, soaking up the sun, and conversing. Adults were interested in outdoor spaces for dining and socializing during their spare time. Meanwhile, teenagers’ preferences revolved around recreational activities and more entertainment venues. tivities and more entertainment venues. Indeed, in 2021, the Office Canopy of Architecture, an architectural firm based in Hangzhou City, Zhejiang Province, collaborated with Xiangban Cultural Tourism to provide a renovation masterplan (Figure 7) for the granary station area in Lianshi Town [55]. Also, in this case study, efforts were made to open walls, activate street corners, and readjust the scale of the empty squares among the abandoned buildings. As previ- ously discussed, similar interventions allow citizens to engage with and utilize cultural relics effectively. y Moreover, the design studio promoted the concept of ‘creating local future scenarios’. This approach involves addressing the needs of specific customer groups and developing innovative, community-oriented solutions through non-standard commercial projects centered around the concept of ‘locality’. Land 2024, 13, 405 13 of 19 m based i sm to pro Figure 7. New granary station masterplan designed by Oﬃce Canopy of Architecture (picture: © Oﬃce Canopy of Architecture). Figure 7. New granary station masterplan designed by Office Canopy of Architecture (picture: © Office Canopy of Architecture). Through a strategi combination of demolition, restoration, and renovation, the goal is to eliminate congestio and scattered elements, creating a continuous space ﬂowing from the riverbank to th Overall, the proposal adheres to the ‘proximity principle’, which, in this case, relies on local merchants’ understanding of residents’ needs. In this sense, cities should actively pur- sue local knowledge and gain a comprehensive understanding of solution configurations at the local level to address smartness [55]. south. Accordingly, the preservation action is enhanced to give value to structures wit historical signiﬁcance (such as the 15 m diameter open-air granary and the ten conic silos) [55]. Consequently, the site’s inherent identity or character is either maintained o To conclude, ‘Lianshi Cuckoo’ (Figure 9) was conceived as a cultural and commercial district targeting local customers in small towns, aiming to create a local identity aligned while incorporating creativity and maintaining a healthy atmosphere. Land 2024, 13, 405 14 of 19 on (Fig- Figure 8. The renovation process (picture: © Oﬃce Canopy of Architecture). Figure 8. The renovation process (picture: © Office Canopy of Architecture). d 2024, 13, x FOR PEER REVIEW 16 of 24, 13, x F Figure 8. The renovation process (picture: © Oﬃce Canopy of Architecture). Figure 8. The renovation process (picture: © Office Canopy of Architecture). Overall, the proposal adheres to the ‘proximity principle’, which, in this case, on local merchants’ understanding of residents’ needs. In this sense, cities should ac pursue local knowledge and gain a comprehensive understanding of solution conﬁ tions at the local level to address smartness [55]. To conclude, ‘Lianshi Cuckoo’ (Figure 9) was conceived as a cultural and comm district targeting local customers in small towns, aiming to create a local identity ali while incorporating creativity and maintaining a healthy atmosphere. Figure 9. Bird’s-eye view of the realized project (picture: © Oﬃce Canopy of Architecture). Figure 9. Bird’s-eye view of the realized project (picture: © Office Canopy of Architecture). Figure 9. Bird’s-eye view of the realized project (picture: © Oﬃce Canopy of Architecture). Figure 9. Bird’s-eye view of the realized project (picture: © Office Canopy of Architecture). 4. Results and Discussion 4. Results and Discussion One of the central aims of the concept of a smart city as well as of a healthy city is to improve the quality of life and the well-being of its citizens. As well-displayed by numer- ous international examples, many cities have decided to use the key features of both the healthy and smart city concepts to reach the targets identiﬁed by the SDGs goals (in par- ti l G l 3 ‘G d H lth d W ll b i ’ d G l 11 ‘S t i bl Citi d C One of the central aims of the concept of a smart city as well as of a healthy city is to improve the quality of life and the well-being of its citizens. As well-displayed by numerous international examples, many cities have decided to use the key features of both the healthy and smart city concepts to reach the targets identified by the SDGs Land 2024, 13, 405 15 of 19 15 of 19 goals (in particular, Goal 3 ‘Good Health and Well-being’ and Goal 11 ‘Sustainable Cities and Communities’). In order to contribute to planning and management choices and to define sustainable strategies for city regeneration and further development, it is vital to set performance indicators able to evaluate the flexible and dynamic condition of cities and citizens, ad- dressing both the two dimensions of health and smart in an urbanized context, considering bottom-up and top-down approaches and initiatives, and monitoring the results and the implementation process. As highlighted by many scholars [67,68], the most important indicators (quantitative and qualitative, linked to individual perception) are related to ecosystems (in the urban environment), health promotion, sustainable mobility, consump- tion of natural resources, green areas, energy production and consumption, public services and facilities, social relationship enhancement, cultural heritage (material and immaterial), community engagement, and so on.i y g g Moreover, the main evident difficulties in the regeneration processes, in particular for former industrial areas, are mainly due to high base costs (decontamination and demolition) and limited financial resources; incapacity to manage the complex and lengthy process of transformation (from the planning phase to the construction and management phase); rigidity of public administration and its slowness in decision-making; fixed regulatory frameworks; planning abilities in all the phase of the process; and a lack of coordination among public bodies, stakeholders, and professionals. 4. Results and Discussion 4. Results and Discussion g p p In Lianshi, the primary focus is on aligning with the activities and consumption preferences of the local residents. Regardless of the project’s return on investment or future urbanization development, prioritizing the incubation of local business and cultural communities is deemed the most cost-effective and optimal choice. This approach is then leveraged to expand the overflow customer base and integrate diverse cultural and entertainment activities, effectively catalyzing the vitality and development of urban public spaces. p p Below, the authors underline the most relevant characteristics that define the Lianshi case study as a healthy and smart city, both in the proposal competition version and in the actual project version: p j Case 1: The Competition Proposal Healthy Aspects: • Public recreation areas: Transforming vacant spaces between buildings into various public recreation areas that promote an active lifestyle and encourage social interaction. These recreation areas can contribute to the physical and mental well-being of the community, providing open spaces for outdoor activities. • Social aggregation: Designed elements along internal paths that encourage social ag- gregation are crucial for promoting mental health and a sense of community. Evoking collective memories through these features strengthens bonds among residents and create a more cohesive social fabric. • Green spaces and connectivity: Creating new pathways and open green spaces en- hances connectivity and promotes a healthy lifestyle. These elements can encourage walking and physical activity, contributing to a more active and sustainable community. Smart Aspects: • Community Condenser: The ‘Community Condenser’ concept as a social catalyst is a smart element. Using local history and existing structures as a cultural motiva- tor demonstrates a smart approach to urban planning, leveraging heritage to drive sustainable urban development. • Heritage Revitalization: The approach of transforming the entire area along the canal into a historic district while preserving distinctive features reflects a savvy integration of the past into the present. This demonstrates how history can be key in creating modern and dynamic urban environments. • Connectivity and ICT hubs: The development of new pathways and the presence of ICT hubs indicate a smart approach to connectivity and technology. Improving Land 2024, 13, 405 16 of 19 circulation through specific signage and open spaces suggests a smart infrastructure that facilitates movement and access to digital resources. circulation through specific signage and open spaces suggests a smart infrastructure that facilitates movement and access to digital resources. 4. Results and Discussion 4. Results and Discussion In conclusion, the proposed intervention harmoniously combines healthy and in- telligent aspects, creating an urban environment that promotes the physical and mental well-being of the community while innovatively leveraging history and technology for sustainable urban development. Case 2: The Project in Real Life Healthy City Elements: • Emphasis on fostering innovation and building healthy environments. • Focus on addressing residents’ daily needs and proposing ‘community commerce’ as a primary function. • Strategic combination of demolition, restoration, and renovation to eliminate conges- tion and create continuous, accessible spaces.i p • Preservation action to give value to structures with historical significance, contributing to heritage conservation.i • Consideration of the significance of sustainability for both innovation-driven urban development and heritage conservation. • Consideration of the significance of sustainability for both innovation-driven urban development and heritage conservation. Smart City Elements: • Utilization of local knowledge and understanding of residents’ needs as part of the ‘proximity principle’. • Implementation of a renovation masterplan that involves activating street corners and readjusting the scale of empty squares, contributing to a smarter urban layout. • Promotion of the concept of ‘creating local future scenarios’ through innovative, community-oriented solutions. • Integration of non-standard commercial projects centered around the concept of ‘local- ity’ aligns with the smart city approach. • Integration of non-standard commercial projects centered around the concept of ‘local- ity’ aligns with the smart city approach. 5. Conclusions Following this path, the qualitative transformation of urban settlements mirrors the following social conditions: healthy and/or smart citizens contribute to the regeneration of healthy and smart cities. and city and territorial management, as well as in the formulation and execution of public policies. It is at the district, neighborhood, and block scales (the smallest scales in the urban planning field) where knowledge, awareness, and inhabitants’ specific actions can substan- tially contribute to achieving these goals. However, this process needs to be integrated into the framework of various local intersectoral public policies and the diverse scales of the materialization of public policies must be taken into account. Following this path, the qualitative transformation of urban settlements mirrors the following social conditions: healthy and/or smart citizens contribute to the regeneration of healthy and smart cities. In conclusion, while smart cities and healthy cities have distinct objectives, the inte- gration of smart and healthy elements into urban planning can lead to more holistic and sustainable city development. The Lianshi Town case study is a valuable example of how these principles can be applied in a real-world context. Author Contributions: Conceptualization, C.P. and E.M.V.; methodology, C.P.; validation, C.P. and E.M.V.; formal analysis, C.P.; investigation, C.P.; writing—original draft preparation, C.P. and E.M.V.; writing—review and editing, C.P. and E.M.V.; supervision, E.M.V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: Data are contained within the article. Data Availability Statement: Data are contained within the article. Acknowledgments: We express our sincere gratitude to the Office Canopy of Architecture for officially supplying us with authentic materials related to the Cuckoo project in Lianshi Town. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Gandy, M. 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The project, operating at the neighborhood scale, demonstrates how the strategy of valuing the existing built environment is one of the most crucial actions to be implemented to satisfy the community’s necessities and help decision-makers reach healthy, smart, and sustainable goals. This is due to the anthropological and social value of the site and its strong relationships with the context and the community. Additionally, the project clearly defines and implements the need to encourage social interaction and accommodate new economic, social, and cultural uses. Overall, the project aims to balance physical and perceptual heritage elements and create new, smart urban spaces where people can live, work, move, and socialize. 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https://openalex.org/W4200025093	https://discovery.ucl.ac.uk/10145795/1/ebc-2021-0034c-2.pdf	English	null	Vesicular dysfunction and pathways to neurodegeneration	Essays in biochemistry	2,021	cc-by	7,885	Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 Review Article Vesicular dysfunction and pathways to neurodegeneration Patrick A. Lewis1,2,3 1Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, United Kingdom; 2Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, United Kingdom; 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, United States of America Dow Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 Introduction Neurodegenerative diseases, characterised by the progressive loss of neurons in the central nervous sys- tem, impact on the lives of millions of people around the world [1]. These disorders span a range of clinical presentations, from disorders characterised primarily by cognitive dysfunction such as Alzheimer’s dis- ease, to those presenting primarily with motor dysfunction such as Parkinson’s disease and Amyotrophic Lateral Sclerosis [2]. A feature common to all of these disorders is a dearth of therapeutic agents that mod- ify the underlying disease process, with current therapies almost exclusively targeting symptoms rather than aetiology [3,4]. As such, developing a deeper understanding of the underlying molecular deficits that drive neuronal cell death is absolutely critical, as gaining insight into these events will open up novel routes to disease-modifying treatments [5]. Building on advances in our comprehension of the underlying genetics of neurodegenerative disease, in particular the identification of Mendelian forms of neurodegen- eration and common risk variants deriving from genome-wide association studies, disruption of cellular processes involved in the trafficking and function of vesicles within the cell has emerged as a convergent pathway across a broad spectrum of neurodegenerative diseases [6]. p y y Patrick A. Lewis1,2,3 1Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, United Kingdom; 2Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, United Kingdom; 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, United States of America 1Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, United Kingdom; 2Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, United Kingdom; 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, United States of America Correspondence: Patrick A. Lewis (plewis@rvc.ac.uk) Cellular control of vesicle biology and trafficking is critical for cell viability, with disruption of these pathways within the cells of the central nervous system resulting in neurodegeneration and disease. The past two decades have provided important insights into both the genetic and biological links between vesicle trafficking and neurodegeneration. In this essay, the pathways that have emerged as being critical for neuronal survival in the human brain will be discussed – illustrating the diversity of proteins and cellular events with three molecular case studies drawn from different neurological diseases. © 2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) Vesicular function in the central nervous system The packaging and trafficking of membrane-bound vesicles is critical for the normal function of eukary- otic cells, responsible for ensuring that cellular components are located in the right place, and at the right time, to carry out physiological roles [7]. The correct regulation of vesicle function and location is required across the full spectrum of eukaryotic cell biology, from energy generation at the mitochondrion through to the regulation of gene expression in the nucleus. This is especially true of the cells of the central nervous system, spanning both neurons and glial cell populations. Neuronal cells, with their extended axons re- quiring long-range vesicle transport, and the critical role played by synaptic vesicles in neurotransmission, are particularly sensitive to disruptions in vesicle trafficking and function [8]. Coupled to their unique en- ergy demands and the low endogenous rate of neurogenesis, it does not require a great deal of additional stress deriving from vesicular dysfunction to push neurons into a degenerative spiral. More recently, there 941 Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 Figure 1. Cellular pathways connecting vesicle biology, trafﬁcking and neurodegeneration, illustrated in a (not to scale) stereotypical neuron (A) Nuclear transport. (B) Regulation of autophagic pathways and lysosomal biology. (C) Endo- and exo-cytic pathways. (D) Endoso- mal vesicle trafficking among the Golgi apparatus, endoplasmic reticulum and the cytoplasm. (E) Axonal transport. (F) Mitochondrial dynamics and damage response. (G) Synaptic function. Downloaded from http://portlandpress.com/essaysbiochem/article-pdf/65/7/941/926957/ebc-2021-0034c.pdf by UK user on 23 February 2022 r pathways connecting vesicle biology, trafﬁcking and neurodegeneration, illustrated in a (not to scale) ron Figure 1. Cellular pathways connecting vesicle biology, trafﬁcking and neurodegeneration, illustrated in a (not to scale) stereotypical neuron (A) Nuclear transport. (B) Regulation of autophagic pathways and lysosomal biology. (C) Endo- and exo-cytic pathways. (D) Endoso- mal vesicle trafficking among the Golgi apparatus, endoplasmic reticulum and the cytoplasm. (E) Axonal transport. (F) Mitochondrial dynamics and damage response. (G) Synaptic function. has been an increasing interest in the role of glial cells, such as astrocytes and microglia, in the neurodegenerative process – with the regulation of vesicular trafficking and function being likewise important for the normal function of these cells [9]. Neurogenetics of vesicular dysfunction g y The neurogenetics of inherited forms of neurodegeneration, and more recently population-based analysis of genetic risk for neurodegenerative disorders through genome-wide association studies, have been a key source of evidence linking disruption of vesicular biology to neuronal cell death [10]. A number of genes that are either mutated in fa- milial neurodegenerative disease or have been identified as risk loci for these disorders have normal functions linked to vesicular biology. Intriguingly, this is true across a range of disorders, including disorders of cognition such as Alzheimer’s disease and frontotemporal dementia, and those where the predominant impact is upon movement such as Parkinson’s disease and the hereditary spastic paraplegias. These disorders are differentiated by their clinical pre- sentations, but also by the distribution of neurodegeneration within the brain and by the neuropathological hallmarks associated with disease (most notably proteinaceous inclusions, such as amyloid plaques and neurofibrillary tangles in the Alzheimer’s brain, and Lewy bodies in Parkinson’s) [11]. © 2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). vignette 1: PINK1, Parkin, mitophagy and the substantia nigra Molecular vignette 1: PINK1, Parkin, mitophagy and the substantia nigra Mitophagy is a specialised form of macroautophagy, a process that is conserved throughout the eukarya as a means to degrade and recycle cellular waste [12]. Macroautophagy is a multistage process, involving the formation of a phagophore (an open, double-membraned vesicle) which then engulfs material targeted for degradation. The en- capsulated material is then trafficked to the lysosomes where the autophagosome fuses with lysosomal vesicles and its contents are broken down. The molecular mechanisms governing the selective targeting and degradation of dys- functional mitochondria first came to light through the discovery of mutations in rare autosomal recessive forms of juvenile parkinsonism, with loss-of-function mutations in the PRKN gene on chromosome 6 identified in sev- eral Japanese kindreds in 1998 and subsequently mutations in the PINK1 gene on chromosome 1 reported in 2004 [13,14]. Mutations in both genes resulted in a similar clinical presentation and disease course, with early onset of symptoms during childhood or adolescence and an extended, motor symptom dominant presentation [15]. The cel- lular functions of Parkin, an E3 ubiquitin ligase, and PTEN-induced kinase 1 (PINK1), a serine/threonine kinase, were linked together by a series of elegant studies in the fruit fly, Drosophila melanogaster, demonstrating that they acted in convergent pathway to manage mitochondrial health [16,17]. Over the course of the last two decades, and through the work of many laboratories, there is now a detailed understanding of how these proteins act to coordinate the targeted degradation of dysfunctional mitochondria [18]. PINK1 is activated by the presence of damaged mito- chondria (this can be induced artificially by the use of chemical stresses such as Carbonyl cyanide m-chlorophenyl hydrazone, which decouples the mitochondrial respiratory chain), and is stabilised at the outer mitochondrial mem- brane [19]. PINK1 then recruits and phosphorylates ubiquitin and then Parkin, leading to the formation of ubiquitin chains on the surface of the damaged mitochondrion. This, in turn, leads to the recruitment of the proteins involved in macroautophagy and the formation of a phagophore in proximity to the mitochondrion, eventually leading to its engulfment by an autophagosome, trafficking to the lysosomes, and degradation. The consequence of losing the function of PINK1 or Parkin in the human brain is localised degeneration of dopaminergic neurons in the substantia nigra, demonstrating a key role for a very specific aspect of vesicular tagging and targeting in neurodegeneration [20]. vignette 1: PINK1, Parkin, mitophagy and the substantia nigra It is of note that this is distinct from specific dysfunction of vesicle trafficking, highlighting that what is in vesicles can be just as important as where those vesicles are. There is also a clear link between the PINK1/Parkin pathway and broader dysfunction of endolysosomal biology, a cellular pathway of increasing interest and importance across the whole spectrum of neurodegenerative diseases [21]. Equally intriguing is the accumulating evidence for a con- vergence of glial mitophagy and neurodegeneration [22,23]. Downloaded from http://portlandpress.com/essaysbiochem/article-pdf/65/7/941/926957/ebc-2021-0034c.pdf by UK user on 23 February 2022 © 2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). © 2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) Pathways and processes Focusing on neuronal cell biology, several critical cellular pathways emerge from the neurogenetics of vesicular dys- function in neurodegenerative disease. These are summarised in Figure 1, and span highly conserved processes op- erating in the soma such as the regulation of mitochondrial biology and waste disposal via the lysosomes, through the trafficking of vesicle up and down the axon, to the regulation of synaptic vesicle function. To discuss and debate these pathways exhaustively would require a textbook, and so in the interests of brevity three case studies, taken from a range of neurodegenerative diseases, will be used to illustrate how a mechanistic understanding of vesicular pathways to progressive neuronal loss is being established. It is also worth pausing to note that, as alluded to above, it is increasingly clear that there is a significant contribution of glial cell function to the 942 Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 neurodegenerative process and that a neuronal centric paradigm for these disorders is a huge oversimplification of what is occurring in the brains of people undergoing neurodegeneration. Unfortunately, how glial biology is disrupted at a pathway level in neurodegenerative diseases remains under explored, and incompletely understood. Molecular vignette 3: the DNAJ proteins, endocytic vesicle trafﬁcking and parkinsonism Endosomal trafficking at the interface of a cell and the outside world, both within the cell and the process of moving material into (endocytosis) and out of (exocytosis) cells, is a requirement for continued cell viability and is central to a host of cellular functions [44]. This is certainly true of cells in the central nervous system, with specific uptake mechanisms playing important roles in neuronal function [45]. As would be expected for a process that is observed in almost all cells, and across a wide range of cellular roles, there are a number of specialised forms of endocytosis [46]. One particular type of endocytosis has come to prominence in context of neurodegeneration is clathrin-mediated endocytosis. Clathrin-mediated endocytosis is a specialised form of endocytosis that involves the invagination of the plasma membrane followed by the formation of a protein cage consisting of clathrin assemblies around the emerging endocytic vesicle. This then buds off into the cytosol, where the clathrin cage is eventually deconstructed and the vesicular contents sent on their way [47]. In the context of neurodegeneration, mutations in DNAJ proteins, a family of chaperones with multiple inputs into the life cycle of a clathrin-coated vesicle, have been identified in a range of disorders presenting with parkinsonism, as well as lysosomal storage disorders (a group of diseases that share a num- ber of aetiological features with parkinsonism) [48]. Autosomal dominant mutations in the DNAJC5 gene, encoding cysteine string protein α, cause a hereditary form of neuronal ceroid lipofuscinosis – a form of lysosomal storage dis- order characterised by the accumulation of lipofuscin within the cells of the central nervous system [49]. This results in neurodegeneration and a complex clinical phenotype, including parkinsonism in some patients. Cysteine string protein α has an important role in ensuring the correct folding and function of SNARE protein complexes, protein assemblies that are required for correct vesicle formation at the cell surface, and alteration of this function with brain cells disrupts endocytic processes. DNAJC6 (encoding auxilin) is a brain expressed gene that is required for the un- coating of clathrin-coated vesicles, coordinating chaperone-mediated disassembly by HSC70. Recessive mutations in DNACJ6 cause a complex young onset form of neurodegeneration, involving parkinsonism as part of its clinical presentation [50,51]. Molecular vignette 2: axonal transport and degeneration of motor neurons neurons Motor neurons, connecting the brain to effector muscles, possess the longest axons in the human nervous system [24]. As such, they are exquisitely dependent upon the anterograde and retrograde trafficking of material, including vesicular structures, between the soma and synaptic terminals – to an extent even greater than is normally the case for neurons [25]. The progressive loss of motor neurons is a defining pathological characteristic for a heterogeneous group of neurological disorders including, amongst others, amyotrophic lateral sclerosis, spinal muscular atrophy and the hereditary spastic paraplegias [26–28]. Across this spectrum of disease, mutations in a large number of genes have been identified with one of the many areas highlighted being proteins with specific functions in vesicular traf- ficking – prominent examples being the SPAST gene involved in the regulation of microtubule dynamics and the ATL1 gene, both of which have been heavily implicated in vesicle trafficking centred around the endoplasmic retic- ulum [29]. Mutations in four genes with direct roles in axonal transport, DCTN1, TUBA4A, KIF1A and KIF5A, have been identified in familial forms of amyotrophic lateral sclerosis (DCTN1, TUBA4A, KIF5A) and hereditary spastic paraplegia (KIF1A and KIF5A) – with further mutations in DYNC1H1 (coding for the dynein heavy chain) linked to spinal muscular atrophy and Charcot-Marie-Tooth disease [30–35]. Mutations in DCTN1, coding for Dy- nactin 1/p150glued, directly impact on the function of the dynactin complex (a large protein complex that works with dynein to transport material along microtubules) [31]. In cellular and animal models, these mutations cause axonal trafficking deficits – with a specific impact on the trafficking of vesicles [36]. This results in the accumulation of vesicles in axons, axononal swelling, disrupted transport and degeneration. For KIF1A and KIF5A, mutations caus- ing hereditary spastic paraplegia – associated with the dysfunction and degeneration of upper motor neurons – and 943 Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 amyotrophic lateral sclerosis alter the function of kinesins, motor proteins that shepherd vesicles up and down the microtubular routes along the axon. Although the molecular consequences of these mutations have not been charac- terised in detail, data from KIF5A knockout mice and in vitro analyses indicating that removal of kinesin heavy chain leads to axonal transport dysfunction and neurodegeneration, implies that the human disease causing mutations are loss-of-function [37,38]. Molecular vignette 2: axonal transport and degeneration of motor neurons Further experiments assessing KIF1A mutations are consistent with a decrease in function [39]. For TUBA4A, coding for α tubulin 4A, rare coding variants and nonsense mutations have been demonstrated to alter the ability of this protein to integrate and form microtubules. The human genome possesses a range of tubulin genes, providing a degree of redundancy in the system for these critical cytoskeletal proteins [40]. It is plausible that mutations in TUBA4A are enriched in amyotrophic lateral sclerosis due to the heightened requirements for axonal transport in motor neurons and a specific role for α tubulin 4A in these cells, however this is yet to be addressed experimentally in any depth. amyotrophic lateral sclerosis alter the function of kinesins, motor proteins that shepherd vesicles up and down the microtubular routes along the axon. Although the molecular consequences of these mutations have not been charac- terised in detail, data from KIF5A knockout mice and in vitro analyses indicating that removal of kinesin heavy chain leads to axonal transport dysfunction and neurodegeneration, implies that the human disease causing mutations are loss-of-function [37,38]. Further experiments assessing KIF1A mutations are consistent with a decrease in function [39]. For TUBA4A, coding for α tubulin 4A, rare coding variants and nonsense mutations have been demonstrated to alter the ability of this protein to integrate and form microtubules. The human genome possesses a range of tubulin genes, providing a degree of redundancy in the system for these critical cytoskeletal proteins [40]. It is plausible that mutations in TUBA4A are enriched in amyotrophic lateral sclerosis due to the heightened requirements for axonal transport in motor neurons and a specific role for α tubulin 4A in these cells, however this is yet to be addressed experimentally in any depth. Dysfunction of the dynein heavy chain, encoded by DYNC1H1, was first implicated in motor neuron survival through the characterisation of a mouse line with a motor phenotype, dubbed the legs-at-odd-angles or loa mouse [41]. This was revealed to have a coding mutation in the dync1h1 gene causing the phenotype. Subsequent investiga- tions revealed mutations in the human DYNC1H1 gene casusing a range of motor neuron disease phenotypes, and investigations using mouse, cellular and in vitro models have revealed specific deficits in the trafficking of vesicles [42,43]. Taken together, these examples provide clear evidence of defined deficits in axonal transport of vesicles across the broad spectrum of diseases of motor neurons. Molecular vignette 2: axonal transport and degeneration of motor neurons As noted at the start of this vignette, in many ways this should not be a surprise given the axonal characteristics of motor neurons and their specific sensibilities, however understand- ing the molecular details of these deficits provides both a window on which genes involved in axonal trafficking are differentially relied upon by motor neurons, as well as potential routes to developing therapies to ameriorate these deficits. © 2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Therapeutic targeting of vesicular dysfunction p g g y A major motivation for investigating the molecular basis of neurodegenerative disease is to facilitate the development of novel therapies for these devastating disorders. How, then, can our growing comprehension of vesicular dysfunc- tion in neurodegenerative diseases aid this process? Drug development for the central nervous system has proven to be an extremely challenging endeavour, with a high failure rate and (to date) very few success stories [58]. One of the challenges facing these efforts is the relatively paucity of drug targets to be investigated, and so the discovery of novel – and potentially tractable – targets associated with membrane biology is of great value to the research community. There has been considerable interest in targeting vesicle trafficking in human diseases for a number of years, with tools developed for a number of aspects of intracellular trafficking [59,60]. With relevance to some of the pathways dis- cussed above, endocytic biology and lysosomal function are examples where substantial efforts have been expended [61,62]. A major obstacle to these efforts is the ubiquitous importance of such trafficking and function, with critical roles across a wide range of organs and physiological processes. Achieving specificity in the central nervous system, and avoiding potentially deleterious on target impacts outside of the brain, remains a major challenge. Downloaded from http://portlandpress.com/essaysbiochem/article-pdf/65/7/941/926957/ebc-2021-0034c.pdf by UK user on 23 February 2022 Conclusions The rapid expansion of genetic analysis for neurodegenerative disease has resulted in an abundance of genes im- plicated in the aetiology of disorders such as Alzheimer’s disease and Parkinson’s disease. By studying the normal function of these genes, as well as examining the consequences of mutations and concomitant dysfunction, we can gain some insights into the cellular processes and pathways that connect variation in these genes to dysfunction of vesicle trafficking and biology in the central nervous system. The examples discussed above come from disparate forms of neurodegenerative disease, and are pertinent to discrete areas of vesicular biology, but it is striking that the location and contents of vesicles (often in the context of protein aggregation and degradation) is a common theme across these disorders. Whether this is emblematic of convergent biology in these disorders, or a form of functional phenocopy, remains a matter of great debate. Key questions remain – most notably why do mutations in genes that code for proteins that appear to have ubiq- uitous function cause specific degeneration of discrete populations of neuronal cells? This is a question that echoes across our current understanding of neurodegeneration, where the basis for selective vulnerability is, at best, unclear [63]. It is indisputable, however, that further investigation and insights into the role of vesicle trafficking dysfunction in the central nervous system will be of great value to future efforts to understand and target neurodegenerative dis- ease [6]. With the application of new technologies and the onward march of human genetics providing ever greater resolution to the genetic architecture and biological basis for these disorders, this is clearly an area to watch. Molecular vignette 3: the DNAJ proteins, endocytic vesicle trafﬁcking and parkinsonism Although the mutations vary in terms of genetic mechanism (spanning clear loss of gene expres- sion through to single coding variants), it is probable that the deficit driving disease is a reduction in auxilin function, thereby disrupting the uncoating and further trafficking of clathrin-coated vesicles [52]. Intriguingly, GAK, which codes for the auxilin paralog GAK, is a candidate gene for a Parkinson’s disease risk locus on chromosome 4 [53]. In addition to coordinating chaperone activity through its J domain, GAK also possesses a protein kinase domain, and is uqiquitously expressed [54,55]. Although GAK has not yet been confirmed as the gene driving association at this locus, this raises the possibility that there is a spectrum of risk associated with clathrin uncoating and Parkinson’s, 944 Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 Essays in Biochemistry (2021) 65 941–948 https://doi.org/10.1042/EBC20210034 with loss of function in DNAJC6 causing a Mendelian form of parkinsonism, and more common (and subtle) vari- ation in GAK associated with heightened risk of Parkinson’s disease. Finally, autosomal dominant coding mutations in DNAJC13, coding for RME-8, have been reported in monogenic forms of Parkinson’s disease [56]. RME-8 has a similar role to auxilin, with its activity centred around coordinating trafficking of vesicles in the endosomal system and trans-Golgi network. It is of note that there are conflicting reports relating to the pathogenicity of DNAJC13 mu- tations, highlighting the need for careful appraisal of genetic risk for human disease, however the biology is certainly intriguing given the other DNAJ proteins involved in neurodegeneration [57]. with loss of function in DNAJC6 causing a Mendelian form of parkinsonism, and more common (and subtle) vari- ation in GAK associated with heightened risk of Parkinson’s disease. Finally, autosomal dominant coding mutations in DNAJC13, coding for RME-8, have been reported in monogenic forms of Parkinson’s disease [56]. RME-8 has a similar role to auxilin, with its activity centred around coordinating trafficking of vesicles in the endosomal system and trans-Golgi network. It is of note that there are conflicting reports relating to the pathogenicity of DNAJC13 mu- tations, highlighting the need for careful appraisal of genetic risk for human disease, however the biology is certainly intriguing given the other DNAJ proteins involved in neurodegeneration [57]. © 2021 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Summary y • Genes identified in Mendelian forms of neurodegeneration and through genome-wide association have highlighted vesicle trafficking as an important area of cell biology in these disorders. • Genes identified in Mendelian forms of neurodegeneration and through genome-wide association have highlighted vesicle trafficking as an important area of cell biology in these disorders. • Cellular characterisation of these genes have revealed deficits in specific cellular trafficking events, including mitophagy, axonal trafficking and endocytosis. • Cellular characterisation of these genes have revealed deficits in specific cellular trafficking events, including mitophagy, axonal trafficking and endocytosis. • The biological basis for these deficits are an area of particular interest with regard to the develop- ment of novel therapeutics for neurodegenerative disorders. • The biological basis for these deficits are an area of particular interest with regard to the develop- ment of novel therapeutics for neurodegenerative disorders. Funding g This work was supported by the Michael J. Fox Foundation [grant number BB/T008709/1-2397254]; Parkinson’s U.K. [grant num- ber PRO-20-15]; the Medical Research Council [grant number MR/N026004/1]; the Biotechnology and Biological Sciences Re- search Council [grant number BB/T008709/1-2397254]; the Aligning Science Across Parkinson’s Research Network; paid consul- tant for Merck Sharp Dohme. Further reading g Clearly, an essay such as this can provide only the briefest of summaries of what is an extensive area of neurodegener- ative research. Fortunately there are a number of excellent reviews addressing vesicular biology in neurodegeneration in great detail, a number of which are cited above. The following, picking up on the case studies included in this essay, are particularly recommended: McWilliams T.G. and Muqit M.M. (2017) PINK1 and Parkin: emerging themes in mitochondrial homeostasis. Curr. Opin. Cell. Biol., 45, 83–91. Opin. Cell. Biol., 45, 83 91. 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https://openalex.org/W2794216324	https://repositorio-aberto.up.pt/bitstream/10216/126511/1/10.3389-fpsyt.2018.00010.pdf	English	null	Validation of the Portuguese Version of Impulsive–Premeditated Aggression Scale in an Inmate Population	Frontiers in psychiatry	2,018	cc-by	6,634	Keywords: aggression, antisocial personality disorder, psychopathy, impulsivity, impulsive–premeditated aggression scale, Barratt Impulsiveness scale, 11th version, Psychopathic Checklist Revised Validation of the Portuguese Version of impulsive–Premeditated aggression scale in an inmate Population Jacinto Costa Azevedo1,2, José Luís Pais-Ribeiro3, Rui Coelho1 and Margarida Figueiredo-Braga1,2 1 Department of Neuroscience and Mental Health, Faculty of Medicine, University of Porto, Porto, Portugal, 2 I3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal, 3 Faculty of Psychology and Educational Sciences, University of Porto, Porto, Portugal Aggression is one of the core symptoms of antisocial personality disorder (ASPD) with therapeutic and prognostic relevance. ASPD is highly prevalent among inmates, being responsible for adverse events and elevated direct and indirect economic costs for the criminal justice system. The Impulsive/Premeditated Aggression Scale (IPAS) is a self-report instrument that characterizes aggression as either predominately impulsive or premeditated. This study aims to determine the validity and reliability of the IPAS in a sample of Portuguese inmates. A total of 240 inmates were included in the study. A principal component factor analysis was performed so as to obtain the construct valid- ity of the IPAS impulsive aggression (IA) and premeditated aggression (PM) subscales; internal consistency was determined by Cronbach’s alpha coefficient; convergent and divergent validity of the subscales were determined analyzing correlations with the Barratt Impulsiveness scale, 11th version (BIS-11), and the Psychopathic Checklist Revised (PCL-R). The rotated matrix with two factors accounted for 49.9% of total variance. IA subscale had 11 items and PM subscale had 10 items. The IA and PM subscales had a good Cronbach’s alpha values of 0.89 and 0.88, respectively. The IA subscale is correlated with BIS-11 attentional, motor, and non-planning impulsiveness dimensions (p < 0.05). The PM subscale is correlated with BIS-11 attentional, motor impulsiveness dimensions (p < 0.05). The PM subscale is correlated with PCL-R interpersonal, lifestyle, and antisocial dimensions (p < 0.05). The IA subscale is not correlated with PCL-R. The Portuguese translated version of IPAS has adequate psychometric properties, allowing the measurement of impulsive and premeditated dimensions of aggression. Citation: Azevedo JC, Pais-Ribeiro JL, Coelho R and Figueiredo-Braga M (2018) Validation of the Portuguese Version of Impulsive–Premeditated Aggression Scale in an Inmate Population. Original Research published: 05 February 2018 doi: 10.3389/fpsyt.2018.00010 Katarina Howner, Karolinska Institutet (KI), Sweden Reviewed by: Giuseppe Sartori, Università degli Studi di Padova, Italy Erich Flammer, ZfP Suedwuerttemberg, Germany Correspondence: Jacinto Costa Azevedo jacintoazevedo@gmail.com Specialty section: This article was submitted to Forensic Psychiatry, a section of the journal Frontiers in Psychiatry Received: 25 October 2017 Accepted: 15 January 2018 Published: 05 February 2018 Edited by: Katarina Howner, Karolinska Institutet (KI), Sweden Edited by: Katarina Howner, Karolinska Institutet (KI), Sweden INTRODUCTION According to the aggression type, individuals may differ in social adjustment, criminal behavior, emotional function, cognitive performance, autonomic response, and treatment outcome (6, 7).h Patients with substance use disorders, depression, bipolar disorder, attention deficit/hyperactivity disorder (AD/HD), as well as suicide attempters and criminal offenders tend to have higher BIS-11 total scores (29). The IA tends to occur in the presence of triggering stimulus that is interpreted as threat or provocation (8). This type of aggression involves affective arousal, leading to a rapid and uncontrolled behavioral response. It has been correlated with information-processing/neurocognitive impairments, abusive home backgrounds, an angry/impulsive/anxious personality, and high psychological stress reactivity (9).h Despite the evidence supporting the notion that persons who display an impulsive aggressive behavior are distinct from their counterparts who present a premeditated behavior, the clini- cal usefulness and generalization is questioned because most studies included violent incarcerated offenders, or psychiatric inpatients. The PM, by contrast, tends to be a planned behavior that has a specific expected goal (8), and it can be explained as a learned behavior (10). It has been correlated with poor parental control, lack of affect, psychopathic personality, and low physiological arousal (9).hi Within the incarcerated population, however, aggression is a relevant daily clinical concern. Therefore, the study of aggres- sion in forensic psychiatric settings should be encouraged in order to find and promote the best clinical practices (30, 31). The classification of an aggressive act or a pattern of aggres- sive behavior allows treatment selection and violence risk management. The IA has a good response to pharmacologic treatment when mood stabilizers and/or antipsychotics are prescribed (11). In the case of PM, on the contrary, the response to pharmacological treatment is generally insufficient (12), and forensic/behavioral strategies are recommended (7). Furthermore, individuals with PM have a higher risk of violent criminal recidivism than those with IA (13).h It is important to use validated instruments in the evaluation of aggressive acts in prisoners in order to improve medical inter- ventions (32). In Portugal, we do not have a validated aggression categorization instrument.h There are two psychometric instruments that allow aggres- sion categorization: the impulsive–premeditated aggression scale (IPAS) and the reactive–proactive aggression questionnaire (2, 33, 34). There is a link between aggressive behavior and psychiatric disorders (14). INTRODUCTION Aggression represents a public health issue. Having a negative impact on individuals and society (1), it can be defined as a “behavioral display in which physical force is used with the intent to harm or damage another individual or object” (2). February 2018 \| Volume 9 \| Article 10 1 Frontiers in Psychiatry \| www.frontiersin.org Azevedo et al. Azevedo et al. IPAS Validity in Portuguese Inmates A clinical definition of aggressive behavior should consider biological, environmental, cultural, and social variables, and their interplay to act as predisposition or eliciting factors (3). The range of behaviors that can be classified as aggressive can vary from verbal aggression to homicide, and it is important to characterize the level of planning, the possible understanding of hypothetical consequences, the presence of frustrations, insults, interpersonal attack, threats, environmental stressors, and associ- ated psychopathologies. Antisocial personality disorder is the only acknowledged psychiatric disorder that confers an increased risk for both IA and PM (20–22), with several studies reporting psychopathy and psychopathic traits as risk factors for PM (23). Psychopathic Checklist Revised (PCL-R) is the gold stand- ard for psychopathy assessment and diagnosis, and separates psychopathic traits into four dimensions: (1) interpersonal, (2) affective, (3) lifestyle, and (4) antisocial (24). f Impulsivity has been related to aggression and is a symptom of ASPD (25). Impulsivity can be a necessary factor for aggression development (26). It can be defined “as a predisposition toward rapid, unplanned reactions to internal or external stimuli without regard to the negative consequences of these reactions to the impulsive individuals or to others” (27). Empirical literature has proposed a dichotomous aggression classification, i.e., impulsive aggression (IA; reactive, affective, or non-planned), and premeditated aggression (PM; proactive, instrumental, predatory, or controlled) (4). The expression “IA” refers to uncontrolled aggressive outbursts that are out of proportion to the provoking event, while “PM” describes aggressive behaviors that are planned, controlled, and/or goal- oriented (5).f Impulsivity can be measured with Barratt Impulsiveness Scale, 11th version (BIS-11), a self-report psychometric instrument that separate impulsivity into three components: (1) acting on the spur of the moment (motor activation), (2) not focusing on the task at hand (attention), and (3) not planning and thinking carefully (lack of planning) (28). Individuals with scores higher than 72 are considered highly impulsive (29). Frontiers in Psychiatry \| www.frontiersin.org INTRODUCTION In the original validation study of IPAS, the sample comprised 93 physically aggressive men recruited in the community with a mean age of 35.9 years, whose principal aggression type was physical assault (n = 80). The aggression was measured through the Lifetime History of Aggression, Buss Perry Aggression Questionnaire, and the Aggression Interview. The principal component analysis identified two factors (IA and PM), which accounted for 16.56 and 14.03% of total variance, respectively. The internal consistency measured by Cronbach’s alpha was 0.77 for the IA factor and 0.82 for the PM factor. Sensitivity for the IA scale was 0.96 and specificity was 0.50. For the PM scale, sensitiv- ity and specificity were 0.60 and 0.96, respectively (2). Individuals with depression, anxiety, psychosis, substance use disorders, hyperactive attention deficit disorder, and personality disorders present a higher frequency of aggressive acts (15). Impulsive aggression has been linked to psychiatric diseases, such as anxiety, depression, personality disorders, and substance use disorders (16). PM has been associated with psychopathic personality traits (17). In particular, antisocial personality disorder (ASPD) is char- acterized by significant irritability, agitation, impulsiveness, and hostility, and aggression represents one of its core symptoms (18). ASPD is highly prevalent among inmates. It is, thus, responsible for adverse events and elevated direct and indirect economic costs in the criminal justice system (19). i All of the validation studies of IPAS reported identical results in principal component analysis, with two factors IA and PM. February 2018 \| Volume 9 \| Article 10 2 Azevedo et al. IPAS Validity in Portuguese Inmates Internal consistency coefficients varied between 0.70 and 0.93 for IA and between 0.66 and 0.90 for PM (Table 1). were explained to the participants, in accordance with the Declaration of Helsinki. Participants were included if they were over 18 years old, had a personal history of aggression in the past 6 months, had been referred to the clinical services for aggressions toward other inmates, were able to read, and if they were capable of providing their written informed consent. Socio-demographic characteristics and forensic history were collected from interviews and clinical records. We applied three psychomet- ric instruments: the IPAS, the BIS-11, and the PCL-R. This sample does not intend to represent all the Portuguese inmate population, as the methods involve a non-probabilistic sample approach. We intend to validate the Portuguese version of the IPAS in inmates and to determine its psychometric properties. INTRODUCTION Thus, we expect to replicate the purported two-factor model of IPAS; we predict that the subscales of the IPAS and the BIS-11 will be positively correlated; we predict that the PM subscales of the IPAS—unlike the IA ones—will be correlated with the PCL-R.h This study is a part of a larger study aiming to characterize biological and psychosocial factors that predict the aggression type in young adult male inmates. February 2018 \| Volume 9 \| Article 10 RESULTS To determine the convergent validity of the IA and the PA subscales of the IPAS, we have utilized BIS-11. The BIS-11 is a self-report questionnaire for assessing general impulsiveness. The current scale version contains 30 items that are coded from 1 (rarely/never) to 4 (almost always/always). The level of impul- siveness is calculated by summing up the scores for each item. All items were defined as identifying impulsiveness within the structure of related personality traits. The factor analysis revealed three components as follows: “attentional impulsiveness,” “motor impulsiveness,” and “non-planning impulsiveness.” Instead of a unique total score, the study of the individual contribution of each component is recommended. Findings report an acceptable/high internal consistency (Cronbach’s alpha 0.79–0.82) of the scale when applied to forensic samples (28). The structural properties of the BIS-11 were replicated in Portuguese speaking samples (44). Psychometric Instruments Impulsive–Premeditated Aggression Scaleh structure and maximize the reliability of factor interpretation, an Oblimin rotation was used so as to obtain an oblique fac- tor solution of the original factors (50). Internal consistency was measured using Cronbach’s alpha (51). Convergent and divergent validity of the IPAS was tested by examining the Pearson’s product-moment correlations with the standard- ized measures of impulsivity and personality (BIS-11 and PCL-R). Confirmatory factorial analysis was tested by calculat- ing goodness-of-fit indice, robust method (52).h The IPAS is a 30-item self-report questionnaire used to classify aggressive acts occurring over the previous 6 months. Items are scored within a five-point Likert scale (1 = Strongly Disagree to 5 = Strongly Agree). The scale differentiates two factors: IA and PM, with the weighted score allowing for the categorization of the type of aggression. The individual’s level of IA and PM was obtained through the sum of 20 of the 30 items of the IPAS: the IA items (eight items: 3, 5, 7, 8, 9, 21, 24, and 26) and the PM items (12 items: 1, 2, 6, 10, 11, 12, 14, 16, 17, 20, 29, and 30) (2). i The analyses were carried out using IBM SPSS Statistics for Windows, Version 22.0 (Armonk, NY, USA: IBM Corp.), and with the Structural Equation Program, EQS version 6.1 (53). Confirmatory Factorial Analysis i y y To test the hypothesis of two dimensions, we use the confirmatory factor analysis, to test the goodness-of-fit indice, robust method. Results showed a comparative fit index of 0.90, and a root mean square error of approximation (RMSEA) of 0.06, 90% confidence interval (0.05, 0.08). Sample Descriptionh The sample comprised 240 inmates with a mean (±SD) age of 35.4 ± 8.4 years; a total of 67.1% (n = 161) of the participants were single, and 58.3% did not have any children (n = 140). The mean education level of the sample was 6.8 ± 3.2 years. The mean time in prison (time spent behind bars at the time of assessment) of the sample was 109.3 ± 70.6 months. A total of 45.8% (n = 110) of the inmates had been convicted of violent crimes (physical assault, murder, attempted murder). Validation procedures were performed in the total sample (n = 240). Principal Component Analysis p p y We have performed a principal component analysis exploring the original 30 items. First, we inspected the Kaiser–Meyer–Olkin Measure of Sampling Adequacy (KMO = 0.87) and reached the conclusion that it was an appropriate value. Second, we achieved a statistically significant Bartlett’s Test of Sphericity (p < 0.01). Third, we extracted two factors (with an oblimin rotation) with eigenvalues of 7.19 (Factor 1, IA) and 3.29 (Factor 2, PM) accounting for 34.27 and 15.65% of total variance, respectively. The IA factor was composed of ten items: 30, 27, 22, 9, 24, 15, 26, 4, 7, and 13. The PM factor was composed of eleven items: 6, 14, 29, 28, 2, 23, 12, 16, 20, 10, and 1. The items 3, 5, 8, 11, 17, 18, 19, 21, and 25 were excluded from the analysis as they exhibited component loadings inferior to 0.40 (Table 2). Psychopathy Checklist Revised y p y To determine the divergent validity of the IA and the PA sub- scales of the IPAS, we have applied the PCL-R. The PCL-R is the gold standard measure of psychopathy, gathering information from records and a semi-structured interview (24, 25). The 20 items are scored as absent (0), present to some degree (1), or fully present (2), having a maximum total score of 40 points. This instrument may be considered a four-factor model compris- ing interpersonal, affective, lifestyle, and antisocial dimensions (45). The interpersonal and affective dimensions jointly serve as second-order factors representing the core traits of the psycho- pathic personality. Lifestyle and antisocial facets form a super- ordinate factor of social deviance (46). The two second-order factors (Factor 1 and Factor 2) are concomitant with the original factor structure reported for the first edition of the PCL-R (47). The structural properties of the PCL-R were replicated in Portuguese samples for the standard protocol, including record review and structured interview (48). The PCL-R was applied by an experienced psychiatrist (Jacinto Costa Azevedo). Transcultural Adaptation and Validation Our sample was collected at two penitentiary institutions in the North of Portugal. The research protocol was formally approved by the Ethics Committee of Centro Hospitalar de São João, Porto, Portugal (Document number 48.14), and by the hosting institution, the General Direction of Probation and Prison Services. Participation was voluntary and there was no reward for it. The participants were all Portuguese. Written informed consent was obtained after the procedures In order to address the transcultural adaptation and validation of IPAS, an initial translation was carried out by a group of Portuguese experts. The back-translation of this first translated version was then performed by a native English-speaking expert. The first group analyzed the semantic and idiomatic correspond- ence of the translation and the back-translation, and performed a final synthesis translation (43). Table 1 \| Validation studies of Impulsive/Premeditated Aggression Scale (IPAS). Reference Sample Mean agea Principal aggression Aggression measures Internal consis tencyb Total explained variancec n Type Origin IAd PMe IA PM Stanford et al. (2) 93 men Physically aggressive men Community 35.9 86% physical assault LHAQf, BPAQg, AIh 0.77 0.82 16.56 14.03 Kockler et al. (35) 86 men Prisoners Inpatient Forensic hospital 38 83% convicted for violent crimesi, 37% non-guilty by reason of insanity nsj 0.81 0.72 20 13 Conner et al. (36) 61 women, 60 men Patients in treatment for opiate dependence Outpatient Psychiatric Service 41.9 ns LHAQ, BPAQ 0.74 0.75 ns Mathias et al. (37) 24 girls, 42 boys Adolescent s with conduct disorder Community 14.5 ns LHAQ, BPAQ 0.82 0.78 18.5 15.6 Stanford et al. (38) 113 men Men convicted of domestic violence Community 36 Domestic violence LHAQ 0.75 0.86 ns Haden et al. (39) 213 women, 127 men Students Community 19.06 ns BPAQ 0.77 0.81 24 12 Kuyck et al. (40) 149 men, 70 women Prisoners Prison 50% between 25 and 39 75% convicted for violent crimes ns 0.93 0.90 ns Chen et al. (41) 389 women, 262 men Students Community 26.1 ns BPAQ 0.70 0.66 12.9 11.9 Romans et al. (42) 114 women, 49 men Psychiatric patients Outpatient Psychiatric Service 25.8 ns OASk 0.85 0.76 23.3 10.1 aY Table 1 \| Validation studies of Impulsive/Premeditated Aggression Scale (IPAS). kOvert Aggression Scale. February 2018 \| Volume 9 \| Article 10 Frontiers in Psychiatry \| www.frontiersin.org IPAS Validity in Portuguese Inmates Azevedo et al. Internal Consistencyh The internal consistency test of the dimensions obtained in the factor analysis with the present sample revealed a good Cronbach’s alpha value for the IA factor (0.89), and a good Cronbach’s alpha value for the PM factor (0.88) (Table 2). Statistical analyses were conducted following the methodology used in the previous IPAS validation studies in adults. The prin- cipal component analysis was conducted with no assumptions regarding the number of potential factors. Lautenschlager’s (49) tables were used to determine the threshold for significant factors (minimum eigenvalue of 2.0) and item factor load- ings (minimum eigenvalue of 0.40). In order to assess factor Reliability was also tested regarding the original version of the IPAS subscales. Cronbach’s alpha for the IA original subscale was acceptable (0.78), and it was good for the PM subscale (0.85). February 2018 \| Volume 9 \| Article 10 Frontiers in Psychiatry \| www.frontiersin.org 4 IPAS Validity in Portuguese Inmates Azevedo et al. Table 2 \| Factor loadings of Impulsive/Premeditated Aggression Scale (IPAS). IPAS Items Impulsive Premeditated 30. Anything could have set me off prior to the incidents 0.63 – 27. I was in a bad mood the day of the incident 0.66 – 22. I was confused during the acts 0.79 – 9. I feel I lost control of my temper during the acts 0.80 – 24. My behavior was too extreme for the level of provocation 0.72 – 15. I became agitated or emotionally upset prior to the acts 0.72 – 26. I consider the acts to have been impulsive 0.66 – 4. I typically felt guilty after the aggressive acts 0.73 – 7. I usually can’t recall the details of the incidents well 0.50 – 13. I feel some of the incidents went too far 0.72 – 6. I feel my actions were necessary to get what I wanted – 0.58 14. I think the other person deserved what happened to them during some of the incidents – 0.65 29. I am glad some of the incidents occurred – 0.64 28. The acts were a “release” and I felt better afterward – 0.75 2. I felt my outbursts were justified – 0.70 23. Prior to the incidents I knew an altercation was going to occur – 0.55 12. I wanted some of the incidents to occur – 0.80 16. The acts led to power over others or improved social status for me – 0.72 20. Convergent and Divergent Validity Convergent and Divergent Validity g g We have studied the convergence between the IPAS and the BIS-11. The statistically significant correlations between the two scales (IPAS and BIS-11) and between its dimensions are detailed in Table 3, showing correlations with attentional, motor, and non-planning dimensions (2, 5, 28). Correlations were statistically significant, suggesting the existence of convergence between the two scales. According to Bentler and Douglas (52), when the goodness- of-fit and adjusted goodness-of-fit indexes are higher than 0.90, the analyses indicate adequate fit of the models. Also, according to Bentler and Douglas, when the RMSEA is less than. 0.10, the analysis indicates adequate fit of the models. We have studied divergent validity through the analysis of cor- relations between IPAS and PCL-R in a subsample of 134 inmates. hi The statistically significant correlations between the two scales (IPAS and PCL-R) and between its dimensions are detailed in Table 3. The IA subscale is not correlated with PCL-R. The PM subscale is correlated with PCL-R interpersonal, lifestyle, and antisocial dimensions (p < 0.05). i In our study, we have assessed the convergent validity through the analysis of Pearson product-moment correlations coefficients between IPAS and BIS-11. We had significant correlations between the IA subscale and the BIS-11 motor activation, atten- tion, and non-planning dimensions. We had significant correla- tions between the PM subscale and the BIS-11 motor activation, attention dimensions, but not for non-planning dimension. Internal Consistencyh Some of the acts were attempts at revenge – 0.75 10. Sometimes I purposely delayed the acts until a later time – 0.63 1. I planned when and where my anger was expressed – 0.64 Eigenvalues 7.19 3.29 Variance (%) 34.27 15.65 Cronbach’s alpha 0.89 0.88 Table 3 \| Pearson correlations coefficients between Impulsive/Premeditated Aggression Scale (IPAS), Barratt Impulsiveness Scale, 11th version (BIS-11), and Psychopathic Checklist Revised (PCL-R). IPAS subscales Measures Subscales IA PM BIS-11 Total score 0.21 0.20 Attentional 0.14* 0.18* Motor 0.26 0.27 Non-planning 0.15* 0.06 PCL-R Total score 0.01 0.29** Interpersonal −0.14 0.24* Affective −0.14 0.16* Lifestyle −0.07 0.22* Antisocial −0.07 0.21* The study used 240 participants for correlations between IPAS and BIS-11; and a subsample of 134 participants for correlations between IPAS and PCL-R. IA, impulsive aggression; PM, premeditated aggression. p < 0.05; *p < 0.01. capture important aspects of the construct, as well as its relevance to the proposed use. The present results show appropriate validity for the Portuguese version of the IPAS.h The items of the instrument were adapted according to the original version (2). In our sample, IPAS was able to demonstrate the presence of two factors: impulsive and PM. All the authors who studied the validity of IPAS were able to reduce the variability of the scale to two factors: IA and PM factors. This is in line with the literature on human aggression which argues that aggression is not a single construct and should be understood as being composed of two dimensions or catego- rized into two different types: impulsive or PM. f In the analysis of the explained variance described by other authors, we have observed that the IA factor can explain between 12.9% (41) and 24% (39) of the total IPAS variance; and that the PM factor can account for between 10.3% (42) and 15.6% (37) of the total IPAS variance. We have obtained higher values of explained variance (34.3% for the IA factor and 15.7% for the PM factor). In the analysis of the IPAS internal consistency described by other authors, we have observed that it varies between 0.70 (41) and 0.93 (40) for the IA factor; and between 0.66 (41) and 0.90 (40) for the PM factor. Our values of internal consistency and variance can be explained by the homogeneity of the sample. DISCUSSION According to AERA-APA-NCME (54), theoretical and empirical pieces of evidence that support the interpretations of test scores are fundamental so as to indicate the degree to which scores We have found a slightly higher correlation between the total score of BIS-11 and IA than with the PM subscale of IPAS. Our February 2018 \| Volume 9 \| Article 10 Frontiers in Psychiatry \| www.frontiersin.org 5 IPAS Validity in Portuguese Inmates Azevedo et al. significant correlations for AI and PCL-R. Stanford and col- leagues, in 2008, evaluated correlations between Psychopathic Personality Inventory (PPI) and the AI and PM factors, having obtained significantly higher values of PPI in the individuals categorized as premeditated aggressors, that is, having higher PM scores (38).h results were line with Mathias and colleagues who reported significant correlations between the IPAS subscales and BIS-11’s total score (correlations coefficients of 0.39 for AI and 0.26 for PM) (37). However, Stanford and colleagues have reported higher sig- nificant correlations between PM and the total score of BIS-11 (correlation coefficients of 0.21 for AI and 0.38 for PM) (2).i These data are in agreement with the literature on psychopa- thy and the type of aggressiveness externalized by individuals with higher expression of psychopathic personality traits. Psychopathy seems to be a risk factor for PM, mainly in forensic samples (23). Our data reveal significant correlations between the three dimensions of impulsivity measured by BIS-11and IA subscale of IPAS. Similar results were reported by Chen and colleagues that obtained significant values for correlations between IA and the three dimensions of BIS-11 (correlation coefficients of 0.15 for attentional impulsiveness, 0.21 for motor impulsiveness, and 0.12 for non-planning impulsiveness and AI) (41). Regarding the generalization of results, some limitations should be considered. Although we have worked with a large sample, it is exclusively composed by men, therefore we do not know if aggressive women will behave in the same manner. The participants are subject to long sentences, making it difficult to generalize the results so as to include inmates with shorter sen- tences. Also, participation in this kind of research in a forensic facility context can change the way inmates respond to the IPAS. We must take into consideration that the legal circumstances of participants can modify the type of answers. This sample was obtained in a prison for convicted individuals, in Northern Portugal. ETHICS STATEMENT The research protocol was formally approved by the Ethics Committee of Centro Hospitalar de São João, Porto, Portugal (Document number 48.14). Written informed consent was obtained after the procedures had been explained to participants, in accordance with the Declaration of Helsinki. Thus, we can explain the correlations between subscales of IPAS and BIS-11 subscales because they are individuals with greater impulsivity conferred by underlying psychopathology. The third possible explanation, related to the non-planning dimension of impulsivity, is perhaps those impulsive individuals who maintain planning ability are those who can develop and learn premeditated aggressive behavior. These findings support the definition of PM in which individuals with PM plan their action and, thus, have fewer impairments in action planning impulsivity dimension (9). The non-planning dimension of impulsivity is related to working memory (59) and executive functions, namely with the subscale of strategic planning of the executive function index (60).h AUTHOR CONTRIBUTIONS JA and MF-B contributed to the conception of the study. The data analysis was carried out by JA and JP. All authors con- tributed to the interpretation of the data. JA and MF-B wrote the manuscript. All authors revised the content critically and approved the final version. DISCUSSION We do not know whether the scale psychometric characteristics are the same in other forensic samples. In future research, it may be useful to consider tools that can externally quantify the individual acts of aggression, such as the Modified- Overt Aggression Scale. In the case of PM subscale of IPAS, we did not observe significant correlations with the non-planning dimension of BIS-11. The same was reported by Chen and colleagues, who only reported a significant correlation of 0.19 between PM and attentional impulsiveness (41). We can explain these results in three possible ways. First, it is assumed that impulsivity is related aggression, not doing any discrimination between dimensions and type of aggression (33). These results are in accordance with the hypothesis that impul- sivity is related and can be a predisposing factor for aggression (26). On the other hand, we should note that there are impulsive individuals who are not aggressive, and therefore, impulsivity is not the only factor necessary for the development of aggressive- ness (55). Second, since our sample is composed of inmates, we can assume a high prevalence of individuals with substance use disorders and ASPD (56). In these types of pathology, there are a high expression of impulsivity (57, 58).h ACKNOWLEDGMENTS These results support the need for use of appropriated psy- chometric scales for the evaluation of aggression, in order to go beyond the assumption that individuals with higher levels of impulsivity are more likely to display externalizing behavior and thus having a greater probability of showing aggressive behavior.h We would like to thank Letícia Malta, MD, and Nurse João Pinto for their collaboration in the acquisition of the data. We would like to thank the Portuguese General Directorate of Prison Services for authorizing the research. This was the rationale for the use of BIS-11 for the evaluation of convergent validity with IPAS. We have evaluated the divergent validity by analyzing cor- relations between AI and PM factors and PCL-R. As a result, we have obtained significant correlations for PM and PCL-R interpersonal, lifestyle, and antisocial dimensions, and not Frontiers in Psychiatry \| www.frontiersin.org REFERENCES Kendall T, Pilling S, Tyrer P, Duggan C, Burbeck R, Meader N, et al. Guidelines: borderline and antisocial personality disorders: summary of NICE guidance. BMJ (2009) 338(7689):293–5. doi:10.1136/bmj.b93 9. Cima M, Raine A. Distinct characteristics of psychopathy relate to different subtypes of aggression. Pers Individ Dif (2009) 47(8):835–40. doi:10.1016/j. paid.2009.06.031 h 33. Suris A, Lind L, Emmett G, Borman PD, Kashner M, Barratt ES. Measures of aggressive behavior: overview of clinical and research instruments. 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Impulsiveness and executive control of working memory. Pers Individ Dif (2004) 37:417–28. doi:10.1016/j. paid.2003.09.013 50. Jackson JE. Oblimin rotation. In: Armitage P, Colton T, editors. Encyclopedia of Biostatistics. John Wiley & Sons, Ltd (2005). 6 p.h 51. Nunnally JC, Bernstein IH. Psychometric Theory. 3rd ed. New York: McGraw- Hill (1994). 60. Spinella M. Self-rated executive function: development of the executive func- tion index. Int J Neurosci (2005) 115:649–67. doi:10.1080/00207450590524304 52. Bentler P, Douglas G. Significance tests and goodness of fit in the analysis of covariance structures. Psychol Bull (1980) 88:588–606. doi:10.1037/ 0033-2909.88.3.588 Conflict of Interest Statement: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. 53. Bentler PM. EQS 6.1 Structural Equations Program. Encino, CA: Multivariate Software (2004). Copyright © 2018 Azevedo, Pais-Ribeiro, Coelho and Figueiredo-Braga. 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 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. Copyright © 2018 Azevedo, Pais-Ribeiro, Coelho and Figueiredo-Braga. February 2018 \| Volume 9 \| Article 10 REFERENCES 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 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. 54. American Educational Research Association, American Psychological Association, National Council on Measurement in Education. Standards for Educational and Psychological Testing. Washington, DC: American Educational Research Association (1999). 55. Scarpazza C, Sellitto M, di Pellegrino G. Now or not-now? The influence of alexithymia on intertemporal decision-making. Brain Cogn (2017) 114:20–8. doi:10.1016/j.bandc.2017.03.001 February 2018 \| Volume 9 \| Article 10 Frontiers in Psychiatry \| www.frontiersin.org 8
https://openalex.org/W4243455737	https://www.qeios.com/read/IZJ2PG/pdf	English	null	Shading Artifact	Definitions	2,020	cc-by	59	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Shading Artifact National Cancer Institute National Cancer Institute Qeios ID: IZJ2PG · https://doi.org/10.32388/IZJ2PG Source National Cancer Institute. Shading Artifact. NCI Thesaurus. Code C87017. An artifact that is due to a group of channels or views that deviate gradually from the true measurement. 1/1
https://openalex.org/W2922413592	https://europepmc.org/articles/pmc6411939?pdf=render	English	null	Enzymatic origin and various curvatures of metabolic scaling in microbes	Scientific reports	2,019	cc-by	11,192	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 4 November 2018 Accepted: 22 February 2019 Published: xx xx xxxx Enzymatic origin and various curvatures of metabolic scaling in microbes Received: 4 November 2018 Accepted: 22 February 2019 Published: xx xx xxxx Liyan Li & Genxuan Wang The famous and controversial power law is a basal metabolic scaling model mainly derived from the “surface rule” or a fractal transport network. However, this law neglects biological mechanisms in the important active state. Here, we hypothesized that the relative metabolic rate and growth rate of actively growing microbes are driven by the changeable rate of their rate-limiting enzymes and concluded that natural logarithmic microbial metabolism (lnλ) and growth (or biomass) (lnM) are both dependent on limiting resources, and then developed novel models with interdependence between lnλ and lnM. We tested the models using the data obtained from the literature. We explain how and why the scaling is usually curved with the difference between microbial metabolic and growth (or biomass’s) half-saturation constants (KM, Kλ) in the active state and agree that the linear relationship of the power law is a particular case under the given condition: KM = Kλ, which means that the enzyme dynamics may drive active and basal metabolic scaling relationships. Our interdependent model is more general than the power law, which is important for integrating the ecology and biochemical processes. Since Louis Pasteur first performed quantitative studies of microbial growth at the dawn of microbiology in 18571,2, microbes have been subjected to more complete and accurate studies on their growth kinetics, physiol- ogy and metabolism3–5 over a century. Within this historical context, microbial growth dynamics models, which simulate the behaviors of microbial growth dynamics and the development of system architecture6, have been applied to the various large-scale processes7–9. Among these models, the farthest-reaching coarse-grained model is Monod’s equation, an unstructured model, describing functional relationships between the specific growth rate (μ) in a culture and a single essential growth-limiting substrate concentration (Cs). Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 Enzymatic origin and various curvatures of metabolic scaling in microbes Many researchers have used the concept of the Michaelis-Menten equation12 to describe the relationship between metabolism and limiting resources35–37, which like the form of the Monod equation, lacks theoretical support and an underlying biological mechanism. Is there a mechanical and theoretical model for the relationship between microbial metabolism and limiting resources? h l h bl h l h l h l b Scientists have long sought to establishing a universal quantitative theory explaining the correlation between metabolism and growth in organisms. Since the allometric growth relationship was put forward38, increasing numbers of researchers have become interested in this pursuit. In microbiology, microbial growth and metab- olism have been extensively and separately investigated3–5. Generally, researchers investigated the relations of microbial growth (or biomass) to metabolic rate usually using the power equation (Eq. 1), so a linear relation- ship between metabolic rate and biomass in microbes has been detected in many studies16,39. Nevertheless, there has been disagreement about the cause versus effect relationship between these variables over time, leading to the conclusion of four not necessarily mutually exclusive possibilities: metabolism drives growth; growth drives metabolism; growth and metabolism affect each other by reciprocal feedback; and (or) growth and metabolism are similarly, but independently related to a third factor or set of factors40. Every possibility has its own supporters who provide many experimental and theoretical lines of evidence in plants, animals and microbes to support their hypotheses41–45, so it is difficult to disentangle cause vs. effect. Therefore, the questions of whether there is a certain relationship between microbial metabolism and growth and which is the dominant force between them both remain unanswered. Given these unanswered questions and the variation in λ0 and α, will the relationship between microbial metabolism and growth still be linear or follow the power equation?i g In fact, the traditional power law is a model that is specifically used for the basal (or inactive) metabolic rate; thus, the log-log relationship between metabolic rate and body size (or biomass) is linear. If the metabolic rate is active, what is the result? Previously, Delong et al.29 showed that active and inactive metabolic rates scale linearly with body mass based on data collected at the interspecies level. Therefore, to obtain a more general understand- ing of microbial growth and metabolism, we aimed to synthesize data from published studies. Enzymatic origin and various curvatures of metabolic scaling in microbes y p p g g g Metabolism, a collection of chemical transformations for maintaining life in a living cell, includes the biolog- ical processes that enable the exchange of material and energy between the body and the outside world and the self-renewal process of matter and energy in the body3. A long time ago, researchers discovered a certain relation- ship between metabolic rate and body size (or biomass), in which small-bodied organisms generally have higher mass-specific metabolic rates than larger-bodied organisms. The same is true at the unicellular level for free-living single-celled microbes15,16. Much progress has been made in understanding metabolism via allometric studies of many small organisms17–19. Furthermore, the relationship between body size and metabolism known as the power law20, is one of the most fundamental features of life, with scaling as presented in Eq. (3): λ λ = M (3) a 0 λ λ = Ma 0 (3) where λ is the whole-organism basal metabolic rate (in watts or another unit of power), λ0 is a normalization constant that is independent of body size or temperature, M is organismal volume (often expressed in biomass, in kg), and α is a scaling exponent.h In 1932, based on animal data, Kleiber concluded that the exponent α is a constant equal to 3/420. Then, to explain why α is equal to 3/4, the metabolic theory of ecology (MTE) was put forward by Brown, West, Enquist and colleagues21–23. The power law is predominant in much empirical literature, but the MTE is not applicable to microbes5,16, mainly because of the value of α24–26, which increasing lines of evidence suggest that α is not equal to 3/4, for example, in some small unicellular organisms, the value of α is greater than 3/419,27. The scaling expo- nent varies not only between taxa but also between cells of an individual species and between species of the same taxonomic group28,29. It is clear that α is variable and inconsistent with an assumption underlying the MTE28, and λ0 also exhibit a very large range of variation30,31. Both λ0 and α, which shift dynamically in plants, animals and microbes, are convincingly influenced by limiting resources, including water, food, and oxygen etc32–34, as are metabolism and body mass. Enzymatic origin and various curvatures of metabolic scaling in microbes The model may take the following forms10: μ μ = = ⋅ + C dC dt C K C 1 (1) x x s s s max or μ μ = = ⋅ + M dM dt C K C 1 (2) s s s max μ μ = = ⋅ + C dC dt C K C 1 (1) x x s s s max μ μ = = ⋅ + C dC dt C K C 1 (1) x x s s s max μ μ = = ⋅ + M dM dt C K C 1 (2) s s s max (1) or μ μ = = ⋅ + M dM dt C K C 1 (2) s s s max (2) in which Cx is the concentration of microbial cells, M is the biomass of microbial cells, dCx/dt and dM/dt are the microbial growth rate, μmax is the maximum specific growth rate, and Ks is the Monod constant, i. e., the half-saturation constant for the substrate. Many people regarded the Monod equation as a theoretical one describing the relationship between the microbial specific growth rate (or biomass) and limiting resources, as the Monod equation is in the same form as the Michaelis-Menten equation, which is one of the most famous models of enzyme kinetics and includes constants with mechanistic meaning11,12. However, the Michaelis-Menten equation was derived from the mecha- nism of enzymatic reaction, while the Monod equation was developed from a curve-fitting exercise. The Monod equation is purely empirical and lacks a theoretical basis13, so none of the Michaelis-Menten constants, which are appropriate for an enzyme-substrate system, can be applied to a substrate-cell system14. Over the last few decades, numerous researchers have devoted great efforts to proposing many powerful theoretical interpretations to support the Monod equation, such as the thermodynamics of a microbial growth process13, and mass transfer College of Life Sciences, Zhejiang University, Hangzhou, China. Correspondence and requests for materials should be addressed to L.L. (email: Liliyan1995@163.com) or G.W. (email: wanggx@zju.edu.cn) Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 1 www.nature.com/scientificreports/ concentrations14. A new mechanical model of the relationship between microbial growth and limiting resources may be helpful for deepening the understanding of the microbial growth kinetics. concentrations14. A new mechanical model of the relationship between microbial growth and limiting resources may be helpful for deepening the understanding of the microbial growth kinetics. Enzymatic origin and various curvatures of metabolic scaling in microbes In the present study, at the interspecific level, we developed resource-dependent equations of natural logarithmic microbial metabolism (lnλ) and growth (or biomass) (lnM) and then obtained equations interdependent between lnλ and lnM based on the hypothesis that the relative metabolic rate and the relative growth rate in microbes are driven by their own rate-limiting enzymes, which is generally supported by data compiled from many articles on microbes. We found that active metabolic scaling is generally nonlinear, its curvature is derived from the difference between microbial metabolic and growth (or biomass’s) half-saturation constants (KM, Kλ) in the active state, and par- ticularly, for the same values of KM and Kλ, there is a linear scaling relationship. Therefore, we argue that a power law based on the basal metabolism may be the particular dynamics in our new interdependent model, which means that, at least in microbes, enzyme dynamics rather than the surface rule or a fractal resource transport network21,46,47 is a main driver of the active and basal metabolic scaling curvatures. The Enzyme-Driven Metabolic Scaling Model y g The resource-dependent active metabolism model. Metabolism is a collection of chemical transfor- mations and enzyme-catalyzed reactions that perform a variety of functions, ranging from nutrient breakdown to the polymerization of macromolecules. It is reasonable to hypothesize that the relative metabolic rate of microbes (dλ/λ) is constrained by the key enzymatic rate of their metabolism48, under the conditions that other factors are constant over time: λ λ = λ d dv (4) 2 Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 www.nature.com/scientificreports/ where λ is the active metabolic rate, vλ is the key enzymatic rate of the metabolism, dλ is the differential of λ and dvλ is the differential of vλ. Equation (5) on the relationship between the metabolic rate and the key enzymatic rate of metabolism was obtained by integrating Eq (4): where λ is the active metabolic rate, vλ is the key enzymatic rate of the metabolism, dλ is the differential of λ and dvλ is the differential of vλ. where λ is the active metabolic rate, vλ is the key enzymatic rate of the metabolism, dλ is the differential of λ and dvλ is the differential of vλ. λf λ Equation (5) on the relationship between the metabolic rate and the key enzymatic rate of metabolism was btained by integrating Eq. (4): λ = λ ae (5) bv λ = λ ae (5 bv (5) λ = λ aebv where a is the coefficient of transformation, and b is the efficiency of vλ. oefficient of transformation, and b is the efficiency of vλ fifi Eq. (6) on the relationship between the metabolic rate and the concentration of a limiting resource was btained by taking the logarithm and substituting the Michaelis-Menten equation12 into Eq. (5): λ λ = ⋅ + + λ λ λ C K C a ln ln ln (6) s s max (6) where Csλ is the concentration of a limiting substrate, ln λmax = bVλ is the maximum metabolic rate when Csλ approaches saturation and other resources remain constant, Vλ is the maximum rate of the key enzymatic reac- tion of the metabolism in the Michaelis-Menten equation, and Kλ is the half-saturation constant. The resource-dependent growth model. Most of the reactions that occur during microbial growth are enzymatic reactions. The Enzyme-Driven Metabolic Scaling Model It is reasonable to hypothesize that the relative rate of microbial biomass (the specific growth rate) is constrained by the key enzymatic rate of microbial growth in each growth stage under the conditions that other factors are constant over time: μ = = μ dM M dv (7) (7) where μ is the specific growth rate, vμ is the key enzymatic rate in the growth process, M is the biomass, dM /M is the relative rate of change in microbial biomass, and dvμ is the partial differential of vμ.i μf μ Equation (8) on the relationship between the specific growth rate and the key enzymatic rate in the growth rocess was obtained by integrating Eq. (7): μ = μ h v (8A) 1 = μ M ce (8B) h v 2fi μ = μ h v (8A) 1 (8A) = μ M ce (8B) h v 2 = μ M ceh v 2 (8B) where c is the coefficient of transformation, and h1, h2 is the efficiency of the key enzymatic rate in the growth process.i where c is the coefficient of transformation, and h1, h2 is the efficiency of the key enzymatic rate in the growth process.i Equation (9) on the relationship between the specific growth rate and the concentration of a growth-limiting resource was obtained by taking the logarithm and substituting the Michaelis-Menten equation12 into Eqs (8A) and (8B): μ μ = ⋅ + μ μ μ C K C (9A) s s max (9A) = ⋅ + + M M C K C c ln ln ln (9B) sM M sM max (9B) where Csμ, CsM is the concentration of a growth-limiting substrate, μmax = h1Vμ is the maximum rate in the growth process when Csμ approaches saturation and other resources remain constant, ln Mmax = h2VM is the maximum biomass of microbial cells in the growth process when CsM approaches saturation and other resources remain constant, Vμ, VM is the maximum rate of the key enzymatic reaction of growth in the Michaelis-Menten equation, and Kμ, KM is the half-saturation constant. The Enzyme-Driven Metabolic Scaling Model λ λ λ λ λ M K M K K K K K K ln ln , ln M M M M max max max λ λ μ μ = ⋅ + + μ λμ K a ln ln ln (11A) max (11A) μ + λμ K (11A) μ μ λ λ = ⋅ − + − λ μλ a K a (ln ln ) (ln ln ) (11B) max μ μ λ λ = ⋅ − + − λ μλ a K a (ln ln ) (ln ln ) max μ μ λ λ = + − λ μλ a K a (ln ln ) (ln ln ) (11B) max (11B) where λ λ μ = − = − μ μ μ λ λμ λ μ λ K K K K K K K ln ln , , max max max μ μ λ = − = − . λ λ λ μ μλ μ λ μ K K K K K K K , ln max max max λ λ μ = − = − μ μ μ λ λμ λ μ λ K K K K K K K ln ln , , max max max μ μ λ = − = − . λ λ λ μ μλ μ λ μ K K K K K K K , ln max max max λ λ μ = − = − μ μ μ λ λμ λ μ λ K K K K K K K ln ln , , max max max μ μ λ = − = − . λ λ λ μ μλ μ λ μ K K K K K K K , ln max max max μ μ λ = − = − . λ λ λ μ μλ μ λ μ K K K K K K K , ln max max max Eqs (12A) and (12B) were obtained when Kλ = KM, and describe the relationship between metabolism and biomass, i.e., the specific growth rate in microbes. Eqs (12A) and (12B) were obtained when Kλ = KM, and describe the relationship between metabolism and biomass, i.e., the specific growth rate in microbes. The Enzyme-Driven Metabolic Scaling Model λ = + d M e ln ln ln (12A) λ = + d M e ln ln ln (12A) λ = + d M e ln ln ln (12A) λ = + M f g ln ln ln (12B) (12A) λ = + M f g ln ln ln (12B) (12B) where where λ λ = = − . d M e a c M ln ln , ln ln ln ln ln max max max max λ λ = = − . f M g c M a ln ln , ln ln ln ln ln max max max max Eq. (12) is obtained by taking the logarithm of equation (13): λ = eM (13A) d λ = eM (13A) d f λ = eM (13A) d λ = M g (13B) f λ = eMd (13A) λ = M g f λ = M g (13B) f (13B) The form of Equation (13) is similar to that of Eq. (3) and, expressly, the power law is a particular form of the interdependent law for both key enzymatic dynamics with the same half-saturation constant. The form of Equation (13) is similar to that of Eq. (3) and, expressly, the power law is a particular form of the interdependent law for both key enzymatic dynamics with the same half-saturation constant. The Enzyme-Driven Metabolic Scaling Model where Csμ, CsM is the concentration of a growth-limiting substrate, μmax = h1Vμ is the maximum rate in the growth process when Csμ approaches saturation and other resources remain constant, ln Mmax = h2VM is the maximum biomass of microbial cells in the growth process when CsM approaches saturation and other resources remain constant, Vμ, VM is the maximum rate of the key enzymatic reaction of growth in the Michaelis-Menten equation, and Kμ, KM is the half-saturation constant. The natural logarithmic model with interdependence between microbial growth and metabo- lism. Eqs (10A), (10B), (11A) and (11B) were obtained by substituting Eqs (9A) and (9B) into Eq. (6) or vice versa under the conditions of the same types of limiting substrates for both microbial metabolism and growth and Kλ ≠ KM. λ λ = ⋅ − + − + λ M c K M c a ln ln (ln ln ) (ln ln ) ln (10A) M M max 1 1 1 (10A) λ λ = ⋅ − + − + λ λ M M a K a c ln ln (ln ln ) (ln ln ) ln (10B) M max 2 2 2 (10B) where where where λ = = λ λ − − λ λ λ K ln , , M K K K M K M K K max ln ln M M M max max Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 3 www.nature.com/scientificreports/ λ = − = − . λ λ λ λ λ M K M K K K K K K ln ln , ln M M M M max max max λ λ μ μ = ⋅ + + μ λμ K a ln ln ln (11A) max μ μ λ λ = ⋅ − + − λ μλ a K a (ln ln ) (ln ln ) (11B) max λ = − = − . Result The enzyme-driven relationship between a limiting resource and active metabolism. The basic hypothesis that the relative rate of microbial metabolism is constrained by the key enzymatic rate of the metabolism and the predictions (Eq. 5) were supported by the data compiled from several publications. The relationship between enzyme activity and metabolic rate is exponential (Fig. 1). In a soil bacterium, the soil respiration rate increased exponentially with acid phosphatase activity (Fig. 1A); the acid phosphatase activity was measured on day 7, with glucose amendment producing the highest bacterial concentrations (data from Anderson et al.49). The respiratory electron transport system (ETS) activity, which reflects the sum of the activities of nicotinamide adenine dinucle- otide (NADH) oxidoreductase and succinate dehydrogenase increased exponentially with the respiratory oxygen consumption in anaerobic (tryptone-yeast extract-sea salt medium containing nitrogen, TYSN) cultures of a marine bacterium (Pseudomonas perfectomarinus) (Fig. 1B) (data from Packard et al.50). The dehydrogenase activity in two Gray Luvisolic soils had a positive effect on microbial respiration (Fig. 1C); the respiratory activity was determined by incubating the soil samples with a CO2 trap for 10 days (data from Vvsr et al.51). The soil bacterial respiration increased with alkaline phosphatase activity and phosphodiesterase activity (Fig. 1D) (data from Frankenberger and Dick48).h p p y p p y ( g ) ( g ) The resources dependence of metabolic rate (Eq. 6) was tested by data on limiting resources and metabolic rate from several publications (Fig. 2). Natural logarithmic bacterial production increased with chlorophyll a in eastern waters of Hong Kong (Fig. 2A) (data from Yuan et al.52). In sea water, the trajectory of the natural logarithmic bacte- ria’s uptake by bacteria of 14C-glycine with different substrate (glycine) concentrations is curved (Fig. 2B); bacterial samples were incubated for 1 h (data from Manahan and Richardson53). Rhododendron leaves and wood veneers were sampled on days 28, 44, 77, 111, and 144 (data from Burns54). The changes in natural logarithmic microbial res- piration rates formed a curve and were associated with decaying plant litter (rhododendron and wood veneer) with increasing dissolved inorganic nitrogen concentrations (Fig. 2C). Natural logarithmic fungal respiration increased with the ergosterol content (Fig. 2D) in soil samples from vegetation zones (data from Imberger and Chiu55). Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 4 www.nature.com/scientificreports/ Figure 1. Metabolic rate increases exponentially with enzymatic activity in microbes, following Eq. (5). Result (A) Acid phosphatase activity vs soil respiration rate (circle) in a soil bacterium (data from Anderson et al.49). (B) ETS activity vs respiratory oxygen consumption (triangle) in the marine bacterium Pseudomonas perfectomarinus (data from Packard et al.50). (C) Dehydrogenase activity vs microbial respiration (square) in two Gray Luvisolic soil zones (data from Vvsr et al.51). (D) Enzyme activity vs soil bacterial respiration (hollow diamond: alkaline phosphatase, solid diamond: phosphodiesterase) (data from Frankenberger and Dick48). All parameter values are given in SI Appendix Table S2. Figure 1. Metabolic rate increases exponentially with enzymatic activity in microbes, following Eq. (5). (A) Acid phosphatase activity vs soil respiration rate (circle) in a soil bacterium (data from Anderson et al.49). (B) ETS activity vs respiratory oxygen consumption (triangle) in the marine bacterium Pseudomonas perfectomarinus (data from Packard et al.50). (C) Dehydrogenase activity vs microbial respiration (square) in two Gray Luvisolic soil zones (data from Vvsr et al.51). (D) Enzyme activity vs soil bacterial respiration (hollow diamond: alkaline phosphatase, solid diamond: phosphodiesterase) (data from Frankenberger and Dick48). All parameter values are given in SI Appendix Table S2. The enzyme-driven relationship between limiting resources and specific growth rate. The microbial mass-specific growth rate is the relative growth rate of microbial biomass. Equation (8) shows that the microbial specific growth rate or biomass increases linearly or exponentially with enzyme activity respectively (Fig. 3). Figure 3A shows that microbial biomass increased exponentially with dehydrogenase activity in surface samples (0–15 cm) of loam (organic C, 0.72% ; pH, 7.7) soil (data from Dar56). ETS activity increased exponen- tially with bacterial biomass in anaerobic (TYSN) cultures of a marine bacterium (P. perfectomarinus) (Fig. 3B). Biomass was monitored by measuring absorbance (A1cm 600) at 600 nm in a 1 cm cell. TYSN- − NO3 and TYSN- − NO2 represented two phases of respiration in anaerobic cultures of P. perfectomarinus (data from Packard et al.50). The relationship between alkaline phosphatase activity and soil microbial biomass is exponential (Fig. 3C) (Frankenberger and Dick48). The relative growth rate increased linearly with urease activity in the marine microalgae species Prorocentrum minimum (Fig. 3D). Urease activities of P. minimum cultures grown with urea and NH4+ sources at the exponential growth phase were used to obtain this relationship (data from Fan et al.57). The limiting resource-dependent equations of natural logarithmic biomass (Eq. 9A) and specific growth rate (Eq. 9B) were supported by data compiled from many papers. Result There is a curvilinear relationship between limiting resources and specific growth rate or natural logarithmic biomass (Fig. 4). The specific growth rate increased with nutritional capacity following the dynamics described by Eq. (9B) (Fig. 4A). RNA and protein extracted from the medium of strains derived from the Escherichia coli K12 strain MG1655 were used to calculate the nutritional capacity and mass-specific growth rate, respectively (data from Scott et al.58). The relationship between the rela- tive exponential-state growth rate of a marine bacterium (Pseudomonas doudoroffii 70) and Na+ concentration is curvilinear (Fig. 4B). The P. doudoroffii 70 was cultured in minimal medium with succinate was added as a carbon source (data from Wisse and Macleod59). The specific growth rate of a Baltic Sea filamentous cyano- bacterial species (Nodularia spumigena) in the exponential period increased only with salinity varying from 0 to 10 PSU following the dynamics described by Eq. (9B) (Fig. 4C) (data from Rakko and Seppälä60). The spe- cific growth rates of two marine microalgae (Dunaliella tertiolecta and Phaeodactylum tricornutum) exhibited a curvilinear response to photon flux for growth (Fig. 4D) (data from Quigg and Beardall661). Maple leaves were sampled on days 14, 28, 44, 77, and 111 (data from Burns54). The natural logarithmic fungal biomass associated The enzyme-driven relationship between limiting resources and specific growth rate. The microbial mass-specific growth rate is the relative growth rate of microbial biomass. Equation (8) shows that the microbial specific growth rate or biomass increases linearly or exponentially with enzyme activity respectively (Fig. 3). Figure 3A shows that microbial biomass increased exponentially with dehydrogenase activity in surface samples (0–15 cm) of loam (organic C, 0.72% ; pH, 7.7) soil (data from Dar56). ETS activity increased exponen- tially with bacterial biomass in anaerobic (TYSN) cultures of a marine bacterium (P. perfectomarinus) (Fig. 3B). Biomass was monitored by measuring absorbance (A1cm 600) at 600 nm in a 1 cm cell. TYSN- − NO3 and TYSN- − NO2 represented two phases of respiration in anaerobic cultures of P. perfectomarinus (data from Packard et al.50). The relationship between alkaline phosphatase activity and soil microbial biomass is exponential (Fig. 3C) (Frankenberger and Dick48). The relative growth rate increased linearly with urease activity in the marine microalgae species Prorocentrum minimum (Fig. 3D). Urease activities of P. The natural logarithmic model with interdependence between microbial growth and metabolism Th d d l h b b l b l d b fi h The natural logarithmic model with interdependence between microbial growth and metabolism. The interdependent relationship between microbial metabolism and biomass or specific growth rate in natu- ral logarithmic space (Eqs 10A and 10B) was tested by data compiled from Vvsr et al.51 (Fig. 5). In two Gray Luvisolic soil zones of Saskatchewan, microbial biomass and respiration rate exhibited a curvilinear relationship (Fig. 5A,B). At the same locations, the relationship between the fungal biomass and respiration rate also showed a curve similar to that in Fig. 5A–D).hi Three statistical parameters, namely, the goodness of fit (R2), residual sum of squares (RSS), and Akaike’s information criterion (AIC)63,64, are regarded as the criterion with which to determine which model is the best representation of a curve. The lnλ values were regressed with respect to M or lnM using exponential, power and mass-dependent equations using the data shown in Fig. 5A,C. The power equation yielded slightly lower AIC values than mass-dependent functions we proposed here (Eq. 10A) (9.0983 < 10.9416; 1.0294 < 1.1482), but our model produced lower RSS values (1.342 < 1.665; 0.504 < 0.743) and higher R2 values (0.7552 > 0.6963; 0.9081 > 0.8644) (Table 1). Therefore, we argue that our model (Eq. 10A) is better than the traditional power equation (Eq. 3) based on the values of R2, AIC, and RSS. Result minimum cultures grown with urea and NH4+ sources at the exponential growth phase were used to obtain this relationship (data from Fan et al.57).hi p g p p ( ) The limiting resource-dependent equations of natural logarithmic biomass (Eq. 9A) and specific growth rate (Eq. 9B) were supported by data compiled from many papers. There is a curvilinear relationship between limiting resources and specific growth rate or natural logarithmic biomass (Fig. 4). The specific growth rate increased with nutritional capacity following the dynamics described by Eq. (9B) (Fig. 4A). RNA and protein extracted from the medium of strains derived from the Escherichia coli K12 strain MG1655 were used to calculate the nutritional capacity and mass-specific growth rate, respectively (data from Scott et al.58). The relationship between the rela- tive exponential-state growth rate of a marine bacterium (Pseudomonas doudoroffii 70) and Na+ concentration is curvilinear (Fig. 4B). The P. doudoroffii 70 was cultured in minimal medium with succinate was added as a carbon source (data from Wisse and Macleod59). The specific growth rate of a Baltic Sea filamentous cyano- bacterial species (Nodularia spumigena) in the exponential period increased only with salinity varying from 0 to 10 PSU following the dynamics described by Eq. (9B) (Fig. 4C) (data from Rakko and Seppälä60). The spe- cific growth rates of two marine microalgae (Dunaliella tertiolecta and Phaeodactylum tricornutum) exhibited a curvilinear response to photon flux for growth (Fig. 4D) (data from Quigg and Beardall661). Maple leaves were sampled on days 14, 28, 44, 77, and 111 (data from Burns54). The natural logarithmic fungal biomass associated Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 5 www.nature.com/scientificreports/ Figure 2. Metabolic rate increases with the concentration of limiting resources in microbes, following Eq. (6). (A) Bacterial production (BP) vs chlorophyll a in eastern waters (data from Yuan et al.52). (B) Substrate concentration vs the rate of uptake of 14C-glycine by marine bacteria (data from Manahan and Richardson53). (C) Dissolved inorganic nitrogen concentration vs microbial respiration rates associated with decaying rhododendron (hollow square) and wood veneer (solid square) leaf litter (data from Burns54). (D) Soil ergosterol content vs fungal respiration (diamond) in a vegetation zone (data from Imberger and Chiu55). The parameter values are given in SI Appendix Table S2. igure 2. Metabolic rate increases with the concentration of limiting resources in microbes, following Eq. (6). Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 Result A) B t i l d ti (BP) hl h ll i t t (d t f Y t l 52) (B) S b t t Figure 2. Metabolic rate increases with the concentration of limiting resources in microbes, following Eq. (6). (A) Bacterial production (BP) vs chlorophyll a in eastern waters (data from Yuan et al.52). (B) Substrate concentration vs the rate of uptake of 14C-glycine by marine bacteria (data from Manahan and Richardson53). (C) Dissolved inorganic nitrogen concentration vs microbial respiration rates associated with decaying rhododendron (hollow square) and wood veneer (solid square) leaf litter (data from Burns54). (D) Soil ergosterol content vs fungal respiration (diamond) in a vegetation zone (data from Imberger and Chiu55). The parameter values are given in SI Appendix Table S2. Figure 2. Metabolic rate increases with the concentration of limiting resources in microbes, following Eq. (6). (A) Bacterial production (BP) vs chlorophyll a in eastern waters (data from Yuan et al.52). (B) Substrate concentration vs the rate of uptake of 14C-glycine by marine bacteria (data from Manahan and Richardson53). (C) Dissolved inorganic nitrogen concentration vs microbial respiration rates associated with decaying rhododendron (hollow square) and wood veneer (solid square) leaf litter (data from Burns54). (D) Soil ergosterol content vs fungal respiration (diamond) in a vegetation zone (data from Imberger and Chiu55). The parameter values are given in SI Appendix Table S2. p p y oncentration vs the rate of uptake of 14C-glycine by marine bacteria (data from Manahan and Richardson53). C) Dissolved inorganic nitrogen concentration vs microbial respiration rates associated with decaying hododendron (hollow square) and wood veneer (solid square) leaf litter (data from Burns54). (D) Soil gosterol content vs fungal respiration (diamond) in a vegetation zone (data from Imberger and Chiu55). The arameter values are given in SI Appendix Table S2. with decaying plant litter changed with increasing dissolved inorganic nitrogen concentrations (Fig. 4E). The steady-state growth rate of a marine diatom (Thalassiosira pseudonana) increased with irradiance following the dynamics described by Eq. (9B) (Fig. 4F) (data from Berges and Harrison62). Discussion Discussion Our Eqs (5), (6), (8), (9), and (10) satisfactorily characterized microbial data. The analytical results support our hypothesis and predictions (Table 1 and SI Appendix). The relative rates of microbial growth and metabolism are interdependent. The driver of metabolic scaling may be enzymatic dynamics rather than the ratio of surface area to volume or a fractal resource transport network21,46,47.h p The most basic indicator of metabolism is the metabolic rate (λ); the metabolism and body size (body ma biomass, M) of organisms scale as λ ∝ Mα20. In the late 1990s, Rubner first described the quantitative relation Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 6 www.nature.com/scientificreports/ Figure 3. Biomass (growth rate) increases exponentially with enzymatic activity in microbes, following Eqs (8A) and (8B). (A) Dehydrogenase activities vs soil microbial biomass (hollow circle) (data from Dar56). (B) ETS activity vs marine bacterial biomass (hollow triangle: TYSN- − NO3, solid triangle: TYSN- − NO2) (data from Packard et al.50). (C) Alkaline phosphatase activity vs soil microbial biomass (hollow diamond) (data from Frankenberger and Dick48). (D) Urease activity vs the relative growth rate of Phaeodactylum tricornutum (hollow square) (data from Fan et al.57). The parameter values are given in SI Appendix Table S3. Figure 3. Biomass (growth rate) increases exponentially with enzymatic activity in microbes, following Eqs (8A) and (8B). (A) Dehydrogenase activities vs soil microbial biomass (hollow circle) (data from Dar56). (B) ETS activity vs marine bacterial biomass (hollow triangle: TYSN- − NO3, solid triangle: TYSN- − NO2) (data from Packard et al.50). (C) Alkaline phosphatase activity vs soil microbial biomass (hollow diamond) (data from Frankenberger and Dick48). (D) Urease activity vs the relative growth rate of Phaeodactylum tricornutum (hollow square) (data from Fan et al.57). The parameter values are given in SI Appendix Table S3. between metabolic rate and body size38; since then, many mathematical scaling models of metabolism have been developed to explain this allometric relationship. One of the famous models is a fractal-like distribution network (WBE) model21,46,47, in which the physicist West and ecologists Brown and Enquist summarized the circulatory system of animals and the vascular bundle system of plants into a resource supply network (WEB model) with self-similar structure, explaining Kleiber’s law (α = 3/4), in 1997. Another famous model is the metabolic-level boundaries (MLB) model proposed by Glazier which based on physical limits, explains why the exponent α var- ies from 2/3 to 165,66. Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 Discussion In addition, there are many other models to explain the different exponential values, such as efficient transportation networks67, cell optimization growth theory (α = 2/3 to 1)68, structural theory (large endotherm:α = 3/4; ectotherm:α = 2/3; small endotherm:α = 1/3 to 3/4)69, and energy consumption (small and medium animals:α = 3/4; large animals:α = 1)70. First, what these theories have in common is that the relation- ship between metabolism and body mass is linear in the logarithmic space. Because of the limitation of employing linear regression, these methods produce only isolated exponential values (the slopes of the lines). However, we can obtain a continuously changing dynamic for the exponent α because we obtained a curvilinear relationship between microbial metabolism and biomass. When we used operations of partial derivatives with respect to our curve (∂lnλ/∂(lnM-lnc1)), we obtained a continuous exponential data set containing the slopes of all the tangent lines tangent to this curve. Second, these theories mentioned above usually borrowed concepts from mathemat- ical geometry, such as fractal geometry or physical limits21,38,65–70 to explain the allometric relationship and paid relatively little attention to the essence of metabolism, namely, that it is a series of enzymatic reactions. However, we argue that enzyme activities drive the relationship between microbial metabolism and biomass because metabolism and growth are a series of biochemical reactions that furnish the materials and energy necessary for biological growth, development, reproduction and evolution. In addition the data fitted by Eqs (5), (6), (8) and (9) (Figs 1–4) supported our hypothesis and predictions that enzymes drive the relative rate of both microbial metab- olism and growth. We do not deny that various theories proposed above, such as the ratio of surface area to volume or the fractal resource transport network, may also affect scaling dynamics by regulating the energy balance or substrate concentration; we simply note that they do not take into account the importance and driving force of the key enzymes. Furthermore, the investigation of Miettinen and Björklund suggested that the meva- lonate pathway activity which is a metabolic pathway essential for synthesizing isopentenyl pyrophosphate and dimethylallyl pyrophosphate in eukaryotes, archaea, and some bacteria71,72, contributes to the nonlinearity of the scaling between cell size and mitochondrial function73. Furthermore, fundamental aspects of enzyme activities Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 7 www.nature.com/scientificreports/ Figure 4. Discussion The limiting resource dependence of growth rate or natural logarithmic biomass in microbes, following Eqs (9A) and (9B). (A) The nutritional capacity vs the mass-specific growth rate of Escherichia coli K12 strain MG1655 (hollow circle) in medium without antibiotics (data from Scott et al.58). (B) Na+ concentration vs the relative growth rate of the marine bacterium Pseudomonas doudoroffii 70 (hollow triangle) in minimal medium, with succinate as a carbon source (data from Wisse and Macleod59). (C) Salinity vs the relative growth rate of a cyanobacterium, Nodularia. spumigena (hollow square) (data from Rakko and Seppälä60). (D) Photon flux vs the specific growth rate of Dunaliella. tertiolecta (hollow diamond) and Phaeodactylum. tricornutum (solid diamond) (data from Quigg and Beardall661). (E) Dissolved inorganic nitrogen concentration vs fungal biomass associated with maple leaves (solid short line segments) (data from Burns54). (F) Irradiance vs growth rate for the marine diatom Thalassiosira pseudonana (plus sign) (data from Berges and Harrison62). The parameter values are given in SI Appendix Table S3. Figure 4. The limiting resource dependence of growth rate or natural logarithmic biomass in microbes, following Eqs (9A) and (9B). (A) The nutritional capacity vs the mass-specific growth rate of Escherichia coli K12 strain MG1655 (hollow circle) in medium without antibiotics (data from Scott et al.58). (B) Na+ concentration vs the relative growth rate of the marine bacterium Pseudomonas doudoroffii 70 (hollow triangle) in minimal medium, with succinate as a carbon source (data from Wisse and Macleod59). (C) Salinity vs the relative growth rate of a cyanobacterium, Nodularia. spumigena (hollow square) (data from Rakko and Seppälä60). (D) Photon flux vs the specific growth rate of Dunaliella. tertiolecta (hollow diamond) and Phaeodactylum. tricornutum (solid diamond) (data from Quigg and Beardall661). (E) Dissolved inorganic nitrogen concentration vs fungal biomass associated with maple leaves (solid short line segments) (data from Burns54). (F) Irradiance vs growth rate for the marine diatom Thalassiosira pseudonana (plus sign) (data from Berges and Harrison62). The parameter values are given in SI Appendix Table S3. could allow deviations from the traditional power law in principle, as we have proposed: the relationship between microbial metabolism and biomass is curvilinear and driven by their respective key enzymes. Discussion (A,B) In two Gray Luvisolic soil zones of Saskatchewan: the Equation (10) prediction is shown by a solid curved line (hollow diamond) and a dashed curved line (solid diamond) (data from Vvsr et al.51). (C,D) In two Grey Luvisolic soil zone of Saskatchewan: Equation (10) prediction is shown by solid curved line (hollow short line segments) and dash curved line (solid short line segments) (data from Vvsr et al.51). The parameter values that provide this best fit are given in SI Appendix Table S4. Model Calculated equation RSS R2 AIC Exponential function λ = ′ + ′ a b M ln ln Fig. 5A 122.9 0.0656 52.1137 Fig. 5C 2.859 0.4785 14.5047 Power function λ = ′ + ′ a b M ln ln ln Fig. 5A 1.665 0.6963 9.0983 Fig. 5C 0.743 0.8644* 1.0294 Mass-dependent function λ = + ′ ′ ∗ − ′ ′ + − ′ d ln a M b c M b (ln ) (ln ) Fig. 5A 1.342 0.7552* 10.9416 Fig. 5C 0.504 0.9081* 1.1482 Table 1. The comparison of model application results for microbial metabolic rate and biomass. Present: p < 0.01; present: p < 0.001. Table 1. The comparison of model application results for microbial metabolic rate and biomass. Present: p < 0.01; *present: p < 0.001. Semi-logarithmic equations, in which the dependent variable is a natural logarithm, are primarily used in empirical economics74, and to describe the dynamics of some microbes, such as the isothermal semi-logarithmic survival curves of microorganisms and spores75. Nevertheless, our two resource-dependent semi-logarithmic equations, namely, Eqs (6), (9), differ from the research that directly introduced the Michaelis-Menten equation to describe the relationship between metabolic rate and limiting resources34–37. For example, the Michaelis-Menten equation is generally used to directly depict the gross photosynthetic rates relative to irradiance at the surface in aquatic systems35. López-Urrutia et al. extended the MTE to account for the relationship between individual gross photosynthesis and photosynthetically active radiation in the oceans using the Michaelis-Menten equa- tion as well34. Sinsabaugh and Shah36 combined metabolic scaling theory23 and kinetic measures of extracellular enzyme activity to relate bacterial productivity to AppVmax, which is a measure of enzyme abundance (catalytic capacity), and Aguiar-González et al.37 used the biochemical enzyme kinetic model (EKM) of respiratory oxygen consumption based on the substrate control of respiratory electron transfer systems. Discussion ould allow deviations from the traditional power law in principle, as we have proposed: the relationship between microbial metabolism and biomass is curvilinear and driven by their respective key enzymes.h The Monod equation, a microbial growth dynamics models, is an empirical equation and has the same form as the Michaelis-Menten equation11. As we validated here, the forms of the Monod equation and empirical growth law are actually part of the predictions of our hypothesis, that is, Equations (8) and (9). Unsurprisingly, accumu- lated evidences in this field5,10 also supports our hypothesis (Fig. 4). We provide a mechanical explanation for the relationship between microbial growth (biomass) and limiting resources, while the Monod equation did not. Scientific Reports \| (2019) 9:4082 \| https://doi.org/10.1038/s41598-019-40712-5 8 www.nature.com/scientificreports/ Figure 5. The natural logarithmic interdependent relationship between microbial biomass and metabolism, following Eq. (10A) and (10B). (A,B) In two Gray Luvisolic soil zones of Saskatchewan: the Equation (10) prediction is shown by a solid curved line (hollow diamond) and a dashed curved line (solid diamond) (data from Vvsr et al.51). (C,D) In two Grey Luvisolic soil zone of Saskatchewan: Equation (10) prediction is shown by solid curved line (hollow short line segments) and dash curved line (solid short line segments) (data from Vvsr et al.51). The parameter values that provide this best fit are given in SI Appendix Table S4. Figure 5. The natural logarithmic interdependent relationship between microbial biomass and metabolism, f ll i E (10A) d (10B) (A B) I t G L i li il f S k t h th E ti (10) Figure 5. The natural logarithmic interdependent relationship between microbial biomass and metabolism, following Eq. (10A) and (10B). (A,B) In two Gray Luvisolic soil zones of Saskatchewan: the Equation (10) prediction is shown by a solid curved line (hollow diamond) and a dashed curved line (solid diamond) (data from Vvsr et al.51). (C,D) In two Grey Luvisolic soil zone of Saskatchewan: Equation (10) prediction is shown by solid curved line (hollow short line segments) and dash curved line (solid short line segments) (data from Vvsr et al.51). The parameter values that provide this best fit are given in SI Appendix Table S4. Figure 5. The natural logarithmic interdependent relationship between microbial biomass and metabolism, following Eq. (10A) and (10B). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. A schematic of various ∆K values (the difference between Kλ and KM) responding to different scaling curvatures. Figure 6. A schematic of various ∆K values (the difference between Kλ and KM) responding to different scaling curvatures. The long-standing question of metabolic scaling may be resolved by our logarithmic equation with inter- dependence between metabolism and biomass. Equation (10) provides a new mechanical model for quan- titatively analyzing the relationship between microbial metabolism and growth. This equation predicts that double-logarithmic dynamic shifts in the metabolism and biomass of most microbes are curvilinear, rather than linear, as predicted by the MTE and other models predicted them were under the conditions of the same types of limiting substrates in both microbial metabolism and growth and Kλ ≠ KM. Furthermore, the double-logarithmic dynamics would be linear under the conditions of the same types of limiting substrates in both microbial metab- olism and growth and Kλ = KM, scaling as in Equation (11), as occurs in the power law. The smaller the difference between Kλ and KM is, the smaller the scaling dynamic curvature is under an active state (Fig. 6). When K = 0, the metabolic rate is basal, and with a shift in K values, microbes need energy to sustain not only basal metabolism, but also active activities and growth. In reality, numerous datassets of the nonlinear scaling dynamics have been analyzed using the linear regression, which may be able to simplify equations, similar to the famous 3/4 power equation20,21. In fact, Kleiber’s law was obtained by using a strictly controlled basal metabolic rate20, and we pre- sume that one of the reasons for the extensive debates about metabolic scaling is the possibility that the data used to draw conclusions, were collected imprecisely and were not completely basal. The power law is just a particular form of the natural logarithmic interdependent law rather than a general law, and our model may be more general when predicting the scaling dynamics between microbial metabolism and biomass. Conclusions h h d We hypothesized that the relative metabolic rate and growth rate might be driven by their rate-limiting enzymes in actively growing microbes. Active metabolic scaling originates from enzyme-driven processes, and the curva- ture of the scaling may derive from different dynamics of substrate responses between metabolism and growth. There may be a shift in the rate from the enzymatic to individual level because the relative rate of individual metabolism and growth is proportional to the rate of their respective rate-limiting-enzyme process. Thus, we conclude that natural logarithmic microbial metabolism (lnλ) and growth (or biomass) (lnM) are both dependent on limiting resources, thereby developing novel models with interdependence between lnλ and lnM, which can described the various metabolic scaling relationships in an active state with the difference between the microbial metabolic and growth (or biomass) half-saturation constants (KM, Kλ). Moreover, under a basal state with the same values of KM and Kλ, there is a linear scaling relationship. The results indicate that enzymatic dynamics may be the origin of active and basal metabolic scaling, and the traditional power law is a particular case of the interde- pendent models under the condition Kλ = KM. Integrating the scaling law with biochemical processes helps settle various debates on the traditional power law, understand how and why the scaling relationship is usually curved, and identify what deives the degree of curvature. Methods By researching for a large number of publications and by using the software GetData Graph Digitizer 2.22, we obtained relevant data for microbes, including the respiration rate, growth rate, enzyme activity, biomass, and concentration of limited resources (details provided in the Result section and SI Appendix Table S1), to verify our hypotheses and equations; more details are provided in the models and results. The software MATLAB R2017b was used to fit the curve and obtain all the coefficients (SI Appendix Tables S2, S3, S4). 1. Pasteur, L. Mémoire sur la fermentation appelée lactique (Extrait par l’auteur). Mol. Med. 1, 599 (1995). 2. Smith, K. A. Louis pasteur, the father of immunology? Front. Immunol. 3, 68 (2012). 3. Shrivastava, B. Microbial physiology and metabolism. LAP Lambert Academic Publishing, Germany (2012). 4. Sokatch, J. R. Bacterial physiology and metabolism. Q. Rev. Biol. 206 (2008). Discussion These are examples in which the Michaelis-Menten equation was used directly to consider the relations between metabolic rate (λ) and limiting resources. However, we predicted that there is a relationship between lnλ or lnM and limiting resources (Eqs 6 and 9) based on our hypothesis that both the relative metabolic rate (dλ/λ) and growth rate (dM/M)10 are constrained by their own rate-limiting enzymes. Equations (10) and (11) are not completely semi-logarithmic; their correlation is strong when the dependent variables are all in the natural logarithm. We proposed the concept of a relative met- abolic rate or biomass, which is different from the studies mentioned above that directly used the metabolic rate. 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https://openalex.org/W2340652090	https://europepmc.org/articles/pmc4833922?pdf=render	English	null	UNDR ROVER - a fast and accurate variant caller for targeted DNA sequencing	BMC bioinformatics	2,016	cc-by	5,168	© 2016 Park et al. 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. UNDR ROVER - a fast and accurate variant caller for targeted DNA sequencing niel J. Park1,3, Roger Li2, Edmund Lau3, Peter Georgeson3, Tú Nguyen-Dumont1 and Bernard J. * Correspondence: bjpope@unimelb.edu.au 2Department of Computing and Information Systems, The University of Melbourne, Melbourne, Victoria 3010, Australia 3Victorian Life Sciences Computation Initiative, The University of Melbourne, Melbourne, Victoria 3053, Australia Full list of author information is available at the end of the article Abstract Background: Previously, we described ROVER, a DNA variant caller which identifies genetic variants from PCR-targeted massively parallel sequencing (MPS) datasets generated by the Hi-Plex protocol. ROVER permits stringent filtering of sequencing chemistry-induced errors by requiring reported variants to appear in both reads of overlapping pairs above certain thresholds of occurrence. ROVER was developed in tandem with Hi-Plex and has been used successfully to screen for genetic mutations in the breast cancer predisposition gene PALB2. ROVER is applied to MPS data in BAM format and, therefore, relies on sequence reads being mapped to a reference genome. In this paper, we describe an improvement to ROVER, called UNDR ROVER (Unmapped primer-Directed ROVER), which accepts MPS data in FASTQ format, avoiding the need for a computationally expensive mapping stage. It does so by taking advantage of the location-specific nature of PCR-targeted MPS data. Results: The UNDR ROVER algorithm achieves the same stringent variant calling as its predecessor with a significant runtime performance improvement. In one indicative sequencing experiment, UNDR ROVER (in its fastest mode) required 8-fold less sequential computation time than the ROVER pipeline and 13-fold less sequential computation time than a variant calling pipeline based on the popular GATK tool. UNDR ROVER is implemented in Python and runs on all popular POSIX-like operating systems (Linux, OS X). It requires as input a tab-delimited format file containing primer sequence information, a FASTA format file containing the reference genome sequence, and paired FASTQ files containing sequence reads. Primer sequences at the 5′ end of reads associate read-pairs with their targeted amplicon and, thus, their expected corresponding coordinates in the reference genome. The primer-intervening sequence of each read is compared against the reference sequence from the same location and variants are identified using the same algorithm as ROVER. Specifically, for a variant to be ‘called’ it must appear at the same location in both of the overlapping reads above user-defined thresholds of minimum number of reads and proportion of reads. Conclusions: UNDR ROVER provides the same rapid and accurate genetic variant calling as its predecessor with greatly reduced computational costs. Keywords: PCR-MPS, Hi-Plex, ROVER, Targeted sequencing, Massively parallel sequencing, Varia Park et al. BMC Bioinformatics (2016) 17:165 DOI 10.1186/s12859-016-1014-9 Park et al. BMC Bioinformatics (2016) 17:165 DOI 10.1186/s12859-016-1014-9 Open Access SOFTWARE Background features is the ability to define a uniform library size which facilitates the removal of off-target amplification by size selection and, in combination with paired-end sequencing, allows complete overlap of read-pairs for each amplicon. The latter aspect permits a high degree of stringency in both the detection of variants and the filtering of artefacts caused by sequencing errors. Previ- ously, we developed ROVER, a variant calling tool which takes advantage of the overlapping reads pro- duced by Hi-Plex [2], and successfully applied Hi-Plex and ROVER to screening for genetic variants in the coding regions of PALB2, detecting all 60 variants In recent work, we developed a highly multiplexed PCR-based target-enrichment system called Hi-Plex (www.hiplex.org) for massively parallel sequencing (MPS) [1]. Hi-Plex is a simple, low-cost protocol that can achieve highly accurate results. One of its key * Correspondence: bjpope@unimelb.edu.au 2Department of Computing and Information Systems, The University of Melbourne, Melbourne, Victoria 3010, Australia 3Victorian Life Sciences Computation Initiative, The University of Melbourne, Melbourne, Victoria 3053, Australia Full list of author information is available at the end of the article Park et al. BMC Bioinformatics (2016) 17:165 Page 2 of 7 Page 2 of 7 [10]). Furthermore, Amplivar merges overlapping reads (using SeqPrep [11]), whereas UNDR ROVER keeps both reads to test their concordance as part of a strin- gent filtering system. identified by previous mutation screening and produ- cing no false positive calls [3, 4]. ROVER requires as inputs a file describing the gen- omic coordinates of target amplicon regions in tab- delimited format and one or more sequence files in BAM format [5] containing paired-end reads mapped to a reference genome. It produces a list of variants in VCF format [6]. ROVER can detect single nucleotide variants (SNVs) and small insertions and deletions (indels). A variant is only reported by ROVER when it appears at the same position in both of the reads in an overlapping pair. UNDR ROVER was designed to support Hi-Plex tar- geted sequencing but is also compatible with other amplicon-based targeted sequencing systems that retain gene-specific primer sequences in the sequencing reads and for which primer and insert coordinates and primer sequences and paired FASTQ files can be supplied in the formats outlined in our documentation. AmpliSeq- generated data would not be compatible, for example, because the gene-specific primers are largely cleaved during library generation. Implementation UNDR ROVER is implemented in Python 2.7 as a command-line application. Its four mandatory argu- ments are: 1) a tab-delimited format file which associ- ates primer-pairs with their genomic coordinates; 2) a tab-delimited format file which matches primer names to their insert sequences; 3) a FASTA format file con- taining the reference sequence (the primers must have been designed from this reference to ensure that the coordinates agree); 4) one or more pairs of FASTQ files. The main output of UNDR ROVER is a VCF file containing the detected variants. Additionally, it pro- duces two log files which report on the overall execu- tion of the program and the depth to which each amplicon is covered by the reads. Having determined the starting coordinates of a particular read, we can then compare its sequence to the reference. To allow for insertions and deletions, it is necessary to perform a gapped alignment in the style of the Needleman-Wunsch algorithm [9]. How- ever, the complexity of this algorithm is a quadratic function of the length of the aligned sequence and, therefore, expensive to compute for every read in the input. Fortunately, we can often avoid this cost be- cause most reads in the input will be identical to the reference or will only differ by a small number of mismatches. In these cases, a simple linear compari- son of the read to the reference is sufficient. We fall back to the gapped alignment algorithm only when the linear comparison fails. Hi-Plex employs PCR to amplify selected target re- gions of DNA. Larger segments of DNA are split into tiles of a specified narrow size range (typically, the order of 100 nucleotides). The regular tile size facili- tates size selection of the amplified product which in- creases on-target stringency and allows both reads of a pair to overlap the entire tile. Hi-Plex is compatible with short-read sequencing platforms such as Illumina TruSeq (MiSeq and HiSeq instruments, Illumina, San Diego, CA, USA) and Ion Torrent (PGM and Proton instruments, Life Technologies, Carlsbad, CA, USA) [12]. Hi-Plex primers consist of a pool of relatively low concentration (individually) gene-specific primers (GSPs) that seed the PCR, and universal adapter primers that drive the majority of the reaction. GSPs are designed to correspond to the sequences flanking the target inserts. Background A key consideration is that UNDR-ROVER is intended to work with sequencing data exhibiting considerable overlap of read-pairs - as such, it is not recommended for use with systems that do not achieve this. By far the most computationally expensive part of detecting variants with ROVER is the time taken to map (or align) the reads to the reference genome. In one indicative experiment, described below, the time taken for mapping with Bowtie (http://bowtie-bio.sour- ceforge.net/) [7] constituted approximately 78 % of the whole ROVER variant calling pipeline. Read mapping is a standard part of whole-exome and whole-genome DNA sequencing pipelines but, as has been demon- strated previously by Amplivar [8] and as we demon- strate in this paper in the context of Hi-Plex, it can be avoided in PCR-based MPS approaches. This is because the 5′ end of each read begins with a primer sequence whose genomic coordinates are already known. This latter information is determined during primer design. The reads do not need to be mapped to the reference because the primer-pairs identify the genomic coordi- nates of the intervening sequence. Optionally, we can further increase our confidence that a read is mapped to the correct location by checking that at least one of the reads in a pair is identical to the reference for a small sequence following the primer. Implementation Figure 1 illustrates the structure of a Hi-Plex library element in relation to the two overlap- ping reads of a read-pair. Amplivar is based on premises similar to those underlying UNDR ROVER but applies a different mechanism. Amplivar uses primer sequences to associ- ate reads with amplicons and reduces computational overheads by aligning reads as groups (using BLAT Park et al. BMC Bioinformatics (2016) 17:165 Page 3 of 7 Fig. 1 Hi-Plex library structure and overlapping reads. The center rectangle represents the target insert DNA sequence flanked by gene-specific primer (GSP) sites (blue) and adapter sequences (green). The two reads of a pair are shown in yellow. The 5′ end of each read starts with its corresponding gene-specific primer sequence. The insert size is chosen so that both reads overlap the target insert sequence completely. The 3′ ends of reads may extend into the adapter sequence depending on the read length and the presence/absence of insertions/deletions in the template DNA. The diagram is not to scale. Typically, the insert sequence will be significantly longer than the primer sequences Fig. 1 Hi-Plex library structure and overlapping reads. The center rectangle represents the target insert DNA sequence flanked by gene-specific primer (GSP) sites (blue) and adapter sequences (green). The two reads of a pair are shown in yellow. The 5′ end of each read starts with its corresponding gene-specific primer sequence. The insert size is chosen so that both reads overlap the target insert sequence completely. The 3′ ends of reads may extend into the adapter sequence depending on the read length and the presence/absence of insertions/deletions in the template DNA. The diagram is not to scale. Typically, the insert sequence will be significantly longer than the primer sequences UNDR ROVER comprises two main steps: 1) associat- ing reads with their corresponding primer tiles and 2) calling variants. satisfy this requirement are discarded. Some provision to allow incomplete overlap is engineered to accom- modate contexts that preclude the achievement of complete ‘tiling’, such as the presence of genomic in- sertion events or intractable sequences for primer de- sign. For additional stringency, UNDR ROVER can optionally test whether, in at least one of the reads in a pair, the sequence just after the primer sequence is an exact match with the corresponding target reference sequence. Implementation By default, this test will use a sequence of 30 nucleotides, but it can be configured by a command- line argument. Since only one read of a read-pair (at either end) is required to match the expected sequence for a read-pair to contribute to variant calling, variants that are present in the terminal regions are detectable unless they coincide with additional variants at the other end of the read-pair. A primer tile is a contiguous section of the genome which is flanked by a primer-pair. We take advantage of the fact that the 5′ end of each read starts with a GSP sequence for which the coordinates are known. There- fore, the start of each read can be compared to the full set of GSPs to identify its corresponding tile. This com- parison is made efficient by storing primer information in a hash table indexed by primer sequence. As such, the coordinates of each read can be found in time propor- tional to the length of the primer sequence. Hi-Plex primers can vary in length to a small degree, therefore UNDR ROVER stores only the first N bases of primers in the hash table. The value of N is user definable and should be no larger than the length of the shortest pri- mer used in the experiment. This scheme requires that the first N bases of a read is an exact match to its corre- sponding primer sequence and, therefore, does not toler- ate mismatches derived from errors in the sequencing chemistry and/or production of primer oligonucleotides. Reads which do not start with a known primer se- quence are discarded. We have not found this to be a problem in practice due to the high fidelity of modern MPS platforms, especially at the 5′ end of reads. In the example experiment described below, out of a total of over 13 million reads, 84 % matched exactly with a pri- mer sequence. UNDR ROVER compares the expected insert sequence from the reference genome to the part of the read fol- lowing the GSP. In the common case we expect the se- quences to be identical or only have one base mismatch. Therefore, as an optimisation, UNDR ROVER first performs a linear comparison of the two. Implementation If more than one mismatch is detected by the linear comparison, then a gapped-alignment of the two sequences is per- formed using the Needleman-Wunsch algorithm as implemented in the pairwise2 module of BioPython [13]. In the majority of cases the linear comparison is sufficient, thus avoiding the significantly greater cost of gapped alignment most of the time. Additional speed can be achieved (with the possibility of slightly increasing the error rate) optionally by allowing more than one mismatch to occur in the linear comparison if each mismatch is separated by less than or equal to UNDR ROVER uses the same variant calling algo- rithm as its predecessor, ROVER, which requires that both reads in a pair overlap their associated tile by at least a specified percentage (by default 90% of the tile must be overlapped by each read). Reads which do not Park et al. BMC Bioinformatics (2016) 17:165 Page 4 of 7 Page 4 of 7 sample separately. For each sample, all reads in each FASTQ file are associated with their corresponding tile (lines 6 to 12), then variants are called for each tile (lines 13 to 28). Each read is associated with its tile by hashing the first N bases of the sequence and looking up the result in the tile map. Each read in a pair is compared to the corresponding reference sequence and differences between the two are computed (lines 19 and 20) using the approach described earlier. Dif- ferences which appear on both read pairs are retained (line 21). Frequencies for each variant are computed and the filters described above are applied. Each SNV is genotyped by computing a pileup of bases at the position of the variant. The most likely genotype is computed by comparing the expected distribution of DNA bases for a given pileup coverage size to the ac- tual distribution of bases. The expected distribution is computed from the ploidy of the putative genotype and a very simple error model, which assumes a constant read error rate which defaults to 1/500, but can be overridden as a command line parameter. By default, UNDR ROVER assumes a diploid genotype model, but this can be overridden to a haploid model via a com- mand line argument. The distance between the ex- pected and actual base frequency distributions are computed using a statistical G-Test which is based on a log-likelihood ratio. Implementation The genotype with the smallest dis- tance to the observed data is taken to be best explan- ation for the observed data. Genotyping can add extra time to variant calling and is therefore only performed when an optional command line argument is set. a specified number of bases. We use the term thor- ough when no more than one mismatch in the linear comparison is permitted, and the term fast for the more lenient option. As with its predecessor, UNDR ROVER only calls variants which appear in both reads of a pair such that 1) the frequency of the variant-pair is above a mini- mum absolute value, 2) the variant-pair occurs above a minimum percentage of all read-pairs overlapping the amplicon, and 3) each read of a pair overlaps the target amplicon by a user-defined minimum percent- age. All of these conditions can be adjusted by com- mand line arguments. Optionally, UNDR ROVER can filter out any bases which do not meet a minimum base-quality score. y We illustrate UNDR ROVER’s algorithm with the pseudo-code in Fig. 2. The algorithm is realised by the GET_VARIANTS procedure which takes four parame- ters: 1) a sequence of paired-end FASTQ files, one pair for each input sample; 2) the list of tile coordinates as- sociated with primer names; 3) a list of primer names associated with corresponding DNA sequences; 4) a reference genome sequence. The output is a VCF file containing variants and associated metadata such as frequency count, genotype and whether they passed various filtering tests. A hash table mapping primers to their correspond- ing tiles is intialised (line 3). The keys of the hash table are length N prefixes of the primer DNA se- quences. The values associated with each key contain the genomic coordinates of the tile plus the reference sequence at the same location. Primer pairs are con- nected by having the entry for the reverse primer point back to the entry for its forward partner. Each pair of FASTQ files is processed in sequence (lines 5 to 29); UNDR ROVER calls variants in each input Results We have demonstrated the performance of UNDR ROVER by comparing it to its predecessor ROVER using a previously published dataset [3]. Hi-Plex was Fig. 2 Pseudo code for variant calling algorithm employed by UNDR ROVER Park et al. BMC Bioinformatics (2016) 17:165 Page 5 of 7 Page 5 of 7 used to screen 95 blood-derived DNA samples targeting the protein coding and some flanking intronic and un- translated regions of PALB2 and XRCC2 using 60 primer-pairs in the PCR. The resulting library was se- quenced on a MiSeq instrument (Illumina) producing 95 pairs of FASTQ files (190 files in total) with an aver- age file size of 23 MiB. Previous application of ROVER to this dataset (aligned to the entire human genome (hg19) using bowtie2-2.1.0) accurately detected all 60 variant occurrences identified through mutation screen- ing and assigned no false positive calls. Application of UNDR ROVER to the same data set yielded the same set of called variants in both thorough and fast modes. Future experiments will seek to validate UNDR ROVER using additional data sets that, similar to the data set used in this study, have been extensively characterised by Sanger sequencing. used to screen 95 blood-derived DNA samples targeting the protein coding and some flanking intronic and un- translated regions of PALB2 and XRCC2 using 60 primer-pairs in the PCR. The resulting library was se- quenced on a MiSeq instrument (Illumina) producing 95 pairs of FASTQ files (190 files in total) with an aver- age file size of 23 MiB. Previous application of ROVER to this dataset (aligned to the entire human genome (hg19) using bowtie2-2.1.0) accurately detected all 60 variant occurrences identified through mutation screen- ing and assigned no false positive calls. Application of UNDR ROVER to the same data set yielded the same set of called variants in both thorough and fast modes. Future experiments will seek to validate UNDR ROVER using additional data sets that, similar to the data set used in this study, have been extensively characterised by Sanger sequencing. genotyping. Approximately 78 % of the ROVER pipeline time is constituted by read alignment with Bowtie. This highlights the significant performance gains possible by avoiding the alignment step. Discussion Discussion The following command line illustrates a typical invoca- tion of UNDR ROVER: We also applied the GATK HaplotypeCaller (version 3.4-46) [14] variant calling software to the same sam- ples, after aligning the FASTQ files to the reference with Bowtie. We instructed GATK to only call variants in the targeted regions. GATK called the same variants as ROVER and UNDR ROVER plus two additional calls which appear to be false positives caused by sequencing artefacts. Both ROVER and UNDR ROVER were able to filter out these false positives because the artefacts did not appear on both reads in the affected read pairs. The GATK command used to call variants is shown below: where the input files coords.tsv and seqs.txt provide the primer coordinates and DNA sequences, respect- ively, hg19.fa is the reference DNA sequence and sam- ple1_r1.fastq and sample_r2.fastq contain the input DNA reads. The output variant calls are written to the file results.vcf. Below is a short example primer coordinates file illus- trating two pairs of primers: Results In summary, for this indi- cative experiment, we see that UNDR ROVER is able to achieve between a 2-fold and 8-fold performance im- provement compared to its predecessor, and up to 13- fold improvement over a pipeline based on GATK, whilst producing the same set of variant calls. Conclusions UNDR ROVER provides a computationally more effi- cient alternative to ROVER and other standard variant calling pipelines for the detection of genetic variants from Hi-Plex-generated datasets while maintaining a high level of accuracy. Acknowledgements This work was supported by the Australian National Health and Medical Research Council (NHMRC) (APP1025879 and APP1029974), the National Institute of Health, USA (RO1CA155767), Cancer Council Victoria (APP1066612) and by a Victorian Life Sciences Computation Initiative (VLSCI) grant (number VR0182), on its Peak Computing Facility, an initiative of the Victorian Government. TN-D is a Susan G. Komen for the Cure Postdoctoral Fellow. RL was supported by the Undergraduate Research Opportunities Program (UROP), VLSCI and the Department of Pathology at The University of Melbourne. The authors would like to thank Benjamin Rubinstein from The University of Melbourne for his input into genotyping algorithms. All participants provided written informed consent for participation in the study. This study was approved by The University of Melbourne Human Research Ethics Committee. Author details 1 Other requirements: PyVCF, Pyfaidx, BioPython and SciPy libraries 1Genetic Epidemiology Laboratory, School of Biomedical Sciences, Medical Building, The University of Melbourne, Melbourne, Victoria 3010, Australia. 2Department of Computing and Information Systems, The University of Melbourne, Melbourne, Victoria 3010, Australia. 3Victorian Life Sciences Computation Initiative, The University of Melbourne, Melbourne, Victoria 3053, Australia. Programming language: Python Programming language: Python Total sequential computing time of the GATK pipeline, ROVER and UNDR ROVER (thorough, genotyping and fast) when applied to 95 Hi-Plex samples targeting PALB2 and XRCC2 with 60 primer-pairs in the PCR. The computing time for the GATK and ROVER pipelines are decomposed into alignment with Bowtie (blue), conversion of alignment file from SAM to BAM format (yellow), indexing and sorting of BAM file (grey), and variant calling (light red for GATK and green for ROVER). Computing times for UNDR ROVER are shown for both the thorough mode (brown) and the fast mode with SNV genotyping (orange), and the fast mode without SNV genotyping (purple) Fig. 3 Runtime comparison of GATK, ROVER and UNDR ROVER. Total sequential computing time of the GATK pipeline, ROVER and UNDR ROVER (thorough, genotyping and fast) when applied to 95 Hi-Plex samples targeting PALB2 and XRCC2 with 60 primer-pairs in the PCR. The computing time for the GATK and ROVER pipelines are decomposed into alignment with Bowtie (blue), conversion of alignment file from SAM to BAM format (yellow), indexing and sorting of BAM file (grey), and variant calling (light red for GATK and green for ROVER). Computing times for UNDR ROVER are shown for both the thorough mode (brown) and the fast mode with SNV genotyping (orange), and the fast mode without SNV genotyping (purple) RL, EL, PG and TN-D contributed to the writing of the manuscript. All authors read and approved the final manuscript. 1. Nguyen-Dumont T, Hammet F, Mahmoodi M, Pope BJ, Giles GG, Hopper GG, Southey MC, Park DJ. Abridged adapter primers increase the target scope of Hi-Plex. Biotechniques. 2014;58(1):33–6. The first column indicates the chromosome of the tar- geted sequence. The second and third columns indicate the start and end coordinates of the target tile. The fourth and fifth columns indicate the unique symbolic names of the forward and reverse primers for a target tile. Below is a short example primer sequences file, with entries corresponding to the gene-specific portions of primers from the example coordinates file above: Figure 3 compares the total sequential computing time for UNDR ROVER against the GATK and ROVER pipelines (including read alignment with Bow- tie) when applied to the entire set of 95 samples. We applied UNDR ROVER in three different modes to show the performance implications of different settings. The performance tests were executed on a single node of an IBM iDataPlex cluster with 16 core 2.7GHz CPUs and 256GB of RAM using the Red Hat Enterprise Linux ver- sion 6 operating system. Total sequential computation times for computing variants in all 95 Hi-Plex input samples were 9535 s for the GATK-based pipeline, 5721 s for the ROVER-based pipeline, 2480 s for UNDR ROVER in thorough mode without genotyping, 736 for UNDR ROVER in fast mode with genotyping, and 690 s for UNDR ROVER in fast mode without The first column indicates the unique symbolic name of the primer and the second column indicates the pri- mer DNA sequence written in the 5′ to 3′ direction. Pri- mer coordinates are matched with primer sequences via their unique symbolic names. Park et al. BMC Bioinformatics (2016) 17:165 Page 6 of 7 Fig. 3 Runtime comparison of GATK, ROVER and UNDR ROVER. Total sequential computing time of the GATK pipeline, ROVER and UNDR ROVER (thorough, genotyping and fast) when applied to 95 Hi-Plex samples targeting PALB2 and XRCC2 with 60 primer-pairs in the PCR. The computing time for the GATK and ROVER pipelines are decomposed into alignment with Bowtie (blue), conversion of alignment file from SAM to BAM format (yellow), indexing and sorting of BAM file (grey), and variant calling (light red for GATK and green for ROVER). Computing times for UNDR ROVER are shown for both the thorough mode (brown) and the fast mode with SNV genotyping (orange), and the fast mode without SNV genotyping (purple) Fig. 3 Runtime comparison of GATK, ROVER and UNDR ROVER. Project name: UNDR ROVER Project name: UNDR ROVER Project name: UNDR ROVER Project home page: https://github.com/bjpop/undr_rover Usage instructions: http://bjpop.github.io/undr_rover Operating systems: POSIX-like operating systems (OS X, Linux) Operating systems: POSIX-like operating systems (OS X, Linux) Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Abbreviations GSP: gene specific primer; MPS: massively parallel sequencing; SNV: single nucleotide variant; VCF: variant call format. Received: 31 July 2015 Accepted: 6 April 2016 References N BJP, DJP and RL designed the overall algorithm. BJP, EL and PG designed and implemented the genotyping algorithm. BJP and RL carried out the implementation. RL and TN-D conducted testing of the software. BJP, DJP, 1. Nguyen-Dumont T, Hammet F, Mahmoodi M, Pope BJ, Giles GG, Hopper GG, Southey MC, Park DJ. Abridged adapter primers increase the target scope of Hi-Plex. Biotechniques. 2014;58(1):33–6. Page 7 of 7 Park et al. BMC Bioinformatics (2016) 17:165 14. GATK [https://www.broadinstitute.org/gatk/]. Accessed 14 Apr 2016. Park et al. BMC Bioinformatics (2016) 17:165 Park et al. BMC Bioinformatics (2016) 17:165 2. Pope BJ, Nguyen-Dumont T, Hammet F, Park DJ. ROVER variant caller: read- pair overlap considerate variant-calling software applied to PCR-based massively parallel sequencing datasets. Source Code Biol Med. 2014;9(1):3. 3. Nguyen-Dumont T, Teo ZL, Pope BJ, Hammet F, Mahmoodi M, Tsimiklis H, Sabbaghian N, Tischkowitz M, Foulkes WD, Giles GG, et al. Hi-Plex for high- throughput mutation screening: application to the breast cancer susceptibility gene PALB2. BMC Med Genet. 2013;6(1):48. susceptibility gene PALB2. BMC Med Genet. 2013;6(1):48. 4. Nguyen-Dumont T, Hammet F, Mahmoodi M, Tsimiklis H, Teo ZL, Li R, Pope BJ, Terry MB, Buys SS, Daly M, et al. Mutation screening of PALB2 in clinically ascertained families from the Breast Cancer Family Registry. Breast Cancer Res Treat. 2015;149(2):547–54. 5. Sequence Alignment/Map Format Specification, Version 1. [http://samtools. github.io/hts-specs/SAMv1.pdf]. Accessed 14 Apr 2016. 6. The Variant Call Format (VCF) Version 4.2 Specification. 6. The Variant Call Format (VCF) Version 4.2 Specification. [h github.io/hts-specs/VCFv4.2.pdf]. Accessed 14 Apr 2016. github.io/hts-specs/VCFv4.2.pdf]. Accessed 14 Apr 2016. 7. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3):R25. 8. Hsu AL, Kondrashova O, Lunke S, Love CJ, Meldrum C, Marquis-Nicholson R, Corboy G, Pham K, Wakefield M, Waring PM, et al. AmpliVar: mutation detection in high-throughput sequence from amplicon-based libraries. Hum Mutat. 2015;36(4):411–8. 9. Needleman SB, Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970; 48(3):443–53. 10. Kent WJ. BLAT–the BLAST-like alignment tool. Genome Res. 2002 10. Kent WJ. BLAT–the BLAST-like alignment tool. Genome Res. 2002;12(4):656–64. 11. SeqPrep [https://github.com/jstjohn/SeqPrep]. Accessed 14 Apr 2016. 11. SeqPrep [https://github.com/jstjohn/SeqPrep]. Accessed 14 Apr 2016. SeqPrep [https://github.com/jstjohn/SeqPrep]. Accessed 14 Apr 2016 12. Nguyen-Dumont T, Pope BJ, Hammet F, Mahmoodi M, Tsimiklis H, Southey MC, Park DJ. Cross-platform compatibility of Hi-Plex, a streamlined approach for targeted massively parallel sequencing. Anal Biochem. 2013;442(2):127–9. 13. Cock PJ, Antao T, Chang JT, Chapman BA, Cox CJ, Dalke A, Friedberg I, Hamelryck T, Kauff F, Wilczynski B, et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009;25(11):1422–3. 14. GATK [https://www.broadinstitute.org/gatk/]. Accessed 14 Apr 2016. Park et al. 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https://openalex.org/W3044520325	https://www.nature.com/articles/s41598-020-69159-9.pdf	English	null	Transport pathways across the West African Monsoon as revealed by Lagrangian Coherent Structures	Scientific reports	2,020	cc-by	10,910	Transport pathways across the West African Monsoon as revealed by Lagrangian Coherent Structures Coumba Niang1,2, Ana Maria Mancho1, Víctor José García‑Garrido3, Elsa Mohino4, Belén Rodriguez‑Fonseca4 & Jezabel Curbelo1,5 The West African Monsoon (WAM) system is the main source of rainfall in the agriculturally based region of the Sahel. Understanding transport across the WAM is of crucial importance due to the strong impact of humidity and dust pathways on local cloud formation. However, the description of this transport is challenging due to its 3D complex nature. Lagrangian Coherent Structures (LCS) simplify transport description across the WAM by providing a geometrical partition of the troposphere into domains. Air parcels within each domain have similar dynamical characteristics. LCS make it possible to achieve an integrated vision of transport pathways across this system. Using this approach we unveil new connections in the WAM system. In particular, we identify transport pathways between the Tropical Easterly Jet (TEJ) and the African Easterly Jet (AEJ). Furthermore, the clockwise circulation associated with the divergent upper part of the Sahara heat low is clearly delimitated. Additionally, we show the presence of mixing regions in the AEJ and the lower part of the TEJ that are linked to pathways to sources of dust and humidity. The West African climate has been recognized as one of the hotspots in the Earth’s climate system1, 2. It is domi- nated by the West African Monsoon (WAM) system, one of the most complex monsoon systems on Earth in which land, ocean and atmosphere are highly coupled. The WAM is crucial for the population of the region. Its variability has a substantial impact on agriculture, livestock farming, water and food resources all of which strongly depend on rainfall, especially in the Sahel. Over this region, accumulated daily rainfall amounts vary from 2 to 16 mm per day during the rainy season3, 4. However, rainfall over the region shows variability at a wide range of time scales: from the decadal droughts during the 1970s and 1980s—which led to widespread famine—to intraseasonal fluctuations5–8. A comprehensive investigation of the WAM features is of prime importance for understanding and predicting the variability at those timescales as well as for the development of the fragile West African economy7. y From an energetic point of view, monsoons are a manifestation of the ITCZ migration over tropical land regions. ITCZ is a facet of the direct overturning circulation exporting energy away from the tropics. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Transport pathways across the West African Monsoon as revealed by Lagrangian Coherent Structures (a) Climatology of 2 m temperature (shaded, in °C) and mean sea level pressure (contour, in hPa) of ERA-Interim averaged for the month of August in the 1979–2015 period. Velocities at 10° W in m/s. (b) Vertical velocity component: positive (negative) values are updraft (downdraft) winds; (c) Zonal velocities: positive (negative) values are westerly (easterly) wind; (d) Meridional velocities: positive (negative) values indicate southerly (northerly) wind. All these data are averaged for the month of August over the 1979–2015 period. The rainbelt band located between 5° N and 15°N is represented in plot (b). In addition, the approximative location of the Tropical Easterly Jet (TEJ), the African Easterly Jet (AEJ) and the monsoon flow are labeled in plot (c) while the southerly Shallow Meridional Cell (SMC) and the InterTropical Discontinuity (ITD) are shown in plot (d). Map in panel (a) is done with Grid Analysis and Display System (GrADS) Version 2.2.0. The URL is http:// cola.gmu.edu/grads/. which, in turn, are key for transporting moisture and energy across the monsoon. From an Eulerian perspective, the average behaviour of such flows can be estimated as the climatology by computing the means of averages of daily flows over long periods. Fig. 1 shows such averaged flows for the month of August in the 1975–2015 time series. This month is considered as the peak of the monsoon over the Sahelian region4. The southerly part of the monsoon flow is visible below 1.5 km up to the Guinean coast at 5° N, where it converges to form a southerly Shallow Meridional Cell (Fig. 1d)13, which could be related to a shallow breeze circulation cell and to frictional deceleration of the flow as it reaches the coast4, 14.h l The rainfall belt develops from 5° N and 15° N, with strong upward motions involving deep convection through the whole tropospheric column (Fig. 1b). Driven by geostrophic adjustment, the African Easterly Jet (AEJ) appears at mid-tropospheric levels (600 hPa, 4 km approximately) (Fig. 1c) and the Tropical Easterly Jet (TEJ) develops at the top of the troposphere (between 250 and 150 hPa, 11 km approximately)15–17. The AEJ is maintained by two diabatically forced circulations: dry convection to the north and moist convection to the south. Transport pathways across the West African Monsoon as revealed by Lagrangian Coherent Structures The seasonal variation of the solar insolation drives the migration of the ITCZ towards the warmer hemisphere, so that the surplus of integrated moist static energy is exported across the Equator9. This simple picture is complicated in the case of monsoons by the presence of land masses, the different heat capacity of which makes the seasonal migration of the monsoon lag behind the sun’s position and also induce zonal asymmetries10. In the case of West Africa, further factors need to be considered: the development of the Atlantic cold tongue close to the Equator during boreal spring and summer, or the presence of the Saharan Heat low (see Fig. 1a) that limits the northward extension of the monsoon4, 11, 12. These factors play a role in shaping the different flows involved in the WAM, 1Instituto de Ciencias Matemáticas, Consejo Superior de Investigaciones Científicas (CSIC), C/ Nicolás Cabrera 15, Campus de Cantoblanco, 28049 Madrid, Spain. 2Laboratoire de Physique de l’Atmosphére et de l’Océan Simón Fongang (LPAO‑SF), Ecole Supérieure Polytechnique (ESP), Université Cheikh Anta Diop, BP 5085, Dakar‑Fann, Senegal. 3Departamento de Física y Matemáticas, Universidad de Alcalá, 28871 Alcalá de Henares, Spain. 4Departamento de Fisica de la Tierra y Astrofisica, Universidad Complutense de Madrid (UCM), Madrid, Spain. 5Departament de Matemàtiques, Universitat Politècnica de Catalunya (UPC), 08028 Barcelona, Spain. email: a.m.mancho@icmat.es Scientific Reports \| (2020) 10:12543 \| https://doi.org/10.1038/s41598-020-69159-9 www.nature.com/scientificreports/ Figure 1. (a) Climatology of 2 m temperature (shaded, in °C) and mean sea level pressure (contour, in hPa) of ERA-Interim averaged for the month of August in the 1979–2015 period. Velocities at 10° W in m/s. (b) Vertical velocity component: positive (negative) values are updraft (downdraft) winds; (c) Zonal velocities: positive (negative) values are westerly (easterly) wind; (d) Meridional velocities: positive (negative) values indicate southerly (northerly) wind. All these data are averaged for the month of August over the 1979–2015 period. The rainbelt band located between 5° N and 15°N is represented in plot (b). In addition, the approximative location of the Tropical Easterly Jet (TEJ), the African Easterly Jet (AEJ) and the monsoon flow are labeled in plot (c) while the southerly Shallow Meridional Cell (SMC) and the InterTropical Discontinuity (ITD) are shown in plot (d). Map in panel (a) is done with Grid Analysis and Display System (GrADS) Version 2.2.0. The URL is http:// cola.gmu.edu/grads/. Figure 1. Transport pathways across the West African Monsoon as revealed by Lagrangian Coherent Structures In the mid-troposphere, the anticyclonic circulation associated with the diverging flow at the top of the Sahara heat Low (SHL) contributes to the maintenance of the African Easterly Jet (AEJ) and modulates its intensity18.h The ITCZ is a convergence zone between humid air coming from the ocean and dry air coming from the Sahara. The demarcation line over land is called the intertropical discontinuity (ITD), shown in Fig. 1d. Above it, another shallow meridional circulation develops related to the SHL. Its lower southerly branch transports moisture across the Sahel, while its upper northerly branch transports dry and warm air, limiting the northward Scientific Reports \| (2020) 10:12543 \| https://doi.org/10.1038/s41598-020-69159-9 www.nature.com/scientificreports/ Figure 2. Particle trajectories of the WAM system. (a) A set of 54 particle trajectories that evolve forwards and backwards in time for a period of τ = 15 days from a regular grid in the plane with constant longitude 10° W; (b) five clusters of 10 particles each, that evolve forwards and backwards in time for a period of τ = 15 days from regions identified with Lagrangian Coherent Structures on the plane with constant longitude 10° W. Maps in this figure are done with MATLAB R2018b. Figure 2. Particle trajectories of the WAM system. (a) A set of 54 particle trajectories that evolve forwards and backwards in time for a period of τ = 15 days from a regular grid in the plane with constant longitude 10° W; Figure 2. Particle trajectories of the WAM system. (a) A set of 54 particle trajectories that evolve forwards and backwards in time for a period of τ = 15 days from a regular grid in the plane with constant longitude 10° W; (b) five clusters of 10 particles each, that evolve forwards and backwards in time for a period of τ = 15 days from regions identified with Lagrangian Coherent Structures on the plane with constant longitude 10° W. Maps in this figure are done with MATLAB R2018b. extent of the monsoon12, 19. Understanding transport across the monsoon features described below is the main aim of this document.i From the averaged velocity fields, it is possible to roughly identify transport paths of air masses. Nevertheless, the results differ if Eulerian and Lagrangian perspectives are taken into account. Transport pathways across the West African Monsoon as revealed by Lagrangian Coherent Structures For instance, low level monsoon winds into the Guinean coast are expected to bring humidity and moisture into the continent from the Atlantic Ocean, which is a necessary condition for rain to occur inland. The dry air from the Sahara can potentially transport dust, which in turn can act as a catalyst for water vapour condensation and cloud formation20, thereby also contributing to rain formation. Many authors have used different methodologies, mostly based on Eulerian analysis, to identify the sources of moisture over West Africa. Most of these studies investigated the sources of water vapour fluxes into West Africa using different sets of data4,21–24. The Eulerian approaches are used to esti- mate the ratio of advected to recycled moisture and to calculate moisture transport from predetermined source regions, although they are unable to identify the moisture source regions directly. The analysis of transport based on Eulerian features, like those displayed in Fig. 1, do not involve real trajectories of atmospheric particles, there- fore deep insights into transport processes that occur in the prototypical WAM are lacking. Typically, approaches based on trajectories (Lagrangian approaches) that study air masses and moisture transport are performed on daily based flows25–34, in which many of the characteristic Eulerian WAM features just described are absent, since they are only visible in the climatological approach. This is the case for instance of the ITCZ pattern, although other features such as the TEJ are persistent in the daily data. In this way, by averaging over long periods, clima- tologists find simple models that highlight the essential flow elements of WAM. Lagrangian methods applied to daily data have been an effective diagnostic tool to identify the sources of air masses over a target region35. The tracking of particles and their moisture budgets in daily settings suggests that much of moisture comes from local recycling36, although the computation of such moisture budget shows some limitations regarding the trajectory accuracy and the time derivative of the humidity used, since unrealistic fluctuations in humidity could be con- sidered as moisture fluxes leading to systematic errors in the tracked humidity budgets. Trajectory analysis has also been used to examine meteorological phenomena like tropical moisture export37–39. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Many studies of LCS in geophysical contexts have been performed in two-dimensional (2D) settings. For instance, stratospheric flows on the timescale of 10 days are to an initial approximation, adiabatic and frictionless, and thus fluid particles and their trajectories are constrained to remain on surfaces of constant specific potential temperature (isentropic surfaces)46–48. A study on the 2D–3D particle motion transition which is observed when passing from the stratosphere to the troposphere is discussed in49.hl p g p p p The study of transport processes in 3D flows brings into the discussion issues about the 3D visualisation of Lagrangian structures (see e.g.50–53). The methodology used in this study focuses on a Lagrangian method based on the Lagrangian descriptor (LD) known as the M function54–56. This function has been used to visualise the three-dimensional Lagrangian structures in idealised 3D flows51,56,57 and also in atmospheric flows, in the stratospheric polar vortex above Antarctica49,52,53. More recently in the context of the Transition State Theory in Chemistry, LDs have been successfully used to picture phase space structures in high dimensional dynamical systems58–60. y In this context, this paper exploits the Lagrangian technique based on the function M, to describe transport across the summer dynamical features of the West African monsoon. The methodology achieves a partition of the troposphere into regions, containing particles with different origins or fates. This analysis enables the re- examination of all the monsoon elements and their interconnections from a transport perspective by describing how air masses are mutually exchanged and how these exchanges may be linked to rain formation. The paper is organised as follows. First, in section “Data and methodology”, we present the data and the methodology. Afterwards in section “Results”, we discuss the results. Finally, section “Discussion and conclusions” presents the conclusions. Transport pathways across the West African Monsoon as revealed by Lagrangian Coherent Structures When applied to West Africa, this type of Lagrangian approaches have shown that a large proportion of tropospheric air masses located over India between 500 and 300 hPa end up over West Africa, after following a direct path through the TEJ40. h h l f h b h h f pt g p g In this context, the major goal of this paper is to obtain a comprehensive characterisation of Lagrangian trans- port across prototypical WAM features, visible only on averaged velocity fields, with the purpose of identifying the role of each WAM element on global transport, with regard not only to moisture transport, but also to dust or aerosols and to discuss how they may influence rain formation41.hll y yl The study of Lagrangian transport in 3D flows is a complicated task because even in well controlled flows, such as those in lab experiments, it has been demonstrated that fluid parcels follow very intricate paths42. The WAM system described in Fig. 1b–d is a 3D flow that also gives rise to genuine entangled flow paths such as those displayed in Fig. 2a. For the study of transport in geophysical flows, the mathematical theory of dynami- cal systems has played an important role. The pioneering contribution by Aref43 on chaotic advection sparked interest in this perspective, inspired by Poincaré’s work. This perspective is based on geometrical structures that separate regions corresponding to trajectories having qualitatively different dynamical fates. In the fluid mechanics community, these geometrical structures have been referred to as Lagrangian Coherent Structures (LCS)44,45, and act as material barriers that fluid particles cannot cross. Figure 2b illustrates these ideas. For the same WAM flow that displays the complex structure in panel (a), LCS help to identify regions, in which clusters of particles behave similarly, helping to extract order out of the apparent disorder of panel (a). The fact that there exist regions in this panel where green, blue and red parcels are mixed up, indicate that the separating boundaries between them, adopt intricate shapes. Scientific Reports \| (2020) 10:12543 \| https://doi.org/10.1038/s41598-020-69159-9 Data and methodology gy Data. In this study, the ERA-Interim meteorological analysis data from the European Centre for Medium- Range Weather Forecasts (ECMWF)61 available at http://www.ecmwf.int is used. In particular, we extract from this source zonal (u) and meridional (v) wind velocity components, vertical velocity in pressure coordinates ( ω = dP/dt ), temperature, specific humidity, potential vorticity, geopotential height and surface pressure. These physical variables are available every six hours (00:00, 06:00, 12:00, 18:00 UTC) and are obtained for the month of August in the 1979 to 2015 period62. Our focus is on the month of August, because this is the peak of the Monsoon season. The data is daily averaged over this period.hh h y g p The horizontal data resolution is 0.75° × 0.75° and 60 hybrid-sigma levels along the vertical coordinate. The vertical velocity in pressure coordinates, ω , is transformed into a vertical velocity w in m/s, following the pro- cedure described in49. Once the 3-D velocities (u, v, w) are obtained for sigma levels, they are interpolated to 41 height levels ranging from 0 to 20,000 m at 500 m intervals. The Supplementary Information provides the three components of the velocities used in this study, in three NetCDF files. Methodology. We consider atmospheric particle trajectories in three dimensions, x(t) , which evolve according to the dynamical system: (1) dx dt = v(t, x), dx dt = v(t, x), (1) where v(t, x) is the velocity field related to the atmospheric winds displayed in Fig. 1. The specific relationship between this velocity and the wind components is found for instance in Eq. (6) in49. The use of the velocities displayed in Figure 1, which are obtained from time averages, transforms the study of transport described by Eq. (1), into the study of transport in a stationary system, representative of the monsoon circulation, in which v(t, x) = v(x) . The study of this system allows a detailed approach to the study of transport induced by the climatological fields described in the “Introduction” section. where v(t, x) is the velocity field related to the atmospheric winds displayed in Fig. 1. The specific relationship between this velocity and the wind components is found for instance in Eq. (6) in49. The use of the velocities displayed in Figure 1, which are obtained from time averages, transforms the study of transport described by Eq. Data and methodology (1), into the study of transport in a stationary system, representative of the monsoon circulation, in which v(t, x) = v(x) . The study of this system allows a detailed approach to the study of transport induced by the climatological fields described in the “Introduction” section. gi To understand transport processes occurring in Eq. (1) from a geometrical point of view, LCS are visualized with the M function. This function is built from forward and backward trajectories computed from Eq. (1). Mathematically it is given by the expression: (2) M(x0, t0, τ) = t0+τ t0−τ v(x(t; x0), t) dt , (2) where · denotes the Euclidean norm, and t0 and x0 are respectively the initial time and position of the fluid parcel, which is integrated forwards and backward in time for a period τ . The M function is obtained by approxi- mating the integral in Eq. (2) by the sum of the lengths (in the Euclidean space) of the segments linking the position of the integrated particle trajectory at two successive time steps. The numerical implementation of Eqs. (1) and (2) in this article exactly follows the procedure described by Curbelo et al.49. In this work, since v is a stationary field, the function M is also time-independent, which simplifies the analysis of the 3D geometrical structures obtained from it.h The dependence of the function M on τ deserves further discussion. Figure 3 illustrates this dependence by means of a representation of M in the latitude-altitude plane used in Fig. 1c at 10° W. For a small τ value of 2 days, Fig. 3a shows that the appearance of M is smooth and closely follows the contours of the zonal velocity, which are overlapped. Figure 3b displays M for a longer τ period of 15 days. The emergence of singular features is visible. These sharp changes, highlighted in the Figure with the red tone, mark boundaries between regions in which particles have different qualitatively behaviours, and are related to material barriers that particles do not cross. A heuristic argument on why material barriers should be traced out by singular features of M is found in55. The function M measures the lengths of curves traced by trajectories, so it is expected to change abruptly Scientific Reports \| (2020) 10:12543 \| https://doi.org/10.1038/s41598-020-69159-9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Data and methodology Evaluation of the M function at the latitude-altitude plane placed at 10° W using different integration periods. (a) τ = 2 days. Contours of the zonal velocity are overlapped; (b) τ = 15 days. The overlapped red tone highlights the singular features that emerge at higher τ values. Figure 3. Evaluation of the M function at the latitude-altitude plane placed at 10° W using different integration periods. (a) τ = 2 days. Contours of the zonal velocity are overlapped; (b) τ = 15 days. The overlapped red tone highlights the singular features that emerge at higher τ values. at the boundaries of regions comprising trajectories with qualitatively different evolutions, since this is exactly what the barriers separate. Mancho et al.56 provide further details about this. Patterns in Fig. 3b are used to select the clusters of particles displayed in Fig. 2b. They are the LCS that help to extract order structures by identifying regions with an homogeneous transport behaviour. In practice, the integration period necessary to display the required LCS depends on the characteristics of each velocity field. The function M reflects the transport history of fluid parcels, and in highly chaotic systems, this history is expected to be increasingly complex for longer τ intervals, which in turn will be reflected in a more complex structure of the function M. We have verified that τ = 15 days is a sufficient choice for our data, and from the physical point of view this is consistent with the time of 30 days used by atmospheric fluid parcels to travel during the month of monsoon peak across the region. Further discussions on the effect of the choice of τ on the structure of M may be found in55,56. Th h f h l h fi d b d l h l f y The choice of this Lagrangian tool versus others is justified because, despite its simple physical interpreta- tion, its potential for highlighting diverse types of dynamical structures has been rigorously proven in selected examples57,63,64. In particular, the “singular features” just discussed are related to dynamical structures linked to highly contracting or expanding regions (connected to mathematical objects called hyperbolic trajectories), while regions with smooth patterns are linked to non-dispersive regions (connected to mathematical objects called tori) that hold matter together. This two-fold capacity has been exploited in the stratospheric polar vortex context in52,53. Data and methodology In contrast, other approaches based on tools such as Finite Time Lyapunov Exponents (FTLE), also used in 3D stratospheric studies65, highlight only structures related to highly contracting or expanding regions.h p g g y g y g p g g The M function contains fully 3D Lagrangian structures, and Fig. 3b just captures the intersection of these structures with the vertical slice. Alternatively, in the fluid mechanics context, considerable efforts have been made in representations of 3D LCS, to fully compute them as 2D surfaces embedded in 3D spaces (see for instance65,66). This way of representing LCS, which is technically complicated, could also have been adopted in our approach. However, this has not been considered because it would have provided not new transport information to what has already been obtained from our simplest perspective, which consists solely of visualising slices of M with different orientations in the geographical domain. On the other hand, our viewpoint has been conjointly adopted in chemical contexts to visualise dynamical structures in high dimensional systems58–60, where it also has been proven to be effective. Results d Contour lines of the zonal velocity are overlapped; (b) backwards and forwards time evolution of the fluid parcels placed in different domains of the AEJ (red, blue, green) and Gulf of Guinea convective cell (cyan) and the Monsoon flow (orange). Time arrows marking the time direction are placed over the trajectories. A blue line marks the horizontal plane at 0.2 km. Maps in this figure are done with MATLAB R2018b. Figure 4. (a) A partition of the AEJ by means of the function M (LD) which indicates different particle origins. Contour lines of the zonal velocity are overlapped; (b) backwards and forwards time evolution of the fluid parcels placed in different domains of the AEJ (red, blue, green) and Gulf of Guinea convective cell (cyan) and the Monsoon flow (orange). Time arrows marking the time direction are placed over the trajectories. A blue line marks the horizontal plane at 0.2 km. Maps in this figure are done with MATLAB R2018b. Rainfall variability is related to the intensity and position of the AEJ67–69. The findings shown in Fig. 4a and b indicate that moisture can be brought into the AEJ from the Gulf of Guinea by red-like trajectories. Of course, humidity transportation also requires evaporation at the ocean surface, which is where temperature plays also a role. Additionally, dust coming from inland lower levels can be transported by blue-like trajectories into the AEJ. Both ingredients can contribute to cloud formation and our analysis confirms this capacity. The area of the partitions achieved by the different grey tones, visible in the function M in the AEJ, provides a measure of the mixing proportion of these components in this WAM feature. This proportion also has direct connections to cloud formation. Finally, the forward evolution of the trajectories confirms the transport of these elements into the Atlantic, with the subsequent impact on cloud formation on that region and potentially on hurricane formation. Indeed, African easterly waves (AEW) propagating through the AEJ are noted for being precursors to tropical cyclones in the tropical Atlantic70. Trajectory analysis and dynamical systems tools in this setting have provided a useful framework in which to describe transport of humid and dry air masses71, 72, which are known to play a role in the tropical cyclogenesis formation.h The very dark feature observed in Fig. Results d Equipped with the tool described in the previous section, we build a geometrical pattern/template on the target region which allows us to perform a comprehensive analysis of air masses sources and fates that affect the major precipitation over West Africa during the peak of the Monsoon season. Figure 4 illustrates this point in detail for the AEJ. Figure 4a overlaps the contours of the zonal velocities on the function M, which has been computed with τ = 15 days along the vertical and latitude coordinates for longitude 10° W in the same plane as Fig. 1. The broken pattern of M over the closed curves of the AEJ indicates the presence in the jet of fluid parcels with a qualitatively different origin or fate: that is, broken contours separate regions with different evolution, either in the past or in the future. In particular, for this case fluid parcels have different origins but are mixed in the AEJ layer. The convoluted forms within it indicate the way in which air masses are mixed. Indeed, three regions are recognised here: particles placed inside the dark grey zone, in upper levels (blue) that are transported into the AEJ from lower altitudes at northern African latitudes; those in lower levels (red) coming from the Gulf of Guinea, and those in the lighter grey zones (green) that come from higher altitudes and northern latitudes, have penetrated into the African continent from the Atlantic Ocean, have then bent at mid-African longitudes and are captured into the AEJ. Indeed, Fig. 4b confirms this point by showing the time evolution of these fluid particles in a longitude-latitude projection. The flight time for these fluid parcels is of 15 days forwards and backwards. For simplicity, only one green particle trajectory is shown, since the other follows a qualitatively similar path and does not add any information. These individual trajectories, displayed in Fig. 4b, are representative of the behaviour of clusters of fluid parcels. This is confirmed by Fig. 2b, in which the blue, red and green clusters are in fact representing fluid parcels in these three regions, with the same colour code. Scientific Reports \| (2020) 10:12543 \| https://doi.org/10.1038/s41598-020-69159-9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. (a) A partition of the AEJ by means of the function M (LD) which indicates different particle origins. Results d Figure 5. (a) A view of the representation of function M in the latitude-altitude plane at longitude 10° W. Figure 5. (a) A view of the representation of function M in the latitude-altitude plane at longitude 10° W. Contour lines of the zonal velocity are overlapped. The TEJ is recognized at latitude ∼ 8° N and altitude ∼ 10 km. The red rectagle highlights the region zoomed in panel (b); (b) a zoom highlighting the lower part of TEJ where the function M has a particularly rich structure; (c) backwards and forwards time evolution of the fluid parcels placed in different sectors of the TEJ; (d) backwards and forwards time evolution of the fluid parcels placed in folds displayed in panel (b) and in a selected sharp region of panel (a). Maps in this figure are done with MATLAB R2018b. a representative for all the particles placed within each domain. This is confirmed by Fig. 2b in which cyan and orange clusters are linked to the blue and green trajectories of Fig. 5c, respectively.h The red square in Fig. 5a highlights the lower part of the TEJ, where the function M has a particularly rich structure and is zoomed in panel (b). This stratified structure is linked to the foldings of an invariant manifold and indicates the presence of a strongly mixing region, where air masses have different sources. Indeed, the two very close particles (yellow and cyan) located there have opposite origins in the lower part of the troposphere, as confirmed by the longitude–latitude–altitude projection in Fig. 5d, one from southern Africa and the other from the north. Although it is not displayed, it occurs analogously with many other air parcels located in that area; there is no any uniformity in their origins, which are very diverse. Finally, the magenta and orange particles displayed in Fig. 5a are located over a sharp feature in the gray scale, which highlight a boundary for the AEJ. Indeed, the magenta and orange particles are located into a transport pathway from the TEJ to the AEJ. The time evolution of these trajectories displayed in Fig. 5d, confirms that these particles travelled there from the TEJ after circling around the dark feature highlighted with a white arrow in Fig. 5a, and that they evolve into the AEJ. Results d 4a between approximately latitudes 2.5° N and 7.5° N, marks the south- erly Shallow Meridional Cell located over the Gulf of Guinea. The time evolution of the cyan particle allocated in its inner part, displayed in Fig. 4b, confirms that this cell traps particles and that they eventually end up in the interior of the continent. The white arrow in Fig. 4a marks the position of a singular feature highlighting a dynamical boundary, which by warping this region in the Gulf of Guinea area prevents mixing (no transport occurs) with the AEJ. Figure 4b also shows an orange trajectory, which corresponds to a parcel placed in the Monsoon flow. This trajectory penetrates into the continent from the ocean, and then ascends before also end- ing into the AEJ. g Figure 5 provides a similar analysis to the one described above, but for the TEJ case. Panel (a) displays the partition of TEJ in a grey scale, according to function M, overlapped with contours of the zonal velocities. Two regions are identified in dark and light grey tones. Red particles, placed in the dark area are particles trajectories that become straight, while green particles, located in the light area, are particles trajectories that become straight but bend south over the Atlantic towards the Gulf of Guinea, while the blue particle bends towards northern African latitudes. Panel (c) shows a projection in the longitude-latitude-altitude space, which confirms this point. This is consistent with the results discussed by40 in which particles trajectories in the TEJ become straight, although our analysis distinguishes regions in the TEJ with different particle fates. Indeed, each particle is just https://doi.org/10.1038/s41598-020-69159-9 Scientific Reports \| (2020) 10:12543 \| www.nature.com/scientificreports/ Figure 5. (a) A view of the representation of function M in the latitude-altitude plane at longitude 10° W. Contour lines of the zonal velocity are overlapped. The TEJ is recognized at latitude ∼ 8° N and altitude ∼ 10 km. The red rectagle highlights the region zoomed in panel (b); (b) a zoom highlighting the lower part of TEJ where the function M has a particularly rich structure; (c) backwards and forwards time evolution of the fluid parcels placed in different sectors of the TEJ; (d) backwards and forwards time evolution of the fluid parcels placed in folds displayed in panel (b) and in a selected sharp region of panel (a). Maps in this figure are done with MATLAB R2018b. Results d Time arrows marking the time direction are placed over the trajectories. Maps in this figure are done with MATLAB R2018b. Figure 7. Evaluation of the function M for τ = 15 days at 2 km height above the sea level. Red, blue, cyan, magenta, yellow and green represent particles located at different domains of the partition induced by M. Time arrows marking the time direction are located over the trajectories. Maps in this figure are done with MATLAB R2018b. Figure 7. Evaluation of the function M for τ = 15 days at 2 km height above the sea level. Red, blue, cyan, magenta, yellow and green represent particles located at different domains of the partition induced by M. Time arrows marking the time direction are located over the trajectories. Maps in this figure are done with MATLAB R2018b. therefore placed below the convective cell that has been associated to the cyan fluid parcel in Fig. 4. The southern boundary of this structure is located approximately above the Equator, to the North of the cold tongue observed in the sea surface temperature climatological series, displayed in Fig. 1a. The higher pressure associated with the local cold temperature, would imply northwards fluid parcel motions close to the sea level and this is confirmed by the circulation direction of the cyan trajectory in Fig. 4b. Our interpretation is therefore that this dynamical structure could be closely linked to the sea surface temperature pattern.h y p p The computation of the function M on the longitude-latitude plane at 2 km height with τ = 15 days is dis- played in Fig. 7. The grey area where magenta and yellow particles are located is a dynamical structure found above the surface SHL. This area displays a rich M structure, which reflects different fluid parcels origins. The evolution in this region is somewhat more complex than what is described at 200 m, because at this level not all fluid parcels are ascending from the bottom. However, there are also fluid parcels coming from above, describing paths such as the magenta trajectory in Fig. 7, while others, such as the yellow one, are coming from lower heights. To the east of this region, there is a light grey structure where particles like the blue one evolve according to the arrows depicted over the trajectories in Fig. Results d Figure 6 provides a detailed description of the ITCZ as visible from the computation of the function M on a longitude-latitude plane at 200 m height with τ = 15 days. The black arrow in the image points to a line where the ITCZ is placed. The blue particles found on that line rapidly ascend (see Fig. 6b). The function M also displays a triple convergent point highlighted with a blue arrow. The position of this point is within the hottest point of the SHL displayed in Fig. 1a. The emergence of this type of spiral pattern has been reported to appear in convective fluids heated from below with peaked temperature distributions73. The convergent lines separate domains where particles have different origins (not shown), although all particles over these lines end up trapped in the lower parts of the AEJ as illustrated in Fig. 6b. Furthermore, particles located in the flat grey regions surrounding the triple point ascend in a similar way. Consistently with what we have previously described, changes in the grey intensity in this plane denote different behaviours of the particles, and thus the ascending evolution is applicable only to regions sharing the grey colour present in the neighbourhood of the triple point. This image also shows a very pronounced tongue-like structure just over the Gulf of Guinea, which geographically is placed at the position of the southerly Shallow Meridional Cell labelled as SMC in Fig. 1d. This tongue-like feature is the intersection with the horizontal plane of the sharp line that appears at low altitudes in Fig. 4a. For reference a horizontal blue line at 200 m is highlighted in Fig. 4a and shows two intersection points with this sharp feature. The tongue is https://doi.org/10.1038/s41598-020-69159-9 Scientific Reports \| (2020) 10:12543 \| www.nature.com/scientificreports/ Figure 6. Evaluation of the function M for τ = 15 at 200 metres height above the sea level. (a) The horizontal components of the currents are overlapped in this image; (b) the forward time evolution of particles placed along these structures. Time arrows marking the time direction are placed over the trajectories. Maps in this figure are done with MATLAB R2018b. Figure 6. Evaluation of the function M for τ = 15 at 200 metres height above the sea level. (a) The horizontal components of the currents are overlapped in this image; (b) the forward time evolution of particles placed along these structures. Discussion and conclusions In this paper we revisit the description of transport across classical West African Monsoon features from a Lagrangian Coherent Structures perspective. We find that with the support of this tool, which has not been used before in this context, we are able to identify not only the main characteristics flows but also new pathways and connections in the area.h The mathematical tool that we have used to display the dynamical template describing transport are Lagran- gian Descriptors, in particular, the so called function M. With the aid of this tool we are able to identify bounda- ries that separate zones where particles have different qualitative behaviours: that is, different origins or fates. This geometrical representation has advantages with respect to the plain representation of trajectories in the troposphere. Indeed, with the latter approach it is extremely difficult to find structures of order because trajec- tories follow very complex paths that intermingle with each other and information about transport is difficult to extract. On the other hand, our analysis provides a guide that allows a precise selection of initial conditions for comparison purposes, which has enabled us to find new transport routes, as well as providing an integrated vision of transport. Our discussion considers a forward and backward travel time for fluid parcels of τ = 15 days consistent with the transport occurring during the peak month season. During this travel time a rich Lagrangian geometrical structure (LCS) emerges and transport related to some of its features has been discussed.h The integrated transport vision provided by this method has allowed us to identify mixing patterns in the AEJ region that are linked to pathways to sources of humidity and dust. Cloud formation coming from dust sources in West Africa has been discussed by Wiacek et al.75, who found that the mean region of cloud formation is consistent with the AEJ location. There is a strong relationship between the AEJ and Saharan mineral dust (SMD) and AEJ and West African precipitation (WAP). A recent paper76 has found how the combined effect of WAP and SMD determines the changes in the AEJ. Discussion and conclusions Thus, it is very important not only to characterise the AEJ but also the sources of dust and rain.i Our approach has enabled us to progress from these findings by sketching an argument based on the LCS patterns about how to quantify the proportion of these elements of dust and humidity on the AEJ. Since dust is a candidate that may act as a catalyst for water vapor condensation, this finding could lead to important con- sequences in cloud formation. Using this Lagrangian approach, the clockwise circulation associated with the diverging flow at the top of the SHL is clear, and has been found to maintain the African Easterly Jet (AEJ)18. Transport pathways detected from LCS unveil connections between the TEJ and the AEJ. Indeed, after following a circulation pattern in the troposphere above the SHL, trajectories found on the TEJ end up in the AEJ. Note that this connection shows particles subsiding from the TEJ to the AEJ, contrary to what could be inferred from the Eulerian perspective14. Unlike suggestions by other authors4, we have not detected a Shallow Meridional Cell associated with the Saharan heat low, and the connection is found through the AEJ. These connections open up new possibilities for the influence and impact of the TEJ on African rainfall, which is a topic currently under discussion77,78. Additionally, through the analysis of a folding structure visible in the M function at the lower part of the TEJ, connections between the Earth’s surface and the TEJ have been found. It should be pointed out that these particles leave the surface over eastern Africa, rather than over the Gulf of Guinea, as could be inferred from the Eulerian perspective14. Finally, our methodological approach very clearly highlights other well-known pathways in the WAM system. Our results are very promising, since we foresee a great impact on the assessment of drought periods in this region, as well as in other monsoon regions if they are studied from this perspective. References 1. Solomon, S. et al. Climate Change 2007: The Physical Science Basis. Working Group I Contribution to the Fourth Assessment Report of the IPCC, volume 1. 01 (2007).hi f 2. Kirtman, B. & Pirani, A. 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Niang are supported by CSIC Grant COOPB20265. B. Rodriguez-Fonseca and E. Mohino acknowledge the support of Project CGL2017- 86415-R. J. Curbelo acknowledges the support of the RyC project RYC2018-025169-I and the U.S. NSF Grant AGS-1832842. Authors thankfully acknowledge the support of the computer resources provided by ICMAT. g A. M. Mancho, C. Niang, V. J. Garcia Garrido and J. Curbelo acknowledge the support of ONR Grant N00014- 17-1-3003. C. Niang acknowledges Fundacion Mujeres por Africa, ICMAT Severo Ochoa Project SEV-2011-0087 and Fundacion Harambee (Becas Guadalupe) for financial support. A. M. Mancho and C. Niang are supported by CSIC Grant COOPB20265. B. Rodriguez-Fonseca and E. Mohino acknowledge the support of Project CGL2017- 86415-R. J. Curbelo acknowledges the support of the RyC project RYC2018-025169-I and the U.S. NSF Grant AGS-1832842. Authors thankfully acknowledge the support of the computer resources provided by ICMAT. www.nature.com/scientificreports/ Atmos. 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A.M.M., E.M., B.R.F. and C.N. wrote the paper with contributions from V.J.G.G. and J.C. g g p p V.J.G.G. and J.C. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-69159-9. Correspondence and requests for materials should be addressed to A.M.M. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, 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/. © The Author(s) 2020 www.nature.com/scientificreports/ Commun. Nonlinear Sci. Numer. Simul. 18(12), 3530–3557 (2013). 57. Lopesino, C., Balibrea-Iniesta, F., García-Garrido, V. J., Wiggins, S. & Mancho, A. M. A theoretical framework for Lagrangian descriptors. Int. J. Bifurc. Chaos 27, 1730001 (2017).i p f 8. Craven, G. T. & Hernandez, R. Deconstructing field-induced ketene isomerization through Lagrangian descriptors. Phys. Chem Chem. Phys. 18, 4008 (2016). Scientific Reports \| (2020) 10:12543 \| https://doi.org/10.1038/s41598-020-69159-9 Competing interests h p g The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-69159-9. Correspondence and requests for materials should be addressed to A.M.M. Correspondence and requests for materials should be addressed to A.M.M. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International 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. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, 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. 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https://openalex.org/W1530846159	https://grasasyaceites.revistas.csic.es/index.php/grasasyaceites/article/download/597/611/611	Spanish; Castilian	null	El tejido adiposo: algo más que un reservorio de energía	Grasas y aceites	2,009	cc-by	13,230	RESUMEN KEY-WORDS: Adipokines – Brown adipose tissue – Temperature regulation – White adipose tissue. KEY-WORDS: Adipokines – Brown adipose tissue – Temperature regulation – White adipose tissue. El tejido adiposo: algo más que un reservorio de energía 1. INTRODUCCIÓN El tejido adiposo es un reservorio de grasa y también un órgano que contribuye a la estética y a la salud de las per- sonas, tarea que cumple funcionando como un tejido endo- crino. El tejido adiposo blanco, formado por células adiposas uniloculares puede regular la homeostasis orgánica entre el gasto y el consumo de energía. Produce una serie de adipo- kinas que regulan el consumo de alimentos, la utilización de glucosa y de lípidos por parte del organismo. El tejido adipo- so pardo o marrón, estructurado por células multiloculares, contiene numerosas gotas de grasa de pequeño tamaño, las que pueden ser hidrolizadas con mayor facilidad. El tejido participa en la termogénesis adaptativa o facultativa debido a la proteína desacoplante-1 (UCP-1) que inhibe la síntesis de ATP produciendo calor. Con la edad, este tejido se redu- ce y por ello actualmente se considera que una forma de combatir la obesidad es lograr que el tejido adiposo blanco adquiera característica de pardo. Este trabajo revisa las prin- cipales características estructurales y funcionales del tejido adiposo blanco y pardo, con énfasis en sus funciones regu- ladoras y endocrinas. Es común para muchos de nosotros el que a lo largo de nuestra existencia vaya modificándose el peso corporal, particularmente con la acumulación de tejido adiposo donde antes no lo teníamos, con lo cual se modifica, a veces lamentándolo mucho, nuestra imagen física. Sin embargo, sin menospre- ciar el efecto poco estético de la acumulación de grasa, esta se torna más grave cuando cobra im- portancia clínica-patológica impactando muy nega- tivamente nuestra salud. La relación entre el sobre- peso-obesidad y el desarrollo de las enfermedades crónicas no transmisibles es una realidad con gran evidencia científica. Sin embargo, es común aso- ciar al tejido adiposo solo como un reservorio ener- gético. La evidencia actual nos indica que las célu- las que constituyen este tejido, los adipocitos y otras que las acompañan, tienen una gran variedad de funciones, todas ellas muy relacionadas con la regulación de la homeostasis corporal. En esta re- visión abordaremos las funciones del tejido adipo- so, desde un punto de vista más allá que el ser so- lo un reservorio de energía. PALABRAS CLAVE: Adipokinas – Regulación de tempe- ratura – Tejido adiposo blanco – Tejido adiposo pardo. PALABRAS CLAVE: Adipokinas – Regulación de tempe- ratura – Tejido adiposo blanco – Tejido adiposo pardo. Por Alfonso Valenzuela B1,2* y Julio Sanhueza C1 entro de Lípidos, INTA, Universidad de Chile, Casilla 138-11, Santiago, Chile. 2 Facultad de Medicina, Universidad de los Andes, Santiago, Chile. (*Autor para la correspondencia: avalenzu@inta.cl) GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495 DOI: 10.3989/gya.043209 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495 DOI: 10.3989/gya.043209 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495 DOI: 10.3989/gya.043209 REVISIÓN REVISIÓN PALABRAS CLAVE: Adipokinas – Regulación de tempe- ratura – Tejido adiposo blanco – Tejido adiposo pardo. ALFONSO VALENZUELA B Y JULIO SANHUEZA C Las células endote- liales del tejido adiposo, como los fibroblastos y otras células de origen mesenquimal, incluyendo los preadipocitos y los adipocitos maduros, secre- tan un factor de crecimiento similar a la insulina (IGF-1), siendo al parecer una de las principales moléculas reguladoras involucradas en la hiperpla- sia del tejido adiposo durante la embriogénesis y en la infancia (Salans et al., 1973; Hager, 1977; Cinti, 2006). ) Se ha logrado identificar dos etapas de creci- miento acelerado del tejido adiposo blanco, una después del nacimiento, ya mencionada, y otra que se produce durante el predesarrollo puberal, entre los 9 y 13 años de edad. La tasa de proliferación del tejido adiposo decrece en la adolescencia y se presenta un equilibrio relativo hasta la adultez (Sa- lans et al., 1973). Normalmente la expansión del te- jido adiposo se debe principalmente a hipertrofia de las células ya presentes (aumento del tamaño celu- lar), sin un aumento en el número de células (hiper- plasia). Sin embargo, una vez que se desencade- nan los mecanismos que conducen a la obesidad, se produce un aumento no solo del tamaño de las células adiposas, también un aumento del número de células, las que pueden hasta quintuplicar el nú- mero original determinado en la postadolescencia. La disminución del tamaño de las células es más fácil que la disminución del número de adipocitos. Por esta razón en muchos casos los regímenes dietéticos tendientes a disminuir la obesidad en los adultos fracasan, porque solo se logra disminuir el tamaño de los adipocitos y no la cantidad de estos, ( g q ) La importancia del tejido adiposo dentro de las especies presenta una gran variabilidad. En los in- vertebrados el tejido adiposo representa un órgano nutricionalmente importante, particularmente en los insectos, y su importancia disminuye en los arácni- dos, crustáceos y moluscos. En los vertebrados el desarrollo del tejido adiposo es un signo evolutivo en los organismos homeotermos. También su pro- porción en el peso corporal varía notoriamente en- tre las especies (cerca del 40% del peso corporal de los cetáceos lo constituye el tejido adiposo), las reservas grasas de las aves migratorias y las reser- vas de los mamíferos también presentan gran dife- rencia, pero constituyen un ejemplo de la importan- cia en la actividad metabólica de este tejido. El desarrollo del tejido adiposo, en especial del tejido adiposo blanco presenta gran diferencia entre las especies. ALFONSO VALENZUELA B Y JULIO SANHUEZA C te desde 20 a 200 micrómetros de diámetro, lo que significa que pueden en ciertas circunstancias au- mentar hasta 1000 veces su volumen. El resto del tejido está constituido por células sanguíneas, célu- las endoteliales, macrófagos, pericitos y precurso- res de los adipocitos en distintos grados de diferen- ciación, ya sean fibroblastos como precursores primarios y preadipocitos que son células intersti- ciales vacías de lípidos y prontas a transformarse en adipocitos (Poissonnet et al., 1984; Hausman, 1987). La proliferación y activación de macrófagos en el tejido adiposo se ha relacionado con los esta- dos inflamatorios que se asocian con la obesidad como patología. Aunque el origen embrionario de las células grasas no es del todo conocido, las in- vestigaciones realizadas sugieren que la línea adi- pocitaria deriva de un precursor embrionario multi- potencial de estructura fusiforme que posee la capacidad para diferenciarse en distintas células especializadas (adipocitos, condrocitos, osteoblas- tos y miocitos) las que finalmente forman parte de la estructura de diferentes tejidos (Geloen et al., 1989). Los procesos celulares que llevan a la con- versión de las células multipotentes en adipoblas- tos y posteriormente en preadipocitos y adipocitos, son todavía poco conocidos, aunque en la actuali- dad se reconocen mecanismos que permiten apro- ximarnos con mayor certeza sobre lo que ocurre en la formación del tejido adiposo. Las principales fun- ciones del tejido adiposo son: reserva energética, amortiguación-protección ósea y aislamiento térmi- co, aunque como veremos, también ahora se acep- ta que posee funciones endocrinas y paracrinas (Gregoire, 1998; Vázquez-Vela et al., 2008). mamíferos el humano es el que nace con mayor cantidad de tejido adiposo (Hager, 1977). La figura 1 muestra comparativamente el contenido de tejido adiposo de diferentes especies. Histológicamente, el tejido adiposo blanco esta altamente vascularizado (aunque menos que el te- jido adiposo pardo), a tal punto que muchos adipo- citos se encuentran en contacto directo con uno o más capilares. Estos permiten la entrada y salida activa de metabolitos, péptidos y factores no peptí- dicos fundamentales en la regulación de la diferen- ciación y el crecimiento celular. 2. EL TEJIDO ADIPOSO, SU ESTRUCTURA BÁSICA Adipose tissue is a reservoir of energy and also an organ that contributes to the aesthetics and health of human body working as an endocrine tissue. White adipose tissue, which is formed by unilocular adipose cells, can modify organic homeostasis by controlling energy expenditure and consumption and by producing adipokines that regulate food consumption, and carbohydrate and lipid metabolic utilization. Brown adipose tissue is structured by multilocular cells containing many small fat drops that can be easily hydrolyzed. This tissue is involved in adaptative or facultative thermogenesis because it contains the uncoupling-1 protein (UCP-1) which by inhibiting ATP synthesis releases heat. The size of brow adipose tissue is reduced with aging and the most recent future strategies to fight obesity may be by transforming white cells into brown adipose tissue. The present work reviews the main structural and functional characteristics of white and brown adipose tissue with emphasis in its endocrine and regulatory function. Fisiológicamente el tejido adiposo es considera- do como un órgano difuso de gran actividad meta- bólica. Aproximadamente entre el 15 al 25% del pe- so corporal de una persona adulta (hombres 15%-20%, > 25% se considera obesidad; mujeres 20%-25%, > 33% se considera obesidad) está re- presentado por el tejido adiposo siendo este, como lo sabemos, una importante reserva energética (Cinti, 2005). Histológicamente el tejido adiposo, visto como un órgano, no está formado solamente por los adipocitos, ya que estos constituyen aproxi- madamente el 60%-70% de su estructura. El tama- ño de los adipocitos puede variar considerablemen- 437 EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA con lo cual estas células tienden a recuperar su ta- maño original en poco tiempo (Hirsh & Batchelor, 1976). cular) y el citoplasma queda reducido a un fino re- borde de la célula. El núcleo, de estructura oval, desplazado hacia la zona periférica, contiene cro- matina de grano fino y no presenta un nucléolo vi- sible. El adipocito del tejido adiposo blanco posee pocas mitocondrias, un retículo endoplasmático ru- goso y liso de baja densidad membranosa y un complejo de Golgi de pequeño tamaño. La gota de lípido está limitada por pequeños filamentos protei- cos denominados perilipinas (Subramanian et al., 2004). Como ya se mencionó, el tejido adiposo blanco es muy vascularizado, cada célula está en contacto con al menos un capilar. Este tejido está subdividido en pequeños lobulillos por tabiques de tejido conectivo no muy definidos. Esta segmenta- ción es más visible en las zonas donde la función de este tejido es la amortiguación, por ejemplo, la zona glútea, encontrándose tabiques de tejido co- nectivo grueso que dividen la zona en diferentes capas (Cinti, 2006; Garaulet et al., 2006). ) Generalmente el tejido adiposo se deposita en áreas con abundante tejido conectivo laxo, como por ejemplo las capas subcutáneas entre el múscu- lo y la dermis. Sin embargo, también se localiza en forma típica, alrededor de las vísceras, riñones, co- razón y otros órganos internos. Muchos estudios avalan el hecho de que el tejido adiposo no es un órgano homogéneo. Mas aún, se plantea que la ubicación topográfica del tejido adiposo hace que tenga perfiles metabólicos distintos dependiendo de la ubicación anatómica, lo cual lo hace suscep- tible de participar en el desarrollo de ciertas patolo- gías. Por ejemplo, la localización intra abdominal- visceral tiene más efectos patológicos que la localización subcutánea (Garaulet et al., 2006). La figura 2 ejemplifica las diferencias entre la grasa subcutánea y visceral. p ( , ; , ) En el ayuno, las células adiposas blancas libe- ran gradualmente los lípidos almacenados y la va- cuola central disminuye de tamaño. El tejido adipo- so blanco se encuentra distribuido como grasa subcutánea (tejido adiposo subcutáneo) y como panículo adiposo en el mesenterio y en la zona re- troperitoneal (tejido adiposo visceral). 3.1. Tejido adiposo blanco Estructuralmente es unilocular (de estructura in- tracelular uniforme) y no es exactamente de color blanco en los primates, más bien de color amarillo variando a diferentes tonalidades las que depen- den principalmente de la dieta del individuo, mien- tras mayor es el contenido de carotenoides dieta- rios, mas intenso es el color amarillo del tejido. Estructuralmente cada adipocito contiene una gota central grande de triacilglicéridos (estructura unilo- EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA En los niños, una capa de grasa uniforme cubre todo el cuerpo, mientras que en los adultos el tejido se acumula en algunas zonas específicas, siendo ésta distribución distinta por género: en la mujer suele acumularse en la región mamaria, cadera, región glútea y mus- los, mientras que en el hombre suele acumularse en la nuca, región lumbo-sacra y región glútea. En ciertas zonas corporales, no se produce liberación de lípidos durante el ayuno, como es el caso del te- jido graso correspondiente a la zona periorbicular de los ojos, rodillas, palmas de las manos, plantas de los pies. En los últimos dos casos el tejido adi- poso cumple una función básicamente de tipo me- cánico como elemento de sostén y amortiguación (Mauriege et al., 1999; Kuk & Ross, 2009). 3. TIPOS DE TEJIDO ADIPOSO Aún persiste una de las más antiguas clasifica- ciones del tejido adiposo y que tiene como base la coloración que este adquiere ante tinciones funda- mentales utilizadas en anatomía patológica. Es así como se le clasifica en tejido adiposo blanco y teji- do adiposo pardo o marrón. ALFONSO VALENZUELA B Y JULIO SANHUEZA C Este no puede ser detectado microscópi- camente durante la etapa embrionaria ni en el na- cimiento de la mayoría de los roedores, mientras que sí está presente desde el nacimiento en los co- nejos, cerdos, conejillos de indias y en los huma- nos. El 14% del peso corporal del recién nacido hu- mano esta constituido por el tejido adiposo, el cual comienza su formación a partir de la semana 14 de gestación. El aumento postnatal es más marcado en los primeros 3 meses de vida, probablemente li- gado a la pobre capacidad de termorregulación corporal a esa edad. Curiosamente, dentro de los Figura 1 Porcentaje de grasa corporal de diferentes especies animales al nacer. Porcentaje de grasa corporal al nacer Cerdo Gato Cordero Babuino León marino Ballena Humano 0 5 10 15 20 Porcentaje de grasa corporal al nacer Porcentaje de grasa corporal al nacer Figura 1 Porcentaje de grasa corporal de diferentes especies animales al nacer. Figura 1 Porcentaje de grasa corporal de diferentes especies animales al nacer. 438 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 Tejido adiposo subcutáneo Diferencias entre la grasa subcutánea y visceral Figura 2 Diferencias anatómicas y metabólicas entre la grasa subcutánea y la grasa visceral. Diferencias entre la grasa subcutánea y visceral Subcutánea – Metabolismo muy lento – Predomina en la mujer – Ubicación femoroglutea – Bajo riesgo de patologías – Problemas estéticos Visceral – Metabolismo muy rápido – Predomina en hombres – Rodea a las vísceras – El exceso es de alto riesgo patológico cardiovascular – Problemas estéticos ALFONSO VALENZUELA B Y JULIO SANHUEZA C Figura 3 Diferente expresión fenotípica del exceso de grasa corporal de tipo ginoide y androide. Expresión fenotípica del exceso de grasa corporal (obesidad) Obesidad Ginoide o tipo pera Obesidad Androide o tipo manzana – Distribución principalmente abdominal tanto subcutánea como visceral (omental, me- sentérica y perirrenal) – Tiene alta trascendencia me- tabólica – Se asocia con alta prevalen- cia de patologías cardiovas- culares y metabólicas (diabe- tes) – Es de más fácil modificación anatómica – Distribución femoroglutea – Poca transcendencia meta- bólica – Se asocia a osteoartrosis, varices, lipodistrofia, linfoe- dema, etc. – De difícil modificación anató- mica Expresión fenotípica del exceso de grasa corporal (obesidad) Tejido adiposo visceral Expresión fenotípica del exceso de grasa corporal (obesidad) También es conocido como tejido adiposo intra- peritoneal. Se le subdivide en omental y mesentéri- co y se ubica en las regiones profundas de la cavi- dad abdominal rodeando las vísceras. Constituye el 5%-10% del tejido adiposo total en mujeres y hombres, respectivamente, siendo similar este por- centaje en individuos de peso normal y obesos (Se- thi & Vidal-Puig, 2007). Las mujeres, en la edad adulta y postmenopausia tienden a aumentarlo más que los hombres (Kuk & Ross, 2009). Metabó- licamente es más activo que el tejido adiposo sub- cutáneo en relación a los procesos de lipogénesis y de lipólisis, lo cual está determinado por una abundancia de receptores adrenérgicos lipolíticos y antilipolíticos (ver más adelante). Posee, además, receptores para glucocorticoides, con lo cual las si- tuaciones de estrés crónico tienen gran impacto so- bre los depósitos de grasa en este tejido, pudiéndo- se producir estímulo de su acumulación, o por el contrario, de su movilización (aumento o baja de peso en condiciones de estrés físico y/o emocio- nal). Desde el punto de vista vascular, está sujeto a drenaje portal, con lo cual los ácidos grasos que se liberan por lipólisis llegan directamente al hígado, constituyendo así un aporte directo de energía pa- ra el metabolismo general. Sin embargo, un exceso de drenaje de ácidos grasos desde el tejido adipo- so visceral al hígado, también facilita el desarrollo de insulinorresistencia hepática, hiperinsulinemia, dislipidemia e hiperglicemia. Su asociación con una mayor prevalencia de patologías como la diabetes 2, hipertensión, dislipidemia y cardiovasculares, ya es evidente (Bakker et al., 2004). Jean Vague defi- nió en 1947 la clásica distribución entre grasa ab- dominal o androide (obesidad tipo manzana) y gra- sa femoroglútea o ginoide (obesidad tipo pera), relacionando a la primera con un mayor riesgo de patologías asociadas a lo que hoy identificamos co- mo el síndrome metabólico (Vague, 1947). La figu- ra 3 muestra las diferencias en la expresión fenotí- pica de ambos tipos de obesidad. – Distribución femoroglutea – Distribución femoroglutea – Poca transcendencia meta- bólica Obesidad Ginoide o tipo pera – Se asocia a osteoartrosis, varices, lipodistrofia, linfoe- dema, etc. Tejido adiposo visceral – De difícil modificación anató- mica – Distribución principalmente abdominal tanto subcutánea como visceral (omental, me- sentérica y perirrenal) – Tiene alta trascendencia me- tabólica Obesidad Androide o tipo manzana – Se asocia con alta prevalen- cia de patologías cardiovas- culares y metabólicas (diabe- tes) – Es de más fácil modificación anatómica Figura 3 Diferente expresión fenotípica del exceso de grasa corporal de tipo ginoide y androide. mayor de mitocondrias. Su color varía desde el do- rado al marrón rojizo y sus células tienen forma po- ligonal, exhibiendo un citoplasma más abundante y granuloso. El núcleo es redondeado, está ubicado un poco excéntrico y contiene gránulos de cromati- na bastante gruesos y con un nucléolo visible. En el citoplasma se observan numerosas mitocondrias grandes y redondas que presentan crestas muy juntas. Los demás organelos se encuentran poco desarrollados. El tejido adiposo pardo aparece a la microscopía característicamente lobulado y puede parecer una glándula en cuanto a su aspecto ma- croscópico. Presenta muchos más capilares que el tejido adiposo blanco, y posee numerosas fibras nerviosas entre sus células. El color marrón está dado por los citocromos que forman parte de la ca- dena respiratoria de las numerosas mitocondrias que posee (Cannon & Nedergaard, 2004). Diferencias entre la grasa subcutánea y visceral Es cuantitativamente el más importante consti- tuyendo alrededor de un 80% del total de la grasa corporal. Su metabolismo es más bien lento en comparación con el tejido adiposo visceral. Esto es, los procesos de lipogénesis y lipogenólisis, discuti- dos más adelante, son de poca relevancia. El tejido adiposo subcutáneo de la región abdominal tiene un comportamiento mixto en términos metabólicos ya que puede ser tan activo como el visceral. Su función más destacada es la de aislante térmico y de amortiguación mecánica. También se ha pro- puesto que ejercería un efecto de “amortiguación metabólica” ya que podría de alguna manera amor- tiguar el impacto de los excesos calóricos en el te- jido adiposo visceral, aunque este aspecto no está aún del todo claro (Sethi & Vidal-Puig, 2007; Bak- ker et al., 2004). – Metabolismo muy lento – Predomina en la mujer – Ubicación femoroglutea – Bajo riesgo de patologías – Problemas estéticos Subcutánea – Metabolismo muy rápido – Predomina en hombres – Rodea a las vísceras – El exceso es de alto riesgo patológico cardiovascular – Problemas estéticos Visceral Figura 2 Diferencias anatómicas y metabólicas entre la grasa subcutánea y la grasa visceral. 439 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA Tras el nacimiento, la hipotermia neonatal produce un estímulo del sistema nervioso que inerva el tejido adiposo pardo a través de la ac- tivación de receptores sensibles a la temperatura situados en el hipotálamo y en la médula espinal. Este tejido es estimulado por la noradrenalina secretada por el sistema nervioso autónomo, produ- ciéndose la hidrólisis aumentada de triacilglicéridos de los adipocitos por activación de la lipasa hormo- na sensible (LHS), y luego la oxidación de ácidos grasos por beta oxidación mitocondrial que va acompañada de gran consumo de oxígeno, produ- ciéndose calor durante este proceso, el cual mantie- ne caliente al neonato. Una proteína identificada co- mo termogenina actúa como un factor desacoplante entre la respiración mitocondrial y la fosforilación oxidativa, de modo que se forma menos ATP y se di- sipa mayor cantidad de calor (Bhakoo, 1974; Price, 1994; Stephenson, et al., 2001). La termogenina pertenece a un grupo de proteínas desacoplantes colectivamente conocidas como UCP (UnCoupling Protein), las que juegan una función muy importan- te en el control de la termogénesis, especialmente en los animales que hibernan. Las UCP son 5, la UCP-1 se encuentra solo en el tejido adiposo pardo, la UCP-2 se distribuye en varios tejidos (músculo, ri- ñón, vísceras), la UCP-3 solo se encuentra en el músculo esquelético, y las UCP-4 y UCP-5 se ubi- can en el cerebro. Se ha observado en recién naci- dos con retraso de crecimiento intrauterino y/o con menor edad gestacional, la presencia de hipoglice- mia, hipoxia y trastornos en la producción de calor, lo cual ocasiona inestabilidad térmica (Bianco & Sil- va, 1987; Symonds et al., 1992; Hesselink, 2003). Esto se debe a que el neonato no presenta un ade- cuado desarrollo del tejido adiposo pardo, por lo cual los depósitos de energía en la forma de triacil- glicéridos no son suficientes. El tejido adiposo par- do posee tres tipos de receptores adrenérgicos: Receptores tipo beta: Su activación estimula a la enzima adenilato-ciclasa, lo cual aumenta las concentraciones intracelulares de AMPc, produ- ciendo la activación de una proteina kinasa que a su vez por fosforilación activa a la LHS, iniciando así la liberación de ácidos grasos los que no aban- donan los adipocitos pardos, siendo beta oxidados por sus abundantes mitocondrias. La máxima ex- presión de estos receptotes se produce en el mo- mento del nacimiento y permanecen activos duran- te toda la lactancia (Cannon & Nedergaard, 2004). EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA uso de una termogénesis obligatoria del metabolis- mo celular, y de una disipación calórica superficial, para lo cual se cuenta con el tejido adiposo pardo, el cual funciona como fuente generadora de calor después del parto. Tras el nacimiento, la hipotermia neonatal produce un estímulo del sistema nervioso que inerva el tejido adiposo pardo a través de la ac- tivación de receptores sensibles a la temperatura situados en el hipotálamo y en la médula espinal. Este tejido es estimulado por la noradrenalina secretada por el sistema nervioso autónomo, produ- ciéndose la hidrólisis aumentada de triacilglicéridos de los adipocitos por activación de la lipasa hormo- na sensible (LHS), y luego la oxidación de ácidos grasos por beta oxidación mitocondrial que va acompañada de gran consumo de oxígeno, produ- ciéndose calor durante este proceso, el cual mantie- ne caliente al neonato. Una proteína identificada co- mo termogenina actúa como un factor desacoplante entre la respiración mitocondrial y la fosforilación oxidativa, de modo que se forma menos ATP y se di- sipa mayor cantidad de calor (Bhakoo, 1974; Price, 1994; Stephenson, et al., 2001). La termogenina pertenece a un grupo de proteínas desacoplantes colectivamente conocidas como UCP (UnCoupling Protein), las que juegan una función muy importan- te en el control de la termogénesis, especialmente en los animales que hibernan. Las UCP son 5, la UCP-1 se encuentra solo en el tejido adiposo pardo, la UCP-2 se distribuye en varios tejidos (músculo, ri- ñón, vísceras), la UCP-3 solo se encuentra en el músculo esquelético, y las UCP-4 y UCP-5 se ubi- can en el cerebro. Se ha observado en recién naci- dos con retraso de crecimiento intrauterino y/o con menor edad gestacional, la presencia de hipoglice- mia, hipoxia y trastornos en la producción de calor, lo cual ocasiona inestabilidad térmica (Bianco & Sil- va, 1987; Symonds et al., 1992; Hesselink, 2003). Esto se debe a que el neonato no presenta un ade- cuado desarrollo del tejido adiposo pardo, por lo cual los depósitos de energía en la forma de triacil- glicéridos no son suficientes. El tejido adiposo par- do posee tres tipos de receptores adrenérgicos: uso de una termogénesis obligatoria del metabolis- mo celular, y de una disipación calórica superficial, para lo cual se cuenta con el tejido adiposo pardo, el cual funciona como fuente generadora de calor después del parto. 3.2. Tejido adiposo pardo Topográficamente el tejido adiposo pardo se en- cuentra principalmente alrededor del cuello y los grandes vasos sanguíneos del tórax en los neona- tos para luego ser subsecuentemente reemplazado en los adultos por el tejido adiposo blanco, aunque en los adultos se conserva tejido adiposo pardo, en pequeños cúmulos, dentro del tejido adiposo blanco (Poissonnet et al., 1984). El tejido adiposo pardo permite realizar lo que se conoce como termogéne- sis adaptativa o facultativa, esto es la capacidad que tiene el organismo para responder al frío. q p ( g , ) El tejido adiposo pardo se encuentra muy des- arrollado en los neonatos, constituyendo entre el 2 al 5% del peso corporal y se encuentra entre las es- cápulas, en las axilas, en la región de la nuca y ro- deando a lo largo los vasos sanguíneos grandes. El tejido adiposo pardo se va modificando con la edad transformándose en tejido adiposo blanco. La prin- cipal función del tejido adiposo pardo es mantener la temperatura corporal en el neonato (Asakura, 2004). Los animales superiores deben mantener su temperatura corporal dentro de un margen muy es- trecho. En el desarrollo embrionario y fetal, el nuevo ser se encuentra aislado de la temperatura ambien- tal por lo cual no posee termorregulación. En el mo- mento del nacimiento, el recién nacido debe hacer A diferencia del tejido adiposo blanco, el tejido adiposo pardo es multilocular, esto es formado por múltiples gotitas citoplasmáticas de diferente tama- ño conteniendo triacilglicéridos. Sus células son más pequeñas que las del tejido adiposo blanco, pero contienen una cantidad considerablemente 440 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 4.1. El desarrollo del tejido adiposo El desarrollo y la expansión del tejido adiposo in vivo está regido por una serie de acciones coordina- das de numerosos factores, que en conjunto forman una red integrada y finamente regulada, diseñada primariamente para el mantenimiento del equilibrio energético en el organismo. Factores de tipo genéti- co, epigenéticos, ambientales y farmacológicos, es- tarían involucrados en esta red. Actualmente, una de las principales líneas de investigación en enfer- medades metabólicas es el estudio y la diferencia- ción de las diferentes redes y señales que facilitan las comunicaciones entre los distintos órganos inter- nos (Gregoire, 2001;Valet et al., 2002; Sethi & Vidal- Puig, 2007). En tal sentido, se han logrado importan- tes avances en la comprensión de las bases moleculares del desarrollo del tejido adiposo a tra- vés de las técnicas de clonación y de caracterización de numerosos genes involucrados en la diferencia- ción y metabolismo del adipocito. La transgénesis, rama de la biología molecular encargada del estudio de funciones y mecanismos de acción mediante la manipulación genética, ha mostrado gran utilidad en la identificación de las funciones fisiológicas de los genes que regulan la adipogénesis (Rosen & Mac- Dougald, 2006; Sethi & Vidal-Puig, 2007). ) Aparentemente, después de todo el desarrollo energético del tejido adiposo pardo, éste evolucio- na hacia un estado energético de reposo, que su- pone la acumulación de lípidos en forma de una gran vacuola, dándole un aspecto unilocular a la célula, característico de las células adiposas blan- cas, aunque no hay certeza que se trate de una transformación total. Este tejido permanece en re- poso de por vida, desde el punto de vista termogé- nico. Se ha propuesto que la reconversión de este tejido nuevamente en células pardas, o la transfor- mación de células adiposas blancas en pardas, po- drían constituir un interesante abordaje para el tra- tamiento de la obesidad mórbida. Sin embargo, aún falta mucho por conocer sobre la regulación y la biología molecular de estas células (Cannon & Ne- dergaard, 2004). La figura 5 ejemplifica las diferen- cias morfológicas entre el tejido adiposo blanco y el tejido adiposo pardo. La propia naturaleza difusa del proceso de adipo- génesis in vivo dificulta el estudio del reclutamiento y diferenciación de los adipocitos. 4. HISTOGÉNESIS Y FISIOLOGÍA DEL TEJIDO ADIPOSO mostrado que recién nacidos expuestos a una tem- peratura de 25 grados Celsius aumentan la capta- ción de oxígeno, aumentando la temperatura en la piel y en zonas específicas donde se acumula el te- jido adiposo pardo (Asakura, 2004). Se ha evalua- do el efecto de la exposición al frío en el tejido adi- poso pardo y en la termogénesis en neonatos ovinos, valorando diferentes parámetros como: consumo de oxígeno y la producción de CO2, de- mostrándose el efecto del frío sobre el tejido adipo- so pardo, donde se observa que en los primeros días postnatales se produce el mayor incremento del consumo de oxígeno (de hasta un 40%) y de la temperatura rectal, lo cual denota una actividad ter- mogénica acelerada. De esta forma, se ha conclui- do que, el incremento en la actividad termogénica del tejido adiposo pardo, en recién nacidos ovinos, está relacionado directamente con la exposición al frío en los primeros días de vida. En roedores se ha evidenciado que los principales factores regulado- res de la actividad termogénica del tejido adiposo pardo son la adrenalina, la insulina y la producción de triyodotironina (Bianco & Silva, 1987; Symonds et al., 1992). mostrado que recién nacidos expuestos a una tem- peratura de 25 grados Celsius aumentan la capta- ción de oxígeno, aumentando la temperatura en la piel y en zonas específicas donde se acumula el te- jido adiposo pardo (Asakura, 2004). Se ha evalua- do el efecto de la exposición al frío en el tejido adi- poso pardo y en la termogénesis en neonatos ovinos, valorando diferentes parámetros como: consumo de oxígeno y la producción de CO2, de- mostrándose el efecto del frío sobre el tejido adipo- so pardo, donde se observa que en los primeros días postnatales se produce el mayor incremento del consumo de oxígeno (de hasta un 40%) y de la temperatura rectal, lo cual denota una actividad ter- mogénica acelerada. De esta forma, se ha conclui- do que, el incremento en la actividad termogénica del tejido adiposo pardo, en recién nacidos ovinos, está relacionado directamente con la exposición al frío en los primeros días de vida. En roedores se ha evidenciado que los principales factores regulado- res de la actividad termogénica del tejido adiposo pardo son la adrenalina, la insulina y la producción de triyodotironina (Bianco & Silva, 1987; Symonds et al., 1992). EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA g Receptores tipo alfa 1: Son independientes de la activación de la enzima adenilato-ciclasa. La acti- vación de las proteínas kinasas, que a su vez acti- van a la LHS, se produce a través del aumento del calcio intracelular producido por la activación de es- tos receptores. De esta forma, su acción es com- plementaria a la activación de los receptores beta adrenérgicos (Cannon & Nedergaard, 2004). Receptores tipo alfa 2: Su activación inhibe a la enzima adenilato-ciclasa, con lo cual disminuye la concentración de AMPc, por lo tanto son antiter- mogénicos. Estos receptores disminuyen a medida que se acerca la hora del nacimiento, favorecien- do así la función termogénica del tejido adiposo pardo en el recién nacido, derivada de la acti- vación de los receptores beta y alfa 1 (Cannon & Nedergaard, 2004). La figura 4 esquematiza la res- puesta del tejido adiposo pardo a distintos estímu- los hormonales. El tejido adiposo pardo tiene gran cantidad de mitocondrias con crestas largas y apiladas (lugar donde ocurre la fosforilación oxidativa), por lo tanto está capacitado para una oxidación activa donde la energía liberada no está ligada a la fosforilación oxidativa sino que es empleada en la producción de calor (Argyropoulos & Harper, 2002). El tejido adi- poso pardo aumenta el número de mitocondrias du- rante los últimos días del desarrollo fetal y después del nacimiento estos organelos aumentan conside- rablemente su volumen. Durante la lactancia conti- nua esta actividad, la que comienza a decaer a las pocas semanas después del nacimiento. Se ha de- Diferente respuesta del tejido adiposo pardo a distintos estímulos hormonales Catecolaminas Receptor tipo beta Adenilato ciclasa AMPc LHS LHS LHS Ca2+ Proteína kinasa C Adenilato ciclasa AMPc Proteína kinasa A Proteína kinasa A Receptor tipo alfa 1 Membrana celular Receptor tipo alfa 2 Figura 4 Respuesta metabólica del tejido adiposo pardo al estímulo producido por catecolaminas en receptores de tipo beta, alfa 1 y alfa 2. Diferente respuesta del tejido adiposo pardo a distintos estímulos hormonales Receptor tipo alfa 1 Receptor tipo alfa 2 Receptor tipo beta Figura 4 Figura 4 Respuesta metabólica del tejido adiposo pardo al estímulo producido por catecolaminas en receptores de tipo beta, alfa 1 y alfa 2. 441 ASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 4.1. El desarrollo del tejido adiposo Se han diseñado una serie de estratégicas que han logrado crear lí- neas celulares primarias inducibles capaces de dife- renciarse en adipocitos in vitro, lo cual ha permitido un mejor conocimiento acerca de los mecanismos que regulan su diferenciación celular a adipocito. En general, el mecanismo mejor identificado es la se- cuencia de eventos transcripcionales en cascada que controla la adipogénesis (Farmer, 2006). q p g ( ) Los procesos implicados en la diferenciación de los precursores adipocitarios hasta adipocitos ma- Diferencias morfológicas entre los tipos de tejido adiposo EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA g ( g , ) Durante la fase de crecimiento, tanto las líneas celulares de preadipocitos como los preadipocitos primarios, son morfológicamente similares a los fi- broblastos (Green &. Kehinde, 1975). Una vez que las células han alcanzado la confluencia, el trata- miento con los inductores adecuados de la diferen- ciación conduce a un cambio drástico en la forma de las células. Los preadipocitos se convierten en células de forma esférica que comienzan a acumu- lar lípidos y que van adquiriendo progresivamente las características morfológicas y bioquímicas pro- pias de los adipocitos maduros. El tratamiento ca- paz de inducir la diferenciación varía en los distin- tos modelos celulares descritos. Aunque los preadipocitos de diferentes fuentes son similares en múltiples aspectos, su respuesta a los agentes inductores de la diferenciación varía considerable- mente. Estas diferencias pueden venir determina- das por el diferente estadio de maduración en el que se obtuvieron los preadipocitos. En la mayor parte de los casos se requiere la presencia de insu- lina (Entingh et al., 2003). En algunos casos, la di- ferenciación se ve acelerada tras el tratamiento du- rante 48 horas con dexametasona, un corticoide, o con isobutilmetilxantina (IBMX), un estimulante de la formación de AMPc, y con altas concentraciones de insulina en presencia de suero bovino fetal. Tras ese período de inducción de la diferenciación, no se requiere la presencia de estos inductores de di- ferenciación para el mantenimiento del fenotipo del adipocito maduro (Mandrup & Lane, 1997). Cambios tempranos en la expresión de genes. La expresión de la enzima lipoproteína lipasa (LPL) ha sido considerada a menudo como un signo tem- prano de la diferenciación adipocitaria. La expre- sión de la LPL ocurre, sin embargo, de manera espontánea al alcanzar la confluencia y es independiente de los inductores de la diferencia- ción. Esta circunstancia sugiere que la LPL puede reflejar la etapa de cese del crecimiento más que ser un marcador temprano del proceso de diferen- ciación. Hasta ahora se han descrito 2 familias de factores de transcripción, las C/EBPs (CCAAT/En- hancer Binding Proteins) y el PPAR (Peroxisome Proliferator Activated Receptor ), que han sido identificados como reguladores de la transcripción de genes adipogénicos. Las tiazolidinedionas, fár- macos con acción antidiabética y que actúan como ligandos directos de PPAR, son además potentes y efectivos estimulantes de la adipogénesis. EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA duros han sido ampliamente estudiados utilizando modelos celulares in vitro. Estos han permitido la caracterización de los eventos moleculares y celu- lares que tienen lugar durante la transición de pre- adipocitos indiferenciados, tipo fibroblastos, hasta células grasas redondeadas maduras (Djian et al., 1985; Gregoire et al., 1998; Entingh et al., 2003). Las líneas celulares utilizadas se pueden dividir en 3 categorías: Células embrionarios totipotentes ca- paces de generar todas las líneas celulares; Célu- las multipotentes que pueden dar lugar a miocitos, adipocitos y condrocito; Células ya comprometidas hacia la línea adiposa, que son las denominadas lí- neas celulares de preadipocitos. paso previo a su conversión terminal en adipocitos maduros. Este proceso de diferenciación supone cambios cronológicos en la expresión de numero- sos genes. Así, se van expresando aquellos genes característicos de los adipocitos, al mismo tiempo que se van reprimiendo genes que son inhibitorios para la adipogénesis o que son innecesarios para la función del adipocito maduro. Todos estos cam- bios en la expresión y función de estos genes con- ducen a la adquisición del fenotipo característico del adipocito (Richon et al., 1997; Shao & Lazar, 1997). ) Aunque los fenómenos moleculares implicados en la diferenciación de los adipocitos no son total- mente conocidos, se ha sugerido un modelo que in- cluye varias etapas: p p Se ha logrado también el cultivo de preadipoci- tos primarios, así como la inducción de su transfor- mación en adipocitos maduros, en diversas espe- cies animales, incluido el hombre. Estas células presentan, además, la ventaja que pueden ser ob- tenidas desde varias especies animales en diferen- tes etapas del desarrollo postnatal y de diferentes depósitos grasos. Esto último es muy importante ya que se han observado importantes diferencias mo- leculares y bioquímicas entre los distintos depósi- tos grasos (Gregoire et al., 1998). Inhibición del crecimiento. Una vez alcanzada la confluencia, los preadipocitos sufren inhibición por contacto y cesan su crecimiento y comienzan a ex- hibir algunos de los marcadores tempranos de la di- ferenciación. Expansión clonal: El tratamiento de estas célu- las en las que ha cesado el crecimiento y que ya se encuentran en etapas de diferenciación, las induce a reentrar en el ciclo celular, produciéndose varios ciclos de replicación del DNA y de duplicación celu- lar. Esta expansión mitótica clonal de células com- prometidas es esencial para completar la diferen- ciación terminal para transformarse en adipocitos maduros. EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA Estos fármacos aumentan la sensibilidad a la insulina promoviendo la diferenciación de adipocitos subcu- táneos, permitiendo así la redistribución de grasa visceral, más patogénica, hacia grasa periférica de menor riesgo. Esta puede ser la razón por la que el tratamiento de la diabetes con este tipo de fárma- cos produce aumento del peso de los pacientes, conocido clínicamente como la “paradoja de las tia- zolidinedionas”. Los factores de transcripción C/EBP y C/EBP parecen jugar también un impor- tante papel en la inducción del PPAR (Lekstrom- Himes & Xanthopoulos, 1998; Kowenz-Leutz & Leutz, 1999; Rosen et al., 2000; Hausman & Haus- man, 2006). Diferencias morfológicas entre los tipos de tejido adiposo Figura 5 Diferencias morfológicas entre el tejido adiposo blanco (unilocular) y el tejido adiposo pardo (multilocular). Mitocondria Gran gota de grasa Núcleo Núcleolo Mitocondrias Aparato de Golgi Gotas de grasa Ret. endoplamáticos (liso y rugoso) Ret. endoplamáticos (liso y rugoso) Tejido adiposo blanco Unilocular Tejido adiposo pardo Multilocular Ret. endoplamáticos (liso y rugoso) Aparato de Golgi Mitocondrias Núcleolo Mitocondria Gran gota de grasa Gotas de grasa Tejido adiposo pardo Multilocular Tejido adiposo pardo Multilocular Tejido adiposo blanco Unilocular Tejido adiposo blanco Unilocular Figura 5 Diferencias morfológicas entre el tejido adiposo blanco (unilocular) y el tejido adiposo pardo (multilocular). 442 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 5.2. Lipólisis Durante la lipólisis, los triacilglicéridos almace- nados en el tejido adiposo son hidrolizados hasta ácidos grasos y glicerol. El paso limitante de este proceso está controlado por la LHS. Esta enzima cataliza la hidrólisis de los tracilglicéridos a monoa- cilglicéridos, los que son finalmente degradados por la enzima monoacilglicerol lipasa. La LHS está sujeta a una fina regulación y se activa por fosfori- lación realizada por una kinasa activada por AMPc. La lipólisis es estimulada por todos los agentes que estimulen a la enzima adenilato-ciclasa y aumenten la formación de AMPc, como ocurre con las cateco- laminas que activan los receptores beta adrenérgi- cos. Por el contrario, la lipólisis es inhibida por aquellas hormonas que inhiban la formación de AMPc. Esto ocurre con catecolaminas que actúan a través de los receptotes alfa 2 adrenérgicos. Las catecolaminas tienen, por lo tanto, un efecto dual sobre la lipólisis y por ello su efecto lipolítico neto depende del balance entre la activación de recep- tores beta y alfa 2 adrenérgicos. Otras hormonas inhibitorias de la lipólisis, como es el caso de la in- sulina, actúan a través de receptores que están asociados a la formación de fosfatidilinositol (IP3), otro importante segundo mensajero que regula di- ferentes funciones celulares (Halliwell et al., 1996; Mauriège et al., 1999). Factores reguladores de la diferenciación de los adipocitos Proadipogénica Antiadipogénica Insulina Ácido retinoico Isobutil metilxantina (IBMX) Esteres de forbol Ácidos grasos poliinsaturados Resistina Tiazolidinedionas Prostaglandina F2 Prostaciclina PGI2 Arseniato de sodio Angiotensina Interferon gamma Glucocorticoides Inhibidores mitóticos Hormona de crecimiento Factor de crecimiento epidérmico Factores reguladores de la diferenciación de los adipocitos Proadipogénica Antiadipogénica Insulina Ácido retinoico Isobutil metilxantina (IBMX) Esteres de forbol Ácidos grasos poliinsaturados Resistina Tiazolidinedionas Prostaglandina F2 Prostaciclina PGI2 Arseniato de sodio Angiotensina Interferon gamma Glucocorticoides Inhibidores mitóticos Hormona de crecimiento Factor de crecimiento epidérmico Ácido retinoico Esteres de forbol Resistina Prostaglandina F2 Arseniato de sodio Interferon gamma Inhibidores mitóticos Factor de crecimiento epidérmico Insulina Isobutil metilxantina (IBMX) Ácidos grasos poliinsaturados Tiazolidinedionas Prostaciclina PGI2 Angiotensina Glucocorticoides Hormona de crecimiento Figura 6 Efecto de diferentes hormonas y de sustancias químicas en la diferenciación de los adipositos. Figura 6 Efecto de diferentes hormonas y de sustancias químicas en la diferenciación de los adipositos. ALFONSO VALENZUELA B Y JULIO SANHUEZA C ALFONSO VALENZUELA B Y JULIO SANHUEZA C Eventos tardíos y diferenciación terminal La figura 6 muestra algunos de estos factores. Factores reguladores de la diferenciación de los adipocitos Proadipogénica Antiadipogénica Insulina Ácido retinoico Isobutil metilxantina (IBMX) Esteres de forbol Ácidos grasos poliinsaturados Resistina Tiazolidinedionas Prostaglandina F2 Prostaciclina PGI2 Arseniato de sodio Angiotensina Interferon gamma Glucocorticoides Inhibidores mitóticos Hormona de crecimiento Factor de crecimiento epidérmico Figura 6 Efecto de diferentes hormonas y de sustancias químicas en la diferenciación de los adipositos. 5. EL TEJIDO ADIPOSO COMO ÓRGANO DE ALMACENAMIENTO Una de las funciones tradicionalmente bien es- tudiadas del tejido adiposo es su función como al- macenador y liberador de energía, lo que ocurre a través de dos procesos, la lipogénesis y la lipoge- nólisis. Durante la fase final de la diferenciación, los adi- pocitos en cultivo incrementan marcadamente la li- pogénesis de novo observándose, por lo tanto, un incremento en la expresión y la actividad de las en- zimas implicadas en esta ruta, tales como la enzi- ma sintetasa de ácidos grasos, la enzima málica y la enzima glicerol-3-fosfato deshidrogenasa. Du- rante esta etapa, también aumenta considerable- mente la sensibilidad a la insulina, debido a un gran aumento del número de receptores de insulina y de transportadores de glucosa dependientes de insu- lina (Glut4). La diferenciación de los adipocitos conlleva a un incremento total en el número de receptores adrenérgicos. Además, se produce un aumento de la síntesis de aP2, una proteína fijado- ra de ácidos grasos específica de los adipocitos, y de la perilipina, la proteína asociada en forma peri- férica a las gotas de lípidos. En esta etapa los adi- pocitos comienzan a secretar algunas sustancias endocrinas y paracrinas tales como la leptina, la adipsina, y la angiotensina (Spiegelman et al., 1983; Morrison & Farmer, 2000; Tchkonia et al., 2002). Existe una gran variedad de factores que re- gulan la diferenciación de los adipocitos, tanto de carácter proadipogénico como antiadipogénico. La figura 6 muestra algunos de estos factores. 5. EL TEJIDO ADIPOSO COMO ÓRGANO DE ALMACENAMIENTO Una de las funciones tradicionalmente bien es- tudiadas del tejido adiposo es su función como al- macenador y liberador de energía, lo que ocurre a través de dos procesos, la lipogénesis y la lipoge- nólisis. No se sabe si la localización del tejido adiposo próxima a órganos vitales, como el corazón, ejerce solo funciones de soporte mecánico o cumple un rol metabólico como fuente local de energía. No es sorprendente que el tejido adiposo esté diseñado para responder a cambios nutricionales específi- cos. De hecho, los adipocitos están preparados pa- ra responder tanto a cambios hormonales como nerviosos (Gillum, 1987). 4.2. La diferenciación del adipocito La diferenciación de los adipocitos es un proce- so complejo en el que los preadipocitos deben inte- rrumpir su crecimiento y salir del ciclo celular como 443 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 Figura 6 Efecto de diferentes hormonas y de sustancias químicas en la diferenciación de los adipositos. Eventos tardíos y diferenciación terminal Los triacilglicéridos que contienen estas lipoproteí- nas son hidrolizados hasta ácidos grasos libres y monoacilglicerol por la LPL que se encuentra adhe- rida a los capilares que irrigan el tejido adiposo. Los ácidos grasos son captados por los adipocitos a través de procesos de transporte activo mediados por proteínas transportadoras específicas de áci- dos grasos. Una vez en el interior de la célula, los ácidos grasos son reesterificados a triacilglicéridos y depositados en el interior de la gran gota de gra- sa incluida en los adipocitos. El término lipogénesis de novo significa específicamente la formación de ácidos grasos a partir de algún precursor derivado del adipocito, por ejemplo, la glucosa. En los huma- nos, el almacenamiento de los ácidos grasos en el tejido adiposo depende prácticamente de la libera- ción de los mismos desde las lipoproteínas por la acción de la LPL. Sin embargo, se ha observado que pacientes con deficiencia de LPL son capaces de acumular triacilglicéridos en el tejido adiposo, lo que hace pensar en la existencia de otros mecanis- mos para la acumulación de triacilglicéridos, como lo sería la lipogénesis de novo (Brun et al., 1989; Ranganathan et al., 2006). Eventos tardíos y diferenciación terminal Durante la fase final de la diferenciación, los adi- pocitos en cultivo incrementan marcadamente la li- pogénesis de novo observándose, por lo tanto, un incremento en la expresión y la actividad de las en- zimas implicadas en esta ruta, tales como la enzi- ma sintetasa de ácidos grasos, la enzima málica y la enzima glicerol-3-fosfato deshidrogenasa. Du- rante esta etapa, también aumenta considerable- mente la sensibilidad a la insulina, debido a un gran aumento del número de receptores de insulina y de transportadores de glucosa dependientes de insu- lina (Glut4). La diferenciación de los adipocitos conlleva a un incremento total en el número de receptores adrenérgicos. Además, se produce un aumento de la síntesis de aP2, una proteína fijado- ra de ácidos grasos específica de los adipocitos, y de la perilipina, la proteína asociada en forma peri- férica a las gotas de lípidos. En esta etapa los adi- pocitos comienzan a secretar algunas sustancias endocrinas y paracrinas tales como la leptina, la adipsina, y la angiotensina (Spiegelman et al., 1983; Morrison & Farmer, 2000; Tchkonia et al., 2002). Existe una gran variedad de factores que re- gulan la diferenciación de los adipocitos, tanto de carácter proadipogénico como antiadipogénico. 6. EL TEJIDO ADIPOSO COMO ÓRGANO SECRETOR-ENDOCRINO principales adipokinas secretadas por el tejido adi- poso y sus efectos metabólicos (Vázquez-Vela et al., 2008). El descubrimiento de una serie de sustancias con efectos fisiológicos producidas por los adipoci- tos, permitió el descubrimiento que el tejido adipo- so se comporta como un órgano endocrino, ejer- ciendo variadas acciones reguladoras, siendo capaz de coordinar y administrar cambios en el ba- lance energético y en el estado nutricional general. Los estudios de microscopía electrónica han permi- tido identificar otros tipos de células en el tejido adi- poso, las que al parecer juegan roles importantes en la regulación de la función de este tejido y su interacción con otros tejidos. Se han encontrado pericitos, células endoteliales, monocitos, macrófa- gos y células pluripotenciales (incluyendo preadi- pocitos) como parte del tejido adiposo y que estarían involucradas en la producción de varios factores endocrinos y paracrinos. Los factores secretados por el tejido adiposo, pueden tener efectos en mu- chos sistemas biológicos, incluyendo la homeosta- sis energética (metabolismo de lípidos, carbohidra- tos, control del apetito, termogénesis), sistema inmunológico, función reproductiva, hemostasia y angiogénesis (Ronti et al., 2006; Sethi & Vidal-Puig, 2007). Una función menos conocida de estas sustan- cias es su potencial para mediar eventos paracri- nos-autocrinos en la regulación del desarrollo, ex- pansión y plasticidad del tejido adiposo. Es más, muchas de las mismas adipokinas implicadas en el desarrollo de la insulino resistencia inducida por la obesidad, pueden alterar el metabolismo lipídico y contribuir a alterar la distribución del tejido adiposo corporal. Además de las funciones endocrinas en cuanto al control de la ingesta de alimentos y/o del gasto energético, las adipokinas pueden actuar lo- calmente ejerciendo efectos autocrinos o paracri- nos que pueden alterar las funciones del tejido adi- poso y su capacidad de expansión (Miner, 2004; Sethi & Vidal-Puig, 2007). 5.1. Lipogénesis El tejido adiposo blanco es el mayor reservorio energético del organismo. La principal fuente de triacilglicéridos para alimentar a este tejido provie- ne de los quilomicrones y de las VLDL circulantes. 444 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 Figura 7 Efectos metabólicos de las principales adipokinas secretadas por el tejido adiposo. 7. PAPEL DEL TEJIDO ADIPOSO EN LA SOBREALIMENTACIÓN Y DISREGULACIÓN METABÓLICA La presencia de un aumento sustancial de la masa grasa no explica por si sola los cambios me- tabólicos ni la resistencia insulínica asociada a la obesidad. Esto ha sido demostrado en modelos ex- perimentales con roedores donde se ha podido ge- nerar resistencia periférica a la insulina, aún en au- sencia de tejido adiposo (Kim et al., 2000; Tschöp & Heiman, 2001). De hecho, la pérdida de los depó- sitos grasos exacerba la resistencia insulínica a tra- vés de la lipotoxicidad inducida por la hiperlipemia, lo cual explicaría en parte el porqué se encuentran desórdenes lipodistróficos en personas delgadas. Estas evidencias subrayan la importancia que el te- jido adiposo tenga una serie de mecanismos de control, los que se ejercen aún en personas con cargas nutricionales normales. ) El tejido adiposo también responde a las sobre- cargas nutricionales excesivas alterando la produc- ción de adipokinas. Se entiende por adipokina a to- da proteína secretada por el tejido adiposo cuya producción está alterada en estados de obesidad y que puede afectar las complicaciones metabólicas asociadas a esta condición patológica. Se ha visto que las adipokinas participan en la regulación de la homeostasis de la glucosa en todo el cuerpo (Tray- hurn, 2005). El tejido adiposo sintetiza una variada gama de adipokinas entre las que se cuentan: lep- tina, adiponectina, factor de necrosis tumoral alfa (TNF-), interleukina 6 (IL-6), resistina, proteína 4 que une ácido retinoico (RBP-4), visfatina, omenti- na. Más aún, recientemente se han descubierto otras moléculas como: serpina, lipocalina-2, PAI 1, apelina, chemerina, glicoproteína Zn-2, etc. Todas estas adipokinas en conjunto se encargan de inte- grar tanto la respuesta alimentaria como el gasto energético del organismo. La figura 7 muestra las g Es de conocimiento general que la obesidad se explique por un aumento de la ingesta alimenticia, asociada a una disminución de la actividad física en términos termodinámicos. Sin embargo, se ha ob- servado que la capacidad de expansión y de alma- cenamiento del tejido adiposo está prefijada o limi- tada. ALFONSO VALENZUELA B Y JULIO SANHUEZA C Cuando el tejido adiposo no puede almacenar de manera adecuada una sobrecarga de nutrien- tes, éstos se almacenan en otros lugares como el músculo, hígado, corazón y páncreas. Estos tejidos no están diseñados para almacenar grandes canti- dades de lípidos siendo más susceptibles a los efectos tóxicos de la propia acumulación de grasa (Unger, 1995). Cada órgano presenta su propia respuesta tóxica. Este daño lipotóxico depende de 3 factores: a) de la magnitud y duración del exceso de nutrientes; b) de la funcionalidad de los meca- nismos de transporte y de almacenamiento de áci- dos grasos en los tejidos y; c) de la disminución de la capacidad oxidativa del órgano. podía expandir pasivamente y acomodar cualquier exceso de nutrientes. Actualmente se sabe que el proceso de expansión del tejido adiposo, entendido como hipertrofia e hiperplasia, es un proceso com- plejo que requiere energía. Desde la aparición de la obesidad como epidemia, el tejido adiposo nunca ha estado sometido a tanta presión para acomodar los grandes volúmenes de tejido graso producto del aumento de ingesta alimenticia (Trayhurn, 2005; Vázquez-Vela et al., 2008). podía expandir pasivamente y acomodar cualquier exceso de nutrientes. Actualmente se sabe que el proceso de expansión del tejido adiposo, entendido como hipertrofia e hiperplasia, es un proceso com- plejo que requiere energía. Desde la aparición de la obesidad como epidemia, el tejido adiposo nunca ha estado sometido a tanta presión para acomodar los grandes volúmenes de tejido graso producto del aumento de ingesta alimenticia (Trayhurn, 2005; Vázquez-Vela et al., 2008). q , ) Adicionalmente a la función del tejido adiposo como depósito de energía, se ha visto que este te- jido cumple un papel importante en la regulación de la homeostasis de la glucosa. Esto se puso en evi- dencia en ratones en los que se bloqueó los trans- portadores GLUT4 de los adipocitos, provocando resistencia periférica a la insulina e intolerancia a la glucosa, pero sin alteraciones en la masa de tejido adiposo. Estos hechos revelan que la captación de glucosa por el tejido adiposo juega un rol en la de- tección y administración de adaptaciones en el me- tabolismo de carbohidratos. A nivel de laboratorio, se han desarrollado modelos en roedores en los cuales al producir modificaciones genéticas a nivel de adipocitos se produce obesidad pero sin los componentes propios de esta patología (resisten- cia insulínica, diabetes 2, hígado graso y dislipide- mia). ALFONSO VALENZUELA B Y JULIO SANHUEZA C Estos hallazgos ponen una vez más en evi- dencia la existencia de mecanismos regulatorios finos que operan tanto para determinar la cantidad como la distribución del tejido adiposo (Cinti, et al., 2005; Sethi & Vidal-Puig, 2007). p g La funcionalidad del sistema de transporte de ácidos grasos en los diversos órganos está relacio- nada con las respuestas individuales de cada órga- no a la lipotoxicidad. Existen factores de transcrip- ción, como el SREBP1 C (Steroid Regulatory Element Binding Protein 1), que actúan como vigi- lante contra la lipotoxicidad en los músculos y otros órganos periféricos. La sobre activación de SREBP1 C en éstos tejidos facilita el depósito de triacilglicéridos a largo plazo. Se favorece principal- mente la acumulación de ácidos grasos saturados de cadena larga, los que ejercen un efecto tóxico a nivel de los tejidos. Por otra parte, se ha encontra- do el PPAR, un factor adipogenético propio del tejido adiposo, en órganos como el músculo y el hígado. La sobreactivación de este regulador trans- cripcional estaría también involucrada en la lipoto- xicidad a nivel de músculos e hígado que caracteri- za a la obesidad y a la resistencia a la isulina (Medina-Gomez et al., 2007). 7. PAPEL DEL TEJIDO ADIPOSO EN LA SOBREALIMENTACIÓN Y DISREGULACIÓN METABÓLICA Antes se pensaba que el tejido adiposo se Principales Adipokinas producidas por el tejido adiposo Efecto en Adipokina ingesta de alimentos Efecto titular Leptina Anorexígena Aumenta sensibilidad a insulina en músculo Adiponectina Disminuye o aumenta Aumenta sensibilidad a insulina en músculo según lugar de acción Resistina Anorexígena Reduce capatación de glucosa hepática Interleukina-6 Anorexígena Reduce sensibilidad a insulina en músculo Omentina Sin efecto Aumenta sensibilidad a insulina en músculo Factor de necrosis Anorexígeno Reduce capatación de glucosa hepática tumoral alta (TNF-) Figura 7 Efectos metabólicos de las principales adipokinas secretadas por el tejido adiposo. Principales Adipokinas producidas por el tejido adiposo Efecto en Adipokina ingesta de alimentos Efecto titular Leptina Anorexígena Aumenta sensibilidad a insulina en músculo Adiponectina Disminuye o aumenta Aumenta sensibilidad a insulina en músculo según lugar de acción Resistina Anorexígena Reduce capatación de glucosa hepática Interleukina-6 Anorexígena Reduce sensibilidad a insulina en músculo Omentina Sin efecto Aumenta sensibilidad a insulina en músculo Factor de necrosis Anorexígeno Reduce capatación de glucosa hepática tumoral alta (TNF-) Figura 7 Efectos metabólicos de las principales adipokinas secretadas por el tejido adiposo. Principales Adipokinas producidas por el tejido adiposo 445 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 8. RESPUESTA DEL TEJIDO ADIPOSO A LA SOBREALIMENTACIÓN ( ) La asociación de los tres factores ya menciona- dos determina la calidad y cantidad de los lípidos intracelulares disponibles para regular las señales celulares, lo cual constituye actualmente un terreno de investigación en materia de nuevos enfoques te- rapéuticos para la obesidad. Existen aún controver- sias sobre qué tipos de especies lipídicas están relacionadas con la capacidad de producir lipotoxi- cidad. Generalmente se ha observado que la acu- mulación ectópica de triacilglicéridos en el tejido hepático y muscular es un signo de lipotoxicidad directamente relacionado con la resistencia a la in- sulina asociada a la obesidad. Sin embargo, algu- nos estudios establecen que la distribución ectópi- ca de triglicéridos por sí sola no sería causa de lipotoxicidad. Algunos investigadores consideran que la acumulación de triacilglicéridos en órganos periféricos bajo condiciones de balance energético positivo, reflejarían la capacidad de un determina- do órgano de funcionar como depósito de nutrien- tes. Lo lógico sería pensar que inhibiendo selec- tivamente la absorción de triacilglicéridos y su consecuente depósito tisular, se reduciría el riesgo de lipotoxicidad. Sin embargo, los estudios mues- tran una inducción de un estado más tóxico por la acumulación de otros lípidos más reactivos, hecho que se demostró al bloquear genéticamente la ex- presión (síntesis) del PPAR en el hígado de rato- nes ob/ob (genéticamente obesos), en los que la El tejido adiposo responde a la sobrealimenta- ción mediante un marcado aumento de su masa, a través de mecanismos de hipertrofia e hiperplasia, provocando obesidad clínica y sus repercusiones metabólicas. Una de las complicaciones de mayor prevalencia en la actualidad lo constituye el des- arrollo de insulino resistencia. Existen 2 hipótesis que explican la insulino resistencia inducida por la obesidad (Sethi & Vidal-Puig, 2007). 1. Existiría una deficiencia para acomodar los excesos de energía producidos por la sobrealimen- tación en el organismo. Esto resultaría en el acu- mulo de éstos componentes lipídicos en otros órga- nos como los músculos y el hígado. Este acumulo inapropiado afectaría la respuesta metabólica nor- mal de éstos tejidos al uso metabólico de la gluco- sa producido por la disminución en ellos de los transportadores de tipo GLUT (Herman & Kahn, 2006). 2. La sobrecarga nutricional en el tejido adiposo estimularía cambios cuali y cuantitativos en la pro- ducción de adipokinas. Estas adipokinas actuarían como moduladores de la sensibilidad a la insulina desencadenando resistencia a la insulina local y sistémica (Trayhurn, 2005). EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA ausencia de depósitos de triacilglicéridos en un contexto de balance energético positivo, provoca mayor resistencia insulínica y falla metabólica ge- neralizada (Medina-Gomez et al., 2007; Sethi & Vi- dal-Puig, 2007). Leptina: Una de las principales adipokinas que libera el tejido adiposo blanco. El aumento de la se- creción de esta hormona, conlleva a la disminución de la ingesta de alimentos, al actuar a nivel de hi- potálamo, específicamente en el núcleo paraventri- cular (Cowley et al., 2001). g, ) Un punto clave en el entendimiento del meca- nismo de toxicidad de las diferentes especies lipídi- cas estaría dado por el estudio de mediadores in- tracelulares de resistencia insulínica inducida por lípidos. En la circulación los ácidos grasos libres, producto de la lipólisis, son inductores directos de insulino resistencia y diabetes 2, particularmente en obesos y en pacientes dislipidémicos. Es más, la infusión de lípidos en roedores promueve la glu- coneogénesis y la salida de glucosa del hígado. Los niveles de glicemia permanentemente altos provocan una disminución de la secreción de insu- lina por parte de las células del páncreas a través de mecanismos lipotóxicos aún no bien definidos (Unger, 1995; Bergman & Ader, 2000). Apelina: Se ha descubierto que es capaz de re- gular la contractibilidad cardíaca y al mismo tiempo disminuir la presión sanguínea, pero adicionalmen- te se ha observado que tiene un efecto similar al de la leptina en el sentido que es responsable de dis- minuir la ingesta de alimentos (O’Shea et al., 2003). Aromatasa: Esta es en realidad una enzima que contiene el tejido adiposo que convierte la hormona androstenediona en un estrona. Esta es una hor- mona que además de inducir la acumulación de grasa mamaria, se ha descubierto que su presen- cia en humanos aumenta la acción de la leptina y al mismo tiempo, el transporte de la leptina en el ce- rebro por lo que, indirectamente, disminuye la in- gesta de alimentos (Mattsson & Olsson, 2007). ( g g ) En el tejido muscular y adiposo el aumento de ácidos grasos libres inhibe la sensibilidad a la insu- lina y la captación de glucosa estimulada por la in- sulina. La hipótesis comúnmente aceptada era que la hiperglicemia es provocada por una inhibición de la captación de glucosa por inhibición, por compe- tencia, con los ácidos grasos. EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA Actualmente se pos- tulan mecanismos que provocan cambios a nivel de la composición lipídica de la membrana celular con un marcado aumento de lípidos reactivos en el me- dio intracelular como el diacil glicerol (DAG) y las ceramidas. Estos lípidos reactivos activarían kina- sas como la IRS-I (receptor catalítico de insulina) a través de la fosforilación de residuos de serina, re- duciendo la función normal del receptor y el efecto de la insulina. Con respecto a las ceramidas, se ha visto que ciertos derivados de ceramidas, como los gangliósidos asociados a la membrana, se compor- tan como mediadores importantes en la insulino re- sistencia inducida por lípidos. Es más, actuarían in- hibiendo específicamente la actividad de la enzima glucosil-ceramida sintetasa (la principal enzima en la síntesis de glucoesfingolípidos). Se puede rever- tir la insulino resistencia en numerosos modelos de obesidad a través de la disminución de los niveles de ceramidas. Cabe destacar, además, que las ce- ramidas están importantemente involucradas en el desarrollo de la apoptósis o muerte celular progra- mada (Aerts et al., 2007; Straczkowski et al., 2007). Adiponectina: Se le ha atribuido un rol cardio- protector y específicamente de disminuir la resis- tencia a la insulina y cuya acción se logra al actuar a nivel de tejidos periféricos, como el músculo, en el que favorece la entrada de glucosa. Reciente- mente, los estudios indican un rol doble de la adi- ponectina en cuanto a la ingesta de alimentos, ya que cuando actúa a nivel del núcleo paraventricular disminuye la ingesta y adicionalmente aumenta la actividad de la UCP-1 por lo que el gasto energéti- co también aumenta. Sin embargo, cuando actúa a nivel del núcleo arcuato del hipotálamo, se observa un aumento de la ingesta de alimentos, y disminu- ye el gasto energético. Por ello, actualmente se re- quiere de mayor cantidad de investigaciones sobre el rol de adiponectina a nivel del cerebro (Shklyaev et al., 2003; Qi et al., 2004). Resistina: Estudios en humanos muestran que esta molécula es secretada por linfocitos mononu- cleares y por células estromales que se encuentran dentro del tejido adiposo. La acción comprobada de esta adipokina es la de disminuir transitoriamente la ingesta de alimentos, esto es el lapso entre la in- gesta y los primeros 90 minutos post ingesta (Tovar et al., 2005). EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA , ) Actividad 11-hidroxiesteroide deshidrogenada: Esta es una enzima del tejido adiposo blanco que se encarga de convertir el cortisol en cortisona. Se ha encontrado correlación positiva de la actividad de esta enzima con la cantidad de grasa corporal. Aunque en el sentido estricto, la cantidad de corti- coides aumenta con el estrés, se ha observado una acción dual de estas hormonas. En general, en la mayoría de las personas, un aumento de los corti- coides aumenta el consumo de alimento, pero en un pequeño grupo en iguales circunstancias, dismi- nuye la ingesta de alimentos (Henry & Clarke, 2008). 8. RESPUESTA DEL TEJIDO ADIPOSO A LA SOBREALIMENTACIÓN 446 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 ALFONSO VALENZUELA B Y JULIO SANHUEZA C Bhakoo O. 1974. Minimal rates of oxygen comsumption in small for dates during the first week of life. Arch. Dis. Child. 49, 583-590. tejido en el organismo, sería la de mantener el pe- so, y conjuntamente regular el gasto energético. Por otra parte, otros órganos producen hormonas y/o neurotransmisores que aumentan la ingesta de alimentos siendo la principal y la más importante, la ghrelina que es sintetizada por el estómago, pero además se suman otras moléculas como las endor- finas, encefalinas, adrenalina, por mencionar algu- nas (Murphy & Bloom, 2006) Por consiguiente, un adecuado peso y un gasto energético que permita conservar este peso, dependerá de la integración de las señales que inhiben la ingesta de alimentos, como de aquellas que la inducen, lo que permite en cada momento controlar la homeostasis energética y de la ingesta. Por lo tanto, actualmente, además de considerar la actividad física como un regulador del peso corporal, cada vez toma mayor fuerza el concepto que la obesidad la debemos entender co- mo un desequilibrio en la función del tejido adiposo, es decir un tejido adiposo normal pasa a convertir- se en un tejido adiposo obesogénico. tejido en el organismo, sería la de mantener el pe- so, y conjuntamente regular el gasto energético. Por otra parte, otros órganos producen hormonas y/o neurotransmisores que aumentan la ingesta de alimentos siendo la principal y la más importante, la ghrelina que es sintetizada por el estómago, pero además se suman otras moléculas como las endor- finas, encefalinas, adrenalina, por mencionar algu- nas (Murphy & Bloom, 2006) Por consiguiente, un adecuado peso y un gasto energético que permita conservar este peso, dependerá de la integración de las señales que inhiben la ingesta de alimentos, como de aquellas que la inducen, lo que permite en cada momento controlar la homeostasis energética y de la ingesta. Por lo tanto, actualmente, además de considerar la actividad física como un regulador del peso corporal, cada vez toma mayor fuerza el concepto que la obesidad la debemos entender co- mo un desequilibrio en la función del tejido adiposo, es decir un tejido adiposo normal pasa a convertir- se en un tejido adiposo obesogénico. , Bergman RN, Ader M. 2000. Free fatty acids and patho- genesis of type 2 diabetes mellitus. Trends. Endocri- nol. Metab. 11, 351-356. Brun LD, Gagne C, Julien P, Tremblay A, Moorjani S, Bou- chard C, Lupien PJ. 1989. ALFONSO VALENZUELA B Y JULIO SANHUEZA C Familial lipoprotein lipase- activity deficiency: study of total body fatness and subcutaneous fat tissue distribution. Metabolism 38, 1005-1009. Cannon B, Nedergaard J. 2004. Brown adipose tissue: function and physiological significance. Physiol. Rev. 84, 277-359. Cinti S. 2005. The adipose organ. Prostaglandins Leukot. Essent. Fatty Acids 73, 9-15. Cinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Fa- loia E, Wang S, Fortier M, Greenberg AS, Obin MS. 2005. Adipocyte death defines macrophage localiza- tion and function in adipose tissue of obese mice and humans. J. Lipid. Res. 46, 2347-2355. p Cinti S. 2006. The role of brown adipose tissue in human obesity. Nutr. Metab. Cardiovasc. Dis. 16, 569-574. Cowley MA, Smart JL, Rubinstein M, Cerdán MG, Diano S, Horvath TL, Cone RD, Low MJ. 2001. Leptin activa- tes anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411, 480-484. 10. CONCLUSIONES Djian P, Phillips M, Green H 1985. The activation of spe- cific gene transcription in the adipose conversion of 3T3 cell. J. Cell. Physiol. 124, 554-556. Se sigue investigando activamente en el tejido adiposo y con el paso de los años se han puesto en evidencia una serie de roles y funciones hasta hace poco completamente desconocidas para este teji- do, incluyendo la homeostasis energética (metabo- lismo de lípidos, carbohidratos, control del apetito, termogénesis), el sistema inmunológico, la función reproductiva, la hemostasia y la angiogénesis. Un capítulo de gran actualidad lo constituye el tejido adiposo y su rol tóxico a nivel metabólico (lipotoxici- dad) dada su enorme relevancia clínica y potencial terapéutico-farmacológica. Debemos esperar en los próximos años nuevos descubrimientos sobre este particular e importante tejido, que como ya hemos visto, es algo más que un reservorio energético. Entingh AJ, Taniguchi CM, and Kahn CR. 2003. Bi-direc- tional regulation of brown fat adipogenesis by the in- sulin receptor. J. Biol. Chem. 278, 33377-33383. Farmer SR. 2006. Transcriptional control of adipocyte for- mation. Cell. Metab. 4, 263-273. Garaulet M, Hernandez-Morante JJ, Lujan J, Tebar FJ, Zamora S. 2006. Relationship between fat cell size and number and fatty acid composition in adipose tis- sue from different fat depots in overweight/obese hu- mans. Int. J. Obes. 30, 899-905. Geloen A, Roy PE, Bukowiecki LJ. 1989. Regression of white adipose tissue in diabetic rats. Am. J. Physiol. 257, E547-553. Gillum RF. 1987. The association of body fat distribution with hypertension, hypertensive heart disease, coro- nary heart disease, diabetes and cardiovascular risk factors in men and women aged 18-79 years. J. Chro- nic. Dis. 40, 421-428. 9. EL TEJIDO ADIPOSO PUEDE REGULAR LA INGESTA DE ALIMENTOS Y EL GASTO ENERGÉTICO Como ya se mencionó, el tejido adiposo sinteti- za una gran variedad de adipokinas, las que se in- tegran a la acción de otras moléculas reguladoras que generan otros órganos, como es el caso del cerebro y del estómago, y que en conjunto partici- pan en la regulación de la ingesta y del gasto ener- gético (Murphy & Bloom, 2006). Revisaremos algu- nas moléculas que participan en estos procesos: En resumen, hay evidencia que el tejido adipo- so secreta varias moléculas que tienen la capaci- dad de disminuir la ingesta de alimentos, de tal ma- nera que la tendencia fisiológica natural de este 447 GRASAS Y ACEITES, 60 (5), OCTUBRE-DICIEMBRE, 437-450, 2009, ISSN: 0017-3495, DOI: 10.3989/gya.043209 EL TEJIDO ADIPOSO: ALGO MÁS QUE UN RESERVORIO DE ENERGÍA Henry BA, Clarke IJ. 2008. Adipose tissue hormones and the regulation of food intake. J. Neuroendocrinol. 20, 842-849. 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https://openalex.org/W4389385276	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1011645&type=printable	English	null	Major depressive disorder and bistability in an HPA-CNS toggle switch	PLOS computational biology/PLoS computational biology	2,023	cc-by	11,035	Ben Ron Mizrachi, Avichai Tendler, Omer Karin, Tomer Milo, Dafna Haran, Avi MayoID, Uri AlonID* Dept. Molecular Cell biology, Weizmann Institute of Science, Rehovot, Israel * urialonw@gmail.com Editor: James R. Faeder, University of Pittsburgh, UNITED STATES Received: May 18, 2023 Accepted: November 1, 2023 Published: December 6, 2023 Received: May 18, 2023 Accepted: November 1, 2023 Published: December 6, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pcbi.1011645 Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Major depressive disorder (MDD) is the most common psychiatric disorder. It has a complex and heterogeneous etiology. Most treatments take weeks to show effects and work well only for a fraction of the patients. Thus, new concepts are needed to understand MDD and its dynamics. One of the strong correlates of MDD is increased activity and dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis which produces the stress hormone cortisol. Existing mathematical models of the HPA axis describe its operation on the scale of hours, and thus are unable to explore the dynamic on the scale of weeks that characterizes many aspects of MDD. Here, we propose a mathematical model of MDD on the scale of weeks, a timescale provided by the growth of the HPA hormone glands under control of HPA hor- mones. We add to this the mutual inhibition of the HPA axis and the hippocampus and other regions of the central nervous system (CNS) that forms a toggle switch. The model shows bistability between euthymic and depressed states, with a slow timescale of weeks in its dynamics. It explains why prolonged but not acute stress can trigger a self-sustaining depressive episode that persists even after the stress is removed. The model explains the weeks timescale for drugs to take effect, as well as the dysregulation of the HPA axis in MDD, based on gland mass changes. This understanding of MDD dynamics may help to guide strategies for treatment. OPEN ACCESS Citation: Ron Mizrachi B, Tendler A, Karin O, Milo T, Haran D, Mayo A, et al. (2023) Major depressive disorder and bistability in an HPA-CNS toggle switch. PLoS Comput Biol 19(12): e1011645. https://doi.org/10.1371/journal.pcbi.1011645 OPEN ACCESS Editor: James R. Faeder, University of Pittsburgh, UNITED STATES RESEARCH ARTICLE Major depressive disorder and bistability in an HPA-CNS toggle switch Ben Ron Mizrachi, Avichai Tendler, Omer Karin, Tomer Milo, Dafna Haran, Avi MayoID, Uri AlonID* PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Introduction Major depressive disorder (MDD) is the most common psychiatric syndrome, affecting about 20% of the world population at some point in life [1]. There were 21 million recorded cases of depression in adults in 2020 in the USA, about 8% of the adult population [2]. MDD in adoles- cents and children has risen sharply in the past decade in many countries [3]. People with MDD suffer from depressed mood or sadness, anhedonia, and may suffer from sleeping and eating disorders. The etiology of MDD is complex and heterogeneous, and multiple genetic and environmental factors contribute to its incidence [4]. Treatment for MDD includes medication, psychotherapy, physical exercise, and electro- convulsive therapy (ECT). Treatment is usually effective for only a fraction of the patients [5,6] and often takes several weeks to show effects [7]. In the past five decades few novel therapeutics for MDD have emerged that go beyond the monoamine theory of depression first developed in the 1960s [8]. Among the endocrine, inflammatory, metabolic and neuronal factors that have been explored, the stress hormone cortisol is one of the most significant correlates of MDD [9]. Stress is associated with MDD, and cortisol is higher than controls in most studies of people with MDD [10]. High cortisol is not only a readout of MDD—it can also be causal for depres- sion. This is seen in Cushing’s syndrome in which a tumor induces high levels of cortisol, resulting in depression in over 50% of patients [11–13]. Similarly, prolonged high-dose gluco- corticoid treatment, such as the cortisol analogues dexamethasone (DEX) or prednisone taken for a few weeks or longer, is associated with depression in a dose-dependent manner [14]. Furthermore, the endocrine system that secretes cortisol, the hypothalamic-pituitary-adre- nal (HPA) axis, is dysregulated in many patients with MDD [15]. Enlarged adrenal glands in suicide victims and stressed animals were one of the clues that led to characterization of the HPA axis [16]. HPA dysregulation in patients with MDD is evident in enlarged adrenal glands [17,18], blunted adrenocorticotropic hormone (ACTH) response in corticotropin-releasing- hormone (CRH) tests [19] and elevated ACTH response in DEX-CRH tests [20,21]. MDD also involves dysfunctions of the central nervous system (CNS) such as impaired memory and hippocampal atrophy [22–24]. The CNS and HPA have mutual interactions involved in MDD. The hippocampus and the prefrontal cortex have inhibitory effects on HPA activation [25–27]. benron20/MDD-and-bistability-in-the-HPA-stress- axis. also explains why prolonged stress—but not brief stress—can trigger a sustained depres- sive episode. The model further sheds light on why antidepressant drugs take weeks to take effect. By comprehending the dynamics of depression, this model may provide valu- able insights for guiding treatment strategies in the future. Funding: This project was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 856487). 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. Author summary Major depressive disorder (MDD) is a prevalent psychiatric condition whose mechanisms are not fully understood. Existing treatments often take weeks to be effective and only benefit a portion of patients, necessitating a fresh perspective on understanding MDD. Research has shown a strong association between MDD and the dysregulation of the human stress hormone pathway. However, previous mathematical models of the stress hormone pathway have only explored its dynamics over a few hours or days, not captur- ing the weeks-long timescale relevant to MDD. To address this, we present a new mathe- matical model that incorporates the growth of the stress hormone glands, and their mutually inhibitory interactions with the brain. This model reveals how the brain and the stress axis toggle between normal and depressed states, with slow dynamics over weeks. It Copyright: © 2023 Ron Mizrachi 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: Code files, animations, generated by Mathematica, of the phase portraits under changing u, a or b parameters can be found here: https://github.com/ 1 / 19 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch benron20/MDD-and-bistability-in-the-HPA-stress- axis. Introduction Conversely, high levels of cortisol can have inhibitory effects on these CNS regions. Cortisol is sensed by glucocorticoid receptors (GR) in the hippocampus and prefron- tal cortex, causing reduced neurogenesis, synaptic arbor reduction, neuronal death [28,29], and a long-term decrease in CNS volume [30]. This interplay between the CNS and HPA is believed to be mechanistically important for MDD [26]. There are several open questions concerning the origin and dynamics of MDD. How does prolonged but not acute stress trigger depression? Once triggered, how does depression persist even after the stressor is gone? What is the origin of the timescale of weeks or longer of each depressive episode as well as the timescale for drugs to take effect? What is the origin of the dif- ference between individuals susceptible to MDD and those not susceptible, in which a given 2 / 19 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch stress triggers depression in the former and not the latter? Understanding these questions can help to guide MDD research and treatment. One way to explore these open questions is to use mathematical modeling [31,32]. Some of the previous mathematical models of MDD did not include specific physiological mechanisms. They show recurrence of depressive episodes [33]. More mechanistic models based on the HPA axis describe the dynamics of hormones and their receptors on the scale of hours [34– 43]. Some show the interplay between the CNS and HPA [39], and present bistability dynamics as an explanation of stress vulnerability and MDD [36,41,42,44]. These HPA models do not discuss the timescale of weeks which is seen in many aspects of MDD. To address the dynamics of MDD on the scale of weeks, we consider the interplay between the HPA and the CNS within a minimal mathematical model. The model supplies the relevant timescale of weeks by including the slow growth of HPA glands under control of HPA hor- mones, an approach recently developed in our group [45–47]. We add to this the mutual inhi- bition by the CNS to obtain a new model which describes the mechanism of depression as a bistable system with a slow dynamical timescale of weeks. The model can explain why pro- longed but not acute stress triggers depression, why treatment can take weeks, and why the HPA axis is dysregulated in MDD. Results Toggle switch model of the HPA—CNS system Introduction It also reveals a hysteresis property that makes recovery from depression more difficult than entry into it. Toggle switch model of the HPA—CNS system We present a minimal mathematical model of the interaction between the HPA axis and the CNS processes that inhibit it (Fig 1A). The model has two variables: the functional mass of the cortisol-secreting cells in the adrenal cortex, A, and the inhibitory activity of the CNS, h. The inhibitory activity h is inspired by the hippocampus, which inhibits the hypothalamic inputs to the HPA axis [26]. Our approach is agnostic, however, to the exact nature of the CNS variable h, which could comprise in addition to the hippocampus, also other regions including the pre- frontal cortex [27,48]. The main idea is that cortisol produced by the HPA axis acts through the glucocorticoid receptors (GR) [30,49] to inhibit the inhibitory CNS activity h [26]. This activity, in turn, inhibits the HPA axis. The mutual inhibition creates a toggle-switch circuit: A and h inhibit each other (Fig 1B). Since the adrenal cortex functional mass A changes over weeks, a slow timescale is introduced to the dynamics of the system. The model can be compactly understood by the nullcline method, an approach which sepa- rates the toggle switch into two arms [50,51]. We first treat each arm independently, and then combine them to find the fixed points of the system, given by the crossing points of the nullclines. The conclusions are robust in the sense that they depend only on the qualitative shapes of the nullclines and not on their precise mathematical forms. This is useful for MDD in which many of the physiological mechanisms are not yet fully characterized. In addition to the quali- tative approach, we describe in Methods a specific mathematical model based on a model for the HPA axis [45] together with equations for the CNS inhibitory activity h. The Methods sec- tion details the equations and parameters used to simulate the dynamics. The first nullcline describes the inhibition of the HPA axis by the CNS. It is defined by the adrenal cortex mass steady state, dA dt ¼ 0, as a function of the inhibitory activity h. h inhibits the HPA axis, since a large h reduces CRH [26] the inducer of ACTH which is the growth factor for the adrenal cortex A. If one clamps h at a given value, A changes its functional mass over the timescale of weeks to reach a steady state Ast. Toggle switch model of the HPA—CNS system The higher h, the lower the HPA activity and PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 3 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch B A D adrenal cortex, A C CNS inhibitory activity, h CNS inhibitory activity, h adrenal cortex, A Separatrix h’=0 A’=0 Separatrix h’=0 A’=0 euthymic state only euthymic state depressed state Fig 1. Mathematical model for the interactions between the HPA axis and its inhibitory CNS regions. A) Schematic of HPA and CNS interactions in the model (icons taken from Biorender [52] according to their Academic License Terms [53]. B) The model can be summarized as a toggle switch between CNS inhibitory activity h and the adrenal mass, A. CNS inhibits the hypothalamic input and thus leads to a decrease in A. A releases cortisol, which inhibits CNS through the glucocorticoid receptor GR. C) Nullclines show three fixed points in susceptible individuals. Euthymic and depressed states are marked in black dots. D) In non-susceptible individuals there is only one euthymic fixed point. C and D used identical parameters except stress input u = 0.75 and u = 0.4 respectively. An analogous shift between 3 and 1 fixed points can occur when changing the CNS system parameters to represent genetic variants. Figure created with Biorender. https://doi.org/10.1371/journal.pcbi.1011645.g001 OS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch A B A B A D adrenal cortex, A CNS inhibitory activity, h Separatrix h’=0 A’=0 euthymic state only D adrenal cortex, A C CNS inhibitory activity, h CNS inhibitory activity, h adrenal cortex, A Separatrix h’=0 A’=0 Separatrix h’=0 A’=0 euthymic state only euthymic state depressed state Fig 1. Mathematical model for the interactions between the HPA axis and its inhibitory CNS regions. A) Schematic of HPA and CNS interactions in the model (icons taken from Biorender [52] according to their Academic License Terms [53]. B) The model can be summarized as a toggle switch between CNS inhibitory activity h and the adrenal mass, A. CNS inhibits the hypothalamic input and thus leads to a decrease in A. A releases cortisol, which inhibits CNS through the glucocorticoid receptor GR. C) Nullclines show three fixed points in susceptible individuals. Euthymic and depressed states are marked in black dots. D) In non-susceptible individuals there is only one euthymic fixed point. Toggle switch model of the HPA—CNS system C and D used identical parameters except stress input u = 0.75 and u = 0.4 respectively An analogous shift between 3 and 1 fixed points can occur when changing the CNS system parameters to represent genetic variants Figure created C CNS inhibitory activity, h adrenal cortex, A Separatrix h’=0 A’=0 euthymic state depressed state D Separatrix euthymic state only adrenal cortex, A adrenal cortex, A adrenal cortex, A Fig 1. Mathematical model for the interactions between the HPA axis and its inhibitory CNS regions. A) Schematic of HPA and CNS interactions in the model (icons taken from Biorender [52] according to their Academic License Terms [53]. B) The model can be summarized as a toggle switch between CNS inhibitory activity h and the adrenal mass, A. CNS inhibits the hypothalamic input and thus leads to a decrease in A. A releases cortisol, which inhibits CNS through the glucocorticoid receptor GR. C) Nullclines show three fixed points in susceptible individuals. Euthymic and depressed states are marked in black dots. D) In non-susceptible individuals there is only one euthymic fixed point. C and D used identical parameters except stress input u = 0.75 and u = 0.4 respectively. An analogous shift between 3 and 1 fixed points can occur when changing the CNS system parameters to represent genetic variants. Figure created with Biorender. https://doi.org/10.1371/journal.pcbi.1011645.g001 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 4 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch hence the smaller Ast. This provides a decreasing curve plotted in the phase plane whose axes are A and h (Fig 1C, red line). In the model described in Methods, solving Eq (18) gives a specific form for this nullcline, A / u h so that A drops with h. Note for future reference that adrenal mass A rises with the stress input u. The second arm of the toggle switch, in which A inhibits h, is given by steady state CNS activity hst as a function of A. The steady state is the value of h at which dh dt ¼ 0. Due to the effects of cortisol, h declines with A. Toggle switch model of the HPA—CNS system Since cortisol inhibits the activity of relevant brain regions such as the hippocampus and the prefrontal cortex through the GR receptor, which has a coop- erative (step like) activity dependence on cortisol, we assume that h goes from a high level hhigh to a low level hlow in a step-like manner (Fig 1C, black line) when cortisol produced by A crosses the dissociation constant kd of the GR receptor. The HPA-CNS model gives the steady state solution hst ¼ 1 GRðx3Þ, which is a step-like func- tion due to the high cooperativity of the GR receptor with a Hill coefficient of about 3. The qualitative shapes of the nullclines are more general than the specific model equations and suffice for many of the conclusions below. Model shows bistability between euthymic and depressed states There are two main scenarios for the model nullclines, which correspond to individuals sus- ceptible and resilient to MDD. At the points where the nullclines intersect, the system is at steady state, because both dA dt ¼ 0 and dh dt ¼ 0. In the first scenario, the two nullclines intersect at three fixed points (Fig 1C). One fixed point has low adrenal cortex mass, A, and high CNS inhibition, h, and can be considered as the euthymic or healthy state. A second fixed point has high adrenal mass A and low CNS inhi- bition h, and represents the depressed state, with high cortisol and an enlarged adrenal cortex. In between, there is an unstable fixed point. This system is bistable: depending on initial condi- tions, it reaches either the healthy or the MDD state. There is a basin of attraction for each state. The boundary between these basins is a curve called the separatrix which goes through the unstable fixed point. In the second scenario, the parameters of the nullclines are such that there is only a single intersection point (Fig 1D). This is the healthy state with low A—the MDD state with high A no longer exists. Such individuals are resilient with respect to major depressive disorder. All initial conditions eventually flow to the healthy fixed point. Susceptibility to MDD is partly due to genetics [11,14]. To conceptualize this susceptibility using the model we note that most MDD-related mutations are not in HPA-related genes (except for genes related to GR), and thus we may assume that the A nullcline is similar between individuals. On the other hand, the h nullcline may depend on many genetic variants that affect the CNS. Multi-gene variations might push this nullcline up or down, and contrib- ute to the hereditary component of susceptibility or resilience to MDD [54]. We analyzed which parameters changes most sensitively change the number of fixed points (Table 1). Prolonged but not acute stress can trigger self-sustained depression The model can help understand the differential effects of prolonged and acute stress. Stress can be modeled by an increase in hypothalamic input u. This shifts the A nullcline up and to the right—because at a given level of CNS inhibition, high stress leads to an enlarged adrenal relative to the low stress condition. It also shifts the h nullcline to the left—because higher stress is more likely to activate GR receptors in the CNS. In the bistable case, a large enough Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 5 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch Table 1. HPA-CNS model nominal parameter values and sensitivities. Parameter meaning Nominal value Fold-change for bifurcation to 1 fixed point a CNS inhibition of HPA at low cortisol 1 0.5,1.5 b CNS inhibition of HPA at high cortisol 1 0.5 u stress input 1 0.75,1.5 T GR activation threshold in the CNS 1.5 0.66, 1.33 a1 CRH secretion parameter 0.17 CRHcortisolh/min 0.75, 1.5 b1 CRH removal rate 0.17/min [39] 0.66, 1.33 a2 ACTH secretion parameter 0.035/min/(CRHP) 0.75, 1.5 b2 ACTH removal rate 0.035/min [39] 0.66, 1.33 a3 cortisol secretion parameter 0.0086/min/(ACTHA) 0.7, 1.45 b3 cortisol removal rate 0.0086/min [39] 0.68, 1.42 aP pituitary cell-mass production per unit CRH 0.049/day/CRH 0.85, 1.2 bP pituitary cell-mass removal rate 0.049/day [45] 0.83, 1.17 aA adrenal cell-mass production per unit ACTH 0.099/day/ACTH 0.85, 1.2 bA adrenal cell-mass removal rate 0.099/day [45] 0.83, 1.17 ah CNS production parameter 0.047 h/day 0.65, 1.35 bh CNS removal rate 0.047/day 0.74, 1.54 https://doi.org/10.1371/journal.pcbi.1011645.t001 Table 1. HPA-CNS model nominal parameter values and sensitivities. shift can abolish the healthy fixed point, leaving MDD as the only possible steady-state (Fig 2A1). shift can abolish the healthy fixed point, leaving MDD as the only possible steady-state (Fig 2A1). If stress lasts less than a few weeks, A starts growing but does not reach the separatrix. After stress is over, the nullcline shifts back to produce a healthy fixed point again, and A returns to its healthy size within weeks. No long-term depression develops (Fig 2A2). If stress however lasts longer than a few weeks, A can grow so much that it crosses the origi- nal (pre-stress) separatrix. Now even if stress is over, and the healthy fixed point is reinstated, A continues to flow to the MDD fixed point because it lies within its basin of attraction. Prolonged but not acute stress can trigger self-sustained depression Once it reaches the MDD fixed point, the system stays there even if stress returns to its previous low value. Therefore, the MDD fixed point is self-sustaining, with low inhibition that fuels high cortisol, which in turn reduces inhibition in a vicious cycle (Fig 2A3). This explains why prolonged but not acute stress can cause MDD in susceptible individuals, and why MDD persists even after the triggering stressor is removed. Animations of these dynamics are provided in a link in Methods. https://doi.org/10.1371/journal.pcbi.1011645.t001 Stress must be reduced below baseline to overcome depression due to a hysteresis effect According to the model, MDD can be relieved by sufficiently reducing stress input. If one reduces stress input u, the A nullcline shifts down and to the left, and the h nullcline shifts to the right. This can abolish the MDD fixed point, leaving the euthymic state as the only possibil- ity (Fig 2B1). As before, this shift to low stress must last long enough (weeks) for the dynamics to cross the separatrix and converge to the euthymic fixed point. Otherwise the system returns to the MDD fixed point once stress is heightened (Fig 2B2 and 2B3). Notably, the stress input must be reduced below the previous normal level in order to restore the healthy fixed point. This effect is known as hysteresis, and is caused because the pos- itive feedback in the MDD state requires a larger than normal reduction of stress to compen- sate [51]. 6 / 19 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch C 0.4 0.6 0.8 1.0 1.2 0.5 1.0 1.5 2.0 2.5 stress stress A2 A3 A1 stress reduction, below baseline B1 B2 B3 u2 u1 Fig 2. Dynamics of the model show how prolonged but not acute stress can lead to depression. A1) Stress shifts the nullclines, destroying the euthymic fixed point. A2) Dynamics under brief stress show recovery. A3) Dynamics under prolonged stress reach the depressed state on the timescale of weeks and remain there even after the stress is removed. B1) exiting depression requires stress to be reduced below baseline due to a hysteresis effect. B2) brief relief is not enough to exit depression B3) recovery on the timescale of weeks upon a prolonged stress relief. C) bifurcation plot of adrenal steady state size versus stress input u shows hysteresis. The transition from euthymic state to depressed state ATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch Biology \| https://doi org/10 1371/journal pcbi 1011645 December 6 2023 7 / 19 C stress stress A2 A3 A1 stress reduction, below baseline B1 B2 B3 stress stress A2 A3 A1 stress stress A1 A2 A3 stress reduction, below baseline B1 B2 B3 B2 stress reduction, below baseline B1 B B3 B3 B2 B1 C 0.4 0.6 0.8 1.0 1.2 0.5 1.0 1.5 2.0 2.5 u2 u1 Fig 2. Model explains dysregulation of endocrine tests in MDD The dysregulation of the HPA axis in MDD is characterized by two main endocrine tests, the CRH and DEX-CRH tests. One explanation for the dysregulation involves impaired GR func- tion [29]. Here we offer a different explanation, in which GR is normal and the dysregulation is caused by enlarged glands in MDD. The CRH test involves injecting CRH and measuring the ACTH response. Patients with MDD often show blunted ACTH responses in the CRH test relative to controls [19]. The present model explains this effect. Cortisol inhibits hypothalamic activity through MR and GR receptors, whereas it inhibits the pituitary activity through GR receptors only [49,55]. As a result, the model predicts that at the MDD fixed point, both the pituitary and the adrenal are larger than their euthymic size, and that the adrenal cortex grows more than the pituitary at steady state. To model the CRH test, we add external CRH to the model in a pulse that lasts 30 min, cor- responding to the lifetime of synthetic CRH (normal and synthetic CRH half-lives are about 4 min and 43 min respectively [45]) Control individuals are modeled with baseline stress levels and normal steady state adrenal size A. The CRH pulse causes an ACTH and cortisol response in both control and depressed simula- tions (Fig 3A1 and 3A2). However, the response is blunted in an individual with MDD, modeled with HPA glands that correspond to the MDD fixed point, namely enlarged adrenal and pituitary and high cortisol. The blunting is due to the repressive effect of high endogenous cortisol on the pituitary ACTH secretion that is not compensated by sufficient pituitary gland size. A similar analysis explains the elevated ACTH in DEX-CRH test in depressed patients [20,21]. In this test, the long-lived cortisol analogue dexamethasone (DEX) is given at night. DEX suppresses the HPA axis. The next day, ACTH is measured after a CRH injection. Indi- viduals with MDD show an elevated ACTH response. The model explains this due to their enlarged pituitary corticotroph mass P. The presence of DEX masks the higher cortisol found in MDD patients, and thus ACTH measured in the test corresponds primarily to the pituitary secretion capacity. MDD individuals in the model have larger P, which causes more ACTH to be secreted per CRH unit than controls (Fig 3B1, 3B2). Model explains dysregulation of endocrine tests in MDD We note that these explanations of the tests do not require assuming that the GR receptor pathway is different in people with MDD and in controls. https://doi.org/10.1371/journal.pcbi.1011645.g002 https://doi.org/10.1371/journal.pcbi.1011645.g002 This hysteresis can be seen in a bifurcation plot, showing the steady state of A versus stress u. Starting in the healthy state, stress must cross a threshold u1 to enter the MDD state of high A. However, starting in the MDD state, stress must drop below a lower threshold u2<u1 in order to return to the healthy state of low A (Fig 2D). Hysteresis may explain why in about 30% of Cushing patients with depression, depression does not abate after the tumor is treated and cortisol level decreases [11–13]. We predict these to be patients with pre-existing susceptibility to MDD, namely nullclines that cross at two sta- ble fixed points. Cushing syndrome causes the transition to the depressed state, which remains occupied even after treatment. Stress must be reduced below baseline to overcome depression due to a hysteresis effect Dynamics of the model show how prolonged but not acute stress can lead to depression. A1) Stress shifts the nullclines, destroying the euthymic fixed point. A2) Dynamics under brief stress show recovery. A3) Dynamics under prolonged stress reach the depressed state on the timescale of weeks and remain there even after the stress is removed. B1) exiting depression requires stress to be reduced below baseline due to a hysteresis effect. B2) brief relief is not enough to exit depression B3) recovery on the timescale of weeks upon a prolonged stress relief. C) bifurcation plot of adrenal steady state size versus stress input u shows hysteresis. The transition from euthymic state to depressed state C 0.4 0.6 0.8 1.0 1.2 0.5 1.0 1.5 2.0 2.5 u2 u1 C 0.4 0.6 0.8 1.0 1.2 0.5 1.0 1.5 2.0 2.5 u2 u1 Fig 2. Dynamics of the model show how prolonged but not acute stress can lead to depression. A1) Stress shifts the nullclines, destroying the euthymic fixed point. A2) Dynamics under brief stress show recovery. A3) Dynamics under prolonged stress reach the depressed state on C Fig 2. Dynamics of the model show how prolonged but not acute stress can lead to depression. A1) Stress shifts the nullclines, destroying the euthymic fixed point. A2) Dynamics under brief stress show recovery. A3) Dynamics under prolonged stress reach the depressed state on the timescale of weeks and remain there even after the stress is removed. B1) exiting depression requires stress to be reduced below baseline due to a hysteresis effect. B2) brief relief is not enough to exit depression B3) recovery on the timescale of weeks upon a prolonged stress relief. C) bifurcation plot of adrenal steady state size versus stress input u shows hysteresis. The transition from euthymic state to depressed state PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 7 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch happens for prolonged stress levels of u = u1. To return from the depressed state to the euthymic state, one must decrease stress below u = u2<u1. happens for prolonged stress levels of u = u1. To return from the depressed state to the euthymic state, one must decrease stress below u = u2<u1. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 Discussion We present a simple mathematical model for depression based on a CNS-HPA toggle switch which controls euthymic and depressed states on the timescale of weeks. This slow timescale is due to the turnover rate of the HPA glands. The timescale of the model explains why acute stress usually doesn’t cause depression whereas prolonged stress can. It also explains why drug treatment takes weeks to affect patients. The model shows bistability, in which patients enter a self-sustaining depressed state that lasts even after stress is removed. This is due to hysteresis in which entry into the depressed state is easier than exit from this state. In addition, the model provides a mechanism for blunted CRH and elevated DEX-CRH tests in depression. This mechanism is based on HPA gland growth and differs from standard explanations based on GR receptor dysregulation. One class of MDD drugs that seems to work without the delay of weeks is ketamine-based treatments, which often show effects within a day [60,61]. If one attempts to understand such drugs within the framework of the model, one may assume that they work in the CNS down- stream of cortisol. They mitigate the negative effect of cortisol on behavior, cognition and mood, as indicated in experiments on mice models [61]. Another (nonexclusive) possibility is that ketamine affects the HPA axis directly, lowering cortisol for a given stressor, as found in male mice [62], and in a human case study in which DEX suppression tracked ketamine in a treatment-resistant patient within days [63]. The present model supplies a mechanistic explanation for the dysregulation of the HPA axis observed in MDD—namely blunted CRH and elevated DEX-CRH tests. This explanation differs from the standard explanation in which the GR receptor feedback is weaker in patients with MDD [29]. In the CRH test, ACTH is blunted because the high cortisol in MDD inhibits ACTH production in the pituitary. A subtle point is that this effect depends on the known dis- tribution of cortisol receptors: GR in the pituitary and GR and MR in the hypothalamus. This causes the pituitary to grow in MDD but to a lesser extent than the growth of the adrenal. If both glands grew by the same factor, the CRH test would not be blunted because the large pitu- itary mass would compensate. Model explains timescale of weeks for antidepressant action We now consider the effects of changes in the h nullcline. These can presumably be caused by drugs or behavioral interventions that affect the CNS. A sufficient rise in h should abrogate the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 8 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch A1 B1 B2 A2 Fig 3. Model explains CRH and DEX/CRH test aberrations in MDD based on enlarged gland sizes. A1) Simulation shows blunted ACTH levels in CRH test in depressed patients compared to controls. A2) MDD patients show blunted ACTH in the CRH test (100μg CRH) compared to controls, adapted from Bardeleben at el [19]. B1) Simulation shows elevated ACTH levels in the DEX/CRH test in depressed patients compared to controls. B2) MDD patients show elevated ACTH in the DEX/CRH test (1.5 mg DEX and 100μg CRH 15 hours later), adapted from Heuser at el [21]. A1 B1 B2 A2 A1 B1 A2 A1 Fig 3. Model explains CRH and DEX/CRH test aberrations in MDD based on enlarged gland sizes. A1) Simulation shows blunted ACTH levels in CRH test in depressed patients compared to controls. A2) MDD patients show blunted ACTH in the CRH test (100μg CRH) compared to controls, adapted from Bardeleben at el [19]. B1) Simulation shows elevated ACTH levels in the DEX/CRH test in depressed patients compared to controls. B2) MDD patients show elevated ACTH in the DEX/CRH test (1.5 mg DEX and 100μg CRH 15 hours later), adapted from Heuser at el [21]. https://doi.org/10.1371/journal.pcbi.1011645.g003 MDD fixed point (Fig 4A1) in susceptible individuals. Now the healthy point is the only possi- ble steady state. The slow timescale of the HPA glands, of weeks, indicates that the intervention must last at least a few weeks, otherwise the separatrix will not be crossed, and the dynamics will return to the MDD point (Fig 4A1-4A3). This may explain why SSRIs and other medications for MDD take a few weeks to show effects [56,57]. Interestingly, SSRIs also activate the HPA axis on short timescales [58]. The model suggests they can be effective in the long term because they boost hippocampal health on the slow timescale of weeks [59]; such effects might cause the shift in the h nullcline that produces a therapeutic effect. Model explains timescale of weeks for antidepressant action 9 / 19 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch treatment A2 A1 A3 Fig 4. Dynamics of the model may explain the timescale of weeks for antidepressant action. A1) treatments that enhance CNS inhibitory activity can abolish the depressed fixed point. A2) brief treatment is not effective. A3) prolonged treatment of weeks or more is effective even after treatment ends. https://doi.org/10.1371/journal.pcbi.1011645.g004 A2 treatment A1 A3 A1 Fig 4. Dynamics of the model may explain the timescale of weeks for antidepressant action. A1) treatments that enhance CNS inhibitory activity can abolish the depressed fixed point. A2) brief treatment is not effective. A3) prolonged treatment of weeks or more is effective even after treatment ends. https://doi.org/10.1371/journal.pcbi.1011645.g004 Discussion One upshot of this blunted response is that beta-endorphin secretion from the pituitary, which is thought to be proportional to ACTH secretion since both are produced from the same polypeptide POMC [64], is decreased in MDD. Beta endorphin is a euphoric and analge- sic hormone. Its blunting may thus contribute to anhedonia, because a given HPA stimulus—a PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 10 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch given CRH pulse—causes lower levels of beta endorphin. This blunted response may contrib- ute to the dysphoria and pain sensitivity in MDD [65,66]. In the DEX-CRH test, the extrinsic DEX acts as a cortisol analogue and masks the high endogenous cortisol level in MDD. The test exposes the functional mass of the pituitary, which is enlarged in MDD in our model. As a result, cortisol is elevated in this test in MDD relative to controls. The model also presents a picture of susceptibility to MDD. Individuals vary in terms of their molecular/physiological parameters. This is described by the nullclines of the model. Individuals in whom the nullclines intersect three times have the potential to be stuck in MDD. Individuals for whom the nullclines intersect only once don’t have the potential for MDD, and instead show a return to euthymic state even after prolonged stress. The model may, in principle, offer a way to determine susceptibility, but this would require experimental approaches to quantify the interaction strengths of the CNS and the HPA. One approach may be quantifying emotional reactivity in terms of HPA dynamics [67,68]. In summary, the HPA-CNS toggle switch model offers an understanding of aspects of MDD and their dynamics on the scale of weeks. The model is based on qualitative physiologi- cal facts; future research on the specific mechanisms can help make it more precise. The pres- ent study rationalizes susceptibility to MDD in terms of bistability of the HPA-CNS feedback loop, explains why prolonged but not acute stress can trigger MDD, why many interventions take weeks to work and how HPA dysregulation occurs in MDD. This understanding may provide concepts to guide the development of treatment strategies for MDD. HPA-CNS toggle switch model We model the HPA axis using the model of Karin et al [45]. We denote the input signal to the HPA system as u [69]. This input is inhibited by the CNS activity h [25–27], a novel feature of the present model. The concentrations of the three hormones CRH, ACTH, and cortisol are denoted x1, x2, x3. Their fast time scale dynamics are described by (1–3): dx1 dt ¼ a1 u hMRðx3ÞGRðx3Þ b1x1 ð1Þ dx2 dt ¼ a2 x1 GRðx3Þ P b2x2 ð2Þ dx3 dt ¼ a3x2A b3x3 ð3Þ ð1Þ ð2Þ dx3 dt ¼ a3x2A b3x3 ð3Þ ð3Þ Where P is the total pituitary corticotroph functional mass and A is the functional mass of the cortisol secreting cells in the adrenal cortex. Cortisol inhibits hypothalamic activity through MR and GR receptors, whereas it inhibits the pituitary activity through GR receptors only [33,38]. We assume that the effects of MR and GR are multiplicative and use the same MR and GR expressions as done in Karin et al [45]. MR inhibitory activity is non cooperative so that MR x3 ð Þ ¼ 1 þ x3 KMR, whereas GR inhibitory activity is Hill like with cooperativity coefficient 3 Where P is the total pituitary corticotroph functional mass and A is the functional mass of the cortisol secreting cells in the adrenal cortex. Cortisol inhibits hypothalamic activity through MR and GR receptors, whereas it inhibits the pituitary activity through GR receptors only [33,38]. We assume that the effects of MR and GR are multiplicative and use the same MR and GR expressions as done in Karin et al [45]. MR inhibitory activity is non cooperative so that MR x3 ð Þ ¼ 1 þ x3 KMR, whereas GR inhibitory activity is Hill like with cooperativity coefficient 3 [45,70], so that GR x3 ð Þ ¼ 1 þ x3 KGR 3. We note that at physiological cortisol levels, MR recep- tors tend to be nearly saturated whereas GR receptors are hardly activated [39,71], namely KMR<<x3<<KGR. In this case we can approximate the MR and GR expressions—MR(x3)x3, [45,70], so that GR x3 ð Þ ¼ 1 þ x3 KGR 3. We note that at physiological cortisol levels, MR recep- tors tend to be nearly saturated whereas GR receptors are hardly activated [39,71], namely KMR<<x3<<KGR. GR(x3)1. This provides a simpler set of equations for hormone levels (4–6): dx1 dt ¼ a1 u hx3 b1x1 ð4Þ dx2 dt ¼ a2x1P b2x2 ð5Þ dx3 dt ¼ a3x2A b3x3 ð6Þ dx1 dt ¼ a1 u hx3 b1x1 ð4Þ ð4Þ ð5Þ dx3 dt ¼ a3x2A b3x3 ð6Þ ð6Þ Next we add the equations for the functional mass of the glands. The growth factor of P is x1 [72,73] and the growth factor of A is x2 [74,75], as detailed in Karin et al [45]. Their dynamic is on a slow time scale of weeks, as a result of the pituitary and adrenal cortex turnover rates (7–8), as given by Karin et al [45] dP dt ¼ aPPx1 bPP ð7Þ dA dt ¼ aAAx2 bAA ð8Þ dP dt ¼ aPPx1 bPP ð7Þ ð7Þ dA dt ¼ aAAx2 bAA ð8Þ ð8Þ The main new feature of the present model is the variable h that represents CNS inhibitory activity. We posit a slow time scale of weeks for h in Eq (9) by setting its removal rate appropri- ately. We note that this choice does not affect the conclusions: a timescale of hours or days for h would leave the qualitative results unchanged, and only have mild effects on the dynamics. The relevant CNS regions, hippocampus and frontal cortex, express both high-affinity MR and low-affinity GR receptors for cortisol. The GR generally has inhibitory effects. As mentioned above, MR receptors are nearly saturated by cortisol in its physiological range [71]. The GR receptors have a cooperative response to cortisol which is approximately step-like. We there- fore, for simplicity, model the inhibitory effect of cortisol on the CNS as a step function, defined by a+bΘ(x3>T). Using a Hill-type function instead of a step function leaves the con- clusions unchanged but makes the analytical solution much more difficult. When cortisol level is lower than the threshold T, only MR receptors are activated, causing CNS inhibition by a factor of a. But when cortisol level exceeds the threshold T, both MR and GR are activated, inhibiting CNS by a factor of a+b. dh dt ¼ ah 1 a þ bYðx3 > TÞ bhh ð9Þ ð9Þ Our HPA-CNS model is the set of equations (Eqs 1,2,3,7,8,9) which describe the dynamics of hormones and glands in the HPA-CNS system. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 HPA-CNS toggle switch model In this case we can approximate the MR and GR expressions—MR(x3)x3, PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 11 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch GR(x3)1. This provides a simpler set of equations for hormone levels (4–6): GR(x3)1. This provides a simpler set of equations for hormone levels (4–6): In order to demonstrate the general features of the model and nullclines, we first use a sim- plified version (Eqs 4,5,6,7,8,9). When considering compensation mechanisms where the GR plays an important role, we use the full HPA-CNS model Eq (Eqs 1,2,3,7,8,9). The nominal parameter values for the model are presented in Table 1. These parameters provide 3 fixed points, and provide hormone levels equal to 1. The fold-change of each param- eter required to bifurcate to a single fixed point are also shown. Some parameters cause a bifur- cation both when raised and lowered, as represented by two fold-change values in the table. The equations have two timescales—Eqs (4–6) have a timescale of hours, whereas Eqs (7–9) have a timescale of weeks. Solving the fast equations at quasi-steady-state (qss) gives the fast Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 12 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch time levels of the hormones x1, x2, x3 (10–12): time levels of the hormones x1, x2, x3 (10–12): s x1, x2, x3 (10–12): x1qss ¼ ð a1b2b3 b1a2a3 u hAP Þ 1 2 / ð u hAP Þ 1 2 ð10Þ x2qss ¼ ð a1a2b3 b1b2a3 Pu Ah Þ 1 2 / ð Pu Ah Þ 1 2 ð11Þ x3qss ¼ ð a1a2a3 b1b2b3 APu h Þ 1 2 / ð APu h Þ 1 2 ð12Þ x1qss ¼ ð a1b2b3 b1a2a3 u hAP Þ 1 2 / ð u hAP Þ 1 2 ð10Þ ð10Þ x2qss ¼ ð a1a2b3 b1b2a3 Pu Ah Þ 1 2 / ð Pu Ah Þ 1 2 ð11Þ ð11Þ x3qss ¼ ð a1a2a3 b1b2b3 APu h Þ 1 2 / ð APu h Þ 1 2 ð12Þ ð12Þ here one assumes that gland sizes are constant on the hour timescale. where one assumes that gland sizes are constant on the hour timescale. Solving Eqs 7 and 8 at steady state shows that the slow time steady state of x1 and x2 is quite simple, and independent on the input u and most other model parameters, due to the proper- ties of integral feedback in these equations [45]. x1st ¼ bP aP ð13Þ x2st ¼ bA aA ð14Þ ð13Þ x2st ¼ bA aA ð14Þ ð14Þ We normalize our equations x1≔x1 x1st ; x2≔x2 x2st, so the baseline level is now x1st ¼ x2st ¼ 1. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 In order to demonstrate this slow steady state solution space in a two-dimensional phase plane, we use the pituitary’s steady state size, Pst ¼ bAaPb2 aAbpa2, removing one of the three equations Animation of phase portrait under parameter changes Animations, generated by Mathematica, of the phase portraits under changing u, a or b param- eters can be found here: https://github.com/benron20/MDD-and-bistability-in-the-HPA- stress-axis. stress-axis. GR(x3)1. This provides a simpler set of equations for hormone levels (4–6): h ¼ ð aA bA Þ 1 2Pst u A ¼ ð aA bA Þ 1 2 bAaPb2 aAbpa2 u A / u A ð18Þ dh dt ¼ 0 ! h ¼ ah bh 1 a þ bYðx3 > TÞ ¼ ah bh 1 a þ bYðð APstu h Þ 1 2 > TÞ ð19Þ dA dt ¼ 0 ! h ¼ ð aA bA Þ 1 2Pst u A ¼ ð aA bA Þ 1 2 bAaPb2 aAbpa2 u A / u A ð18Þ ð18Þ dh dt ¼ 0 ! h ¼ ah bh 1 a þ bYðx3 > TÞ ¼ ah bh 1 a þ bYðð APstu h Þ 1 2 > TÞ ð19Þ ð19Þ These two nullclines can generate a bistable system as seen in Fig 1. Now, we explore the compensation mechanisms of the system, using the steady state solu- tion of (2–3) and (7–8). This compensation can be estimated by substituting x1st; x2st in Eqs (2– 3) steady state solutions. We find that Ast / x3 whereas Pst / GR x3 ð Þ ¼ 1 þ x3 KGR 3. Both the pituitary and adrenal cortex grow in order to compensate for high CRH and ACTH levels, but the adrenal cortex grows more than the pituitary. Interventions and drugs that affect the CNS are modeled by increasing CNS inhibitory activity by a factor of D: dh dt ¼ ah D a þ bYðx3 > TÞ bhh ð20Þ ð20Þ GR(x3)1. This provides a simpler set of equations for hormone levels (4–6): 1st 2st Eqs 13 and 14 show that upon a step change in input u, the change in hormone levels x1 and x2 is transient and the pituitary and adrenal glands grow to compensate to return CRH and ACTH to their original baseline levels within weeks. This growth and compensation can be calculated by substituting x1st; x2st in Eqs (5–6). From Eq (5) we find that the adrenal gland steady-state sizes rises linearly with steady-state cortisol Ast / x3. On the other hand, from Eq (6) we find that the pituitary’s steady state size is independent on cortisol level, Pst ¼ bAaPb2 aAbpa2. Below we will show that if we solve the original Eqs (1–3) and consider GR inhibitive effect on the pituitary, we find a compensation mechanism from both adrenal and pituitary glands. A p 2 Below we will show that if we solve the original Eqs (1–3) and consider GR inhibitive effect on the pituitary, we find a compensation mechanism from both adrenal and pituitary glands. Now, if we substitute x1st in Eq (4), we find that the steady state of cortisol is x3st ¼ a1aP b1bP u h / u h : x3st ¼ a1aP b1bP u h / u h : This makes biological sense by having cortisol proportional to its hypothalamic input signal u. To obtain the HPA-CNS model slow time steady state solution, we substitute fast time scale solutions (10–12) into the slow Eqs (7–9): dP dt ¼ aPPð u hAP Þ 1 2 bpP ð15Þ dA dt ¼ aAAð Pu Ah Þ 1 2 bAA ð16Þ dh dt ¼ ah 1 a þ bYðð APu h Þ 1 2 > TÞ bhh ð17Þ ð15Þ ð16Þ ð17Þ In order to demonstrate this slow steady state solution space in a two-dimensional phase plane, we use the pituitary’s steady state size, Pst ¼ bAaPb2 aAbpa2, removing one of the three equations PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 13 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch and allowing a 2D analysis. This allows us to solve dh dt ¼ 0 and dA dt ¼ 0 to find the slow time scale steady state of the system, and to understand the shape of h and A nullclines (18–19): dA dt ¼ 0 ! Author Contributions Conceptualization: Ben Ron Mizrachi, Avichai Tendler, Omer Karin, Tomer Milo, Uri Alon. Data curation: Ben Ron Mizrachi, Dafna Haran, Uri Alon. Formal analysis: Ben Ron Mizrachi, Avichai Tendler, Omer Karin, Tomer Milo, Avi Mayo, Uri Alon. Funding acquisition: Uri Alon. Investigation: Avichai Tendler, Dafna Haran, Avi Mayo. Investigation: Avichai Tendler, Dafna Haran, Avi Mayo. Methodology: Ben Ron Mizrachi, Avichai Tendler, Omer Karin, Avi Mayo, Uri Alon. Methodology: Ben Ron Mizrachi, Avichai Tendler, Omer Karin, Avi Mayo, Uri Alon. Project administration: Uri Alon. Project administration: Uri Alon. Resources: Uri Alon. Resources: Uri Alon. Software: Ben Ron Mizrachi, Avichai Tendler, Avi Mayo. Supervision: Uri Alon. Supervision: Uri Alon. Supervision: Uri Alon. Validation: Tomer Milo, Uri Alon. Visualization: Ben Ron Mizrachi, Avi Mayo. Writing – original draft: Ben Ron Mizrachi, Uri Alon. Writing – original draft: Ben Ron Mizrachi, Uri Alon. Writing – review & editing: Ben Ron Mizrachi, Uri Alon. Writing – review & editing: Ben Ron Mizrachi, Uri Alon. Supporting information The Supporting Information, including all code files and animations can be found here: https://github.com/benron20/MDD-and-bistability-in-the-HPA-stress-axis. S1 File. Hysteresis. (PY) S2 File. CRH test graph. (PY) S3 File. DEX/CRH test graph. (PY) S4 File. CRH test data. (PY) S5 File. DEX/CRH test data. (PY) S6 File. HPA phase portrait. (IPYNB) S7 File. Dynamic of brief stress. (PY) S8 File. Dynamic of prolonged stress. (PY) S2 File. CRH test graph. (PY) S2 File. CRH test graph. (PY) S3 File. DEX/CRH test graph. (PY) S3 File. DEX/CRH test graph. (PY) S4 File. CRH test data. (PY) S5 File. DEX/CRH test data. (PY) S6 File. HPA phase portrait. (IPYNB) S7 File. Dynamic of brief stress. (PY) S8 File. Dynamic of prolonged stress. (PY) PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1011645 December 6, 2023 14 / 19 PLOS COMPUTATIONAL BIOLOGY Major depressive disorder and bistability in an HPA-CNS toggle switch S9 File. 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https://openalex.org/W4214908356	https://vtechworks.lib.vt.edu/bitstream/10919/111262/1/2022-JOM1.pdf	English	null	Analysis of Model Thermal Profile Forecasts Associated with Winter Mixed Precipitation within the United States Mid-Atlantic Region	Journal of operational meteorology	2,022	public-domain	13,181	ABSTRACT Winter mixed-precipitation events across the mid-Atlantic region of the United States from 2013–2014 through 2018–2019 were used to analyze common short-term model forecasts of vertical atmospheric thermal structure. Using saturated forecast soundings of the North American Mesoscale (NAM), higher-resolution nested NAM (NAMnest), and the Rapid Refresh models—corresponding with observed warm-nose precipitation events (WNPEs)—several thermal metrics formed the basis of the analysis of observed and forecast soundings, including Bourgouin positive and negative areas. While the three models accurately forecast the general thermal structure well during WNPEs, a warm bias is evident within each. Well forecast are maximum and minimum temperatures within the warm nose and surface-based cold layer, respectively, but the cold layer is commonly too thin for each of the models, and the warm nose is regularly too thick, particularly within NAM and NAMnest forecasts. Forecasts of a cold layer that is too shallow tend to coincide with observations of stronger synoptic- scale upward motion, a deeper cold surface-based layer, and a higher isentropic surface. Forecasts of a warm nose that is too thick tend to coincide with observations of weaker upward motion, a shallower cold surface- based layer, and a lower isentropic surface across the region. Two-thirds of precipitation-type estimates from model soundings agreed with those derived from observed soundings, with the remaining third predominantly representing a warm bias in precipitation type. damage events in the United States and Canada have been well documented (McCray et al. 2019), while the study of insured property losses in the United States due to freezing rain revealed >$16 billion in damages over a 52-yr period (Changnon 2003). The most ANITA SILVERMAN (Manuscript received 20 June 2021; review completed 2 February 2022) Corresponding author address: Andrew Ellis, 295 West Campus Drive MC0115, Blacksburg, VA 24061 E il lli @ t d E-mail: awellis@vt.edu Analysis of Model Thermal Profile Forecasts Associated with Winter Mixed Precipitation within the United States Mid-Atlantic Region ANDREW W. ELLIS Department of Geography, Virginia Tech, Blacksburg, Virginia STEPHEN J. KEIGHTON NOAA/National Weather Service, Blacksburg, Virginia STEPHANIE E. ZICK Department of Geography, Virginia Tech, Blacksburg, Virginia ANITA SILVERMAN NOAA/National Weather Service, Blacksburg, Virginia Ellis, A. W., S. J. Keighton, S. E. Zick, A. S. Shearer, C. E. Hockenbury, and A. Silverman. 2022: Analysis of model thermal profile forecasts associated with winter mixed precipitation within the United States mid-Atlantic region. J. Operational Meteor., 10 (1), 1-17, doi: https://doi.org/10.15191/nwajom.2022.1001. Analysis of Model Thermal Profile Forecasts Associated with Winter Mixed Precipitation within the United States Mid-Atlantic Region ANDREW W. ELLIS Department of Geography, Virginia Tech, Blacksburg, Virginia STEPHEN J. KEIGHTON NOAA/National Weather Service, Blacksburg, Virginia STEPHANIE E. ZICK Department of Geography, Virginia Tech, Blacksburg, Virginia ANDREW S. SHEARER School of Meteorology, The University of Oklahoma, Norman, Oklahoma CASEY E. HOCKENBURY Department of Geography, Virginia Tech, Blacksburg, Virginia ANITA SILVERMAN NOAA/National Weather Service, Blacksburg, Virginia (Manuscript received 20 June 2021; review completed 2 February 2022) Ellis, A. W., S. J. Keighton, S. E. Zick, A. S. Shearer, C. E. Hockenbury, and A. Silverman. 2022: Analysis of model thermal profile forecasts associated with winter mixed precipitation within the United States mid-Atlantic region. J. Operational Meteor., 10 (1), 1-17, doi: https://doi.org/10.15191/nwajom.2022.1001. forecasts associated with winter mixed precipitation within the United States mid-Atlantic region. J. Operational Meteor., 10 (1), 1-17, doi: https://doi.org/10.15191/nwajom.2022.1001. 1. Introduction Winter mixed-precipitation events, particularly those involving freezing rain, are among the most disruptive and costly weather hazards. Billion-dollar 1 NWA Journal of Operational Meteorology Ellis et al Ellis et al 4 March 2022 Figure 1. Atmospheric sounding at 1200 UTC 18 February 2021 at Blacksburg, VA (KRNK), as portrayed on a skewT–logp thermodynamic diagram. Vertical profiles of air (Ta) and dewpoint (Td) temperatures are represented by the solid, black lines. The 0°C isotherm is in red (Obtained from http://weather.uwyo.edu/ upperair/sounding.html.). Click image for an external version; this applies to all figures and hereafter. obvious impacts are interruption of travel and damage to power infrastructure, which can result in prolonged power loss. For example, an ice accretion of 0.25–1.27 cm (0.1–0.5 in) from an autumn 2018 event across southwestern Virginia within the eastern United States disrupted power for 77% of utility customers in one rural county while triggering 59 traffic accidents on state roads within a 4-h period across a four-county area (Gendreau 2021). Areas of the eastern United States east of the Appalachian Mountains are susceptible to mixed- precipitation events as low-level cold air can become entrenched against the mountains beneath warm flow aloft (Keeter et al. 1995; Rauber et al. 2001; Changnon and Karl 2003). Freezing rain and ice pellets commonly occur when ice crystals aloft fall through a warm (>0°C or 32°F) layer above surface-based cold (<0°C or 32°F) air. The degree to which the ice crystal melts dictates the surface precipitation type, with partial melting leaving an ice nucleus that aids refreezing to an ice pellet, and complete melting requiring the aid of a cold surface for refreezing as accretion, or freezing rain (Forbes et al. 1987). Light freezing rain also can occur in a homogeneously cold atmosphere within which moisture/saturation is shallow, and thus too warm to generate the ice crystals necessary for atmospheric ice formation. Limited moisture and weaker vapor diffusion without ice crystal nuclei generally yields very light freezing rain or drizzle (McCray et al. 2019). Figure 1. Atmospheric sounding at 1200 UTC 18 February 2021 at Blacksburg, VA (KRNK), as portrayed on a skewT–logp thermodynamic diagram. Vertical profiles of air (Ta) and dewpoint (Td) temperatures are represented by the solid, black lines. The 0°C isotherm is in red (Obtained from http://weather.uwyo.edu/ upperair/sounding.html.). Click image for an external version; this applies to all figures and hereafter. 1. Introduction Similar studies have been conducted to evaluate the forecast precipitation phase in multiple versions of the HRRR model (Ikeda et al. 2013, 2017) and for comparing multiple precipitation-type algorithms over multiple winter seasons (Reeves et al. 2014). Such an approach can be useful for understanding systemic biases for a given model (in this case with a focus on mixed- precipitation events in a particular geographic region). The spatial domain for the study is an area of the mid- Atlantic region, east of the Appalachian Mountains, that (November through April) from 2013–2014 through 2018–2019. Still, changes to the models during the study period could influence skill or bias. We reviewed the NCEP documentation of changes to the models and the implementation dates (https://www.nco.ncep.noaa. gov/pmb/changes/), and identified five modifications that could impact the accuracy of thermal profile forecasts. These modifications include: (1) February 2014 (RAP model)—upgrade of data assimilation, upgrade to WRF core, upgrade to microphysics, and new planetary boundary layer scheme; (2) August 2014 (NAM model)—upgrade of microphysics, radiation, and convective parameterization scheme, and changes to data assimilation; (3) August 2016 (RAP model)— changes to assimilation of hydrometeor and mesonet data, and updates to WRF and microphysics package; (4) March 2017 (NAM/NAMnest model)—NAMnest grid spacing change (4 km to 3 km), improved radiation scheme, quantitative precipitation forecast bias correction, changes to data assimilation, and reduction of terrain smoothing; (5) July 2018 (RAP model)—data assimilation upgrade, WRF update, new hybrid vertical coordinate system, improved terrain representation, improved simulation of air temperatures over terrain, and improved microphysics for upper clouds. It is beyond the scope of this study to examine whether a given change alters model performance related to the thermal profile, or to what degree performance is altered. Instead, we analyze the overall model performance with the caveats that continuous model upgrades to operational models can influence any biases that we uncover during the study period. Similar studies have been conducted to evaluate the forecast precipitation phase in multiple versions of the HRRR model (Ikeda et al. 2013, 2017) and for comparing multiple precipitation-type algorithms over multiple winter seasons (Reeves et al. 2014). Such an approach can be useful for understanding systemic biases for a given model (in this case with a focus on mixed- precipitation events in a particular geographic region). 1. Introduction 890 to 690 hPa above surface-based cold layer (946– 890 hPa) with a minimum temperature of –5.3°C. At or close to saturation through about 700 hPa, the thermal profile yielded a mix of freezing rain and ice pellets. For this form of thermal profile, precipitation type is predicated on the temperature and thickness of each of the layers—freezing rain (ice pellets) from a generally warmer (cooler) atmosphere consisting of a thicker (thinner) warm layer and a thinner (thicker) cold layer. Warm southerly flow above a surface cold layer is relatively common within the eastern United States east of the Appalachian Mountains, where cold-air advection directed from the northeast by an anticyclone to the north “dams” cold air against the mountains. Cold-air damming yields ageostrophic flow of cold air southward along the eastern mountain slopes while inducing higher atmospheric pressure with an accumulation of mass and a depth of cold air sufficient for frozen or freezing precipitation. Warm-air advection above the cold layer can result from southerly geostrophic flow, or from southerly flow originating lower in the atmosphere in association with passage of a mid-latitude cyclone, or weaker low-pressure center. While the vertical thermal profile and synoptic atmospheric conditions for mixed-precipitation events in the eastern United States are rather well understood in theory, precipitation-type forecasting remains a practical challenge (Ralph et al. 2005; Ikeda et al. 2017; McCray et al. 2019). As Hux et al. (2001) noted, “the proper forecast of type and duration of winter precipitation is one of the most difficult challenges in operational meteorology.” Forecasts of thermodynamic structure are key, and it is important to understand interregional variability in model and algorithm performance (Robbins and Cortinas 2002). Guidance for forecasters as to how individual model biases might influence model blends of first-guess fields (e.g., warm-nose strength) that determine precipitation-type output is useful (Wandishin et al. 2005). Ikeda et al. Within the thermal profile common to ice-pellet and freezing rain events, the warm air aloft often is referenced as the “warm nose” because of the shape of the profile on a thermodynamic diagram. In the example from 1200 UTC 18 February 2021 at Blacksburg, Virginia (KRNK, Fig. 1), a deep warm layer, with a maximum temperature of 4.4°C, extends from around ISSN 2325-6184, Vol. 10, No. 1. Introduction 1 2 NWA Journal of Operational Meteorology 4 March 2022 Ellis et al 4 March 2022 Ellis et al (2013) evaluated the ability of the National Oceanic and Atmospheric Administration’s (NOAA) High- Resolution Rapid Refresh (HRRR) model to determine precipitation phase as rain, snow, or mixed during the 2010–2011 winter season. They found good qualitative model performance in terms of the spatial coverage in rain–snow transition areas and freezing-precipitation cases. However, HRRR model performance was less skillful quantitatively in regions with mixed precipitation (Ikeda et al. 2013). Their results signaled a need for a deeper diagnosis of model shortcomings in depicting mixed-precipitation events. Subsequently, Ikeda et al. (2017) re-engaged the HRRR model’s ability to forecast mixed-precipitation using surface observations and soundings across two winter seasons. They found the model to forecast the observed temperature profile associated with mixed-precipitation events reasonably well, but they identified a warm bias—particularly at the surface (<2°C) and in cold-air damming situations in the eastern United States (~4°C)—yielding errors in precipitation-type forecasts. (November through April) from 2013–2014 through 2018–2019. Still, changes to the models during the study period could influence skill or bias. We reviewed the NCEP documentation of changes to the models and the implementation dates (https://www.nco.ncep.noaa. gov/pmb/changes/), and identified five modifications that could impact the accuracy of thermal profile forecasts. These modifications include: (1) February 2014 (RAP model)—upgrade of data assimilation, upgrade to WRF core, upgrade to microphysics, and new planetary boundary layer scheme; (2) August 2014 (NAM model)—upgrade of microphysics, radiation, and convective parameterization scheme, and changes to data assimilation; (3) August 2016 (RAP model)— changes to assimilation of hydrometeor and mesonet data, and updates to WRF and microphysics package; (4) March 2017 (NAM/NAMnest model)—NAMnest grid spacing change (4 km to 3 km), improved radiation scheme, quantitative precipitation forecast bias correction, changes to data assimilation, and reduction of terrain smoothing; (5) July 2018 (RAP model)—data assimilation upgrade, WRF update, new hybrid vertical coordinate system, improved terrain representation, improved simulation of air temperatures over terrain, and improved microphysics for upper clouds. It is beyond the scope of this study to examine whether a given change alters model performance related to the thermal profile, or to what degree performance is altered. Instead, we analyze the overall model performance with the caveats that continuous model upgrades to operational models can influence any biases that we uncover during the study period. 1. Introduction The sounding station linked to each location is indicated. and one AWOS (KBCB) station locations (Fig. 1). The sounding station linked to each Figure 2. The locations of the five surface stations (KBCB, KCHO, KGSO, KIAD, KROA) and three upper-air stations (KGSO, KIAD, KRNK; Table 1) across surface elevation (200-m interval) within the mid-Atlantic region. The METAR code, unknown precipitation (UP), was included because of the known difficulty in mixed- precipitation identification by ASOS/AWOS sensors (Landolt et al. 2019). Potential WNPEs included all periods of ≥6 h [a threshold previously used to define a long-duration event (McCray et al. 2020)] with one or more precipitation types (Table 2) coincident with an air temperature ≤1.7°C. For inclusion as a WNPE, ≥50% of the observations within the period needed to meet the weather code and temperature criteria, with ≥10% of observations of a precipitation type other than snow or rain. The observation threshold for snow/rain eliminated events that were predominantly snow, cold rain, or mixed rain/snow. Figure 2. The locations of the five surface stations (KBCB, KCHO, KGSO, KIAD, KROA) and three upper-air stations (KGSO, KIAD, KRNK; Table 1) across surface elevation (200-m interval) within the mid-Atlantic region. The basis of a subjective classification of the synoptic pattern associated with each potential WNPE was the inspection of 3-h surface weather maps from the Weather Prediction Center’s Surface Analysis Archive (https://www.wpc.ncep.noaa.gov/archives/web_pages/ sfc/sfc_archive.php), 12-h surface weather maps from the Storm Prediction Center’s archive (https://www. spc.noaa.gov/obswx/maps/), and archived radar data. Four synoptic patterns conducive to warm-air advection over a cold surface layer, each with some degree of cold-air damming, constituted the classification. 1. Introduction These include: (1) passage of a low-pressure center to the west of the study area (southerly sourced warm advection), (2) Miller-A type low-pressure center evolution (Miller 1946), with cyclone development and emergence from the southeastern United States and passage along the Atlantic Ocean coastline (Atlantic-sourced warm advection), (3) Miller-B type low-pressure center evolution (Miller 1946), with cyclone passage from west- to-east over or to the south of the study area followed by intensification or secondary cyclogenesis near the coastline (Atlantic-sourced and/or southerly sourced warm advection), and (4) high-pressure center passage to the north in a classic cold-air damming scenario east of the Appalachian Mountains beneath isentropic lift of warmer southerly flow aloft (colloquially referred to as used to identify potentially impactful events, this study focuses on those with an identifiable warm nose in the sounding data, which represents the underlying cause of the precipitation type and is the key forecast element. 1. Introduction In this study, we analyze model forecast soundings associated with warm-nose precipitation events (WNPEs) for three numerical weather prediction (NWP) models operated by the National Centers for Environmental Prediction (NCEP): North American Mesoscale (NAM), higher-resolution nested NAM (hereafter, NAMnest), and Rapid Refresh (RAP). These represent the three relatively high-resolution models for which forecast sounding archives are readily available, while they also represent the model solutions used in the short term by the National Weather Service (NWS) National Blend of Models (Craven et al. 2020; https:// www.meted.ucar.edu/education_training/course/52). All three models are generated with Weather Research and Forecasting (WRF) model dynamical solvers. The two NAM models use the WRF Nonhydrostatic Mesoscale Model (WRF-NMM) solver and run four times per day (0000, 0600, 1200, and 1800 UTC), out to 84 h for the NAM and 60 h for the NAMnest (NCEP 2020). The NAM and NAMnest models are run with 12- and 3-km horizontal grid spacings, respectively, with 60 vertical layers. The RAP model uses the Advanced Research WRF (WRF-ARW) core and operates on a 3-km spatial grid with 50 vertical layers and executes four times per day with hourly output to 51 h; the WRF- ARW also runs on an hourly schedule out to 21 h (NCEP 2020). The spatial domain for the study is an area of the mid- Atlantic region, east of the Appalachian Mountains, that is frequently susceptible to winter precipitation-type forecast challenges. Specifically, this study focuses on areas represented by three sounding locations [KRNK; Greensboro, North Carolina (KGSO); and Sterling, Virginia (KIAD); see Fig. 2] and five Automated Surface Observing System (ASOS)/Automated Weather Observing System (AWOS) locations (Table 1, Fig. 2). While surface observations of precipitation type are To focus on contemporary model operation, the period of study is limited to the six cool seasons ISSN 2325-6184, Vol. 10, No. 1 3 NWA Journal of Operational Meteorology Ellis et al NWA Journal of Operational Meteorology 4 March 2022 4 March 2022 Ellis et al Ellis et al Location Station ID Elevation (m) Sounding Station Chantilly, VA KIAD 93 KIAD Charlottesville, VA KCHO 195 KRNK, KIAD Greensboro, NC KGSO 270 KGSO Roanoke, VA KROA 358 KRNK Blacksburg, VA KBCB 654 KRNK Table 1. The four ASOS and one AWOS (KBCB) station locations (Fig. 1). The sounding station linked to each location is indicated. Table 1. The four ASOS and one AWOS (KBCB) station locations (Fig. 1). a. Identification of warm-nose precipitation events Raw Meteorological Aerodrome Reports (METAR) data for each of the five ASOS/AWOS study locations were downloaded from the Iowa Environmental Mesonet (IEM) data archive at Iowa State University (https://mesonet.agron.iastate.edu/request/download. phtml) for November through April for the 6-yr study period 2013–2014 through 2018–2019. Any of nine METAR weather report codes associated with precipitation (Table 2) in combination with a surface air temperature ≤1.7°C (35°F) were used to define the possibility of frozen or freezing precipitation. ISSN 2325-6184, Vol. 10, No. 1 4 4 NWA Journal of Operational Meteorology 4 March 2022 4 March 2022 Ellis et al Ellis et al for both the surface-based cold layer and elevated warm nose, including minimum and maximum cold- and warm-layer temperature, mean temperature, mean wind direction and speed, pressure and height bounds, and the negative and positive areas using the Bourgouin method (Bourgouin 2000). The Bourgouin method can be used to predict probable precipitation type based on thermal condition and depth of the warm and cold portions of the thermal profile, or the areas of the warm (positive area) and cold (negative area) layers on a thermodynamic diagram (e.g., tephigram or skewT– logp). We used the original Bourgouin method because the modified version from Birk et al. (2021) was not available at the time of data analysis. The Bourgouin method requires only mean layer temperature (Tt) and potential temperature (θ) at the top and bottom of the layer. The Bourgouin layer area is calculated as Table 2. The nine METAR weather reports used to identify precipitation and their definitions. The codes may include a qualifying code of freezing (FZ) and modifiers to indicate intensity as heavy (+) or light (-). i y y ( ) g ( ) Code Definition BR Mist DZ Drizzle GS Small Hail/Snow Pellets (<5 mm) IC Ice Crystals PL Ice Pellets RA Rain SG Snow Grains SN Snow UP Unknown Precipitation “overrunning”). Stratification by synoptic pattern was intended to support a more granular analysis of forecast soundings associated with WNPEs. (1) (1) (1) Observational data from the array of soundings within the timeframe of each potential WNPE (and ultimately exhibiting a warm nose coincident with precipitation) were downloaded from the University of Wyoming upper-air data portal (http://weather.uwyo. edu/upperair/sounding.html). a. Identification of warm-nose precipitation events Soundings from KRNK were linked to potential WNPEs at three ASOS/AWOS locations (KBCB, KROA, KCHO), while soundings at KIAD were linked to potential WNPEs at two locations (KIAD, KCHO), and soundings at KGSO were only linked to potential WNPEs at the KGSO ASOS site (Fig. 2). At KCHO, a mixed-precipitation event led to review of coincident soundings at both KRNK and KIAD. As the interest is in WNPEs, only observed soundings exhibiting a warm nose above a freezing layer, and coincident with saturation of the lower atmosphere, were retained for comparison with model soundings. Saturation was defined as the occurrence of precipitation at the co-located ASOS/AWOS site at the time of the sounding or within the preceding hour (coinciding with sounding initiation). where Cp is the specific heat of air at constant pressure (1004 J kg–1 K–1). Applied to the cold and warm layers separately, the computation yields a solution for area on a thermodynamic diagram in units of specific energy (J kg–1). Bourgouin (2000) used precipitation- type observations and a linear relationship between corresponding positive and negative areas to establish a predictor of precipitation type [ice pellets (IP) versus freezing rain (FZRA)] based on the relative sizes of the two areas. A small positive area and large negative area supports ice pellets, while a large positive area and small negative area supports freezing rain. The Bourgouin thresholds for determining probable precipitation type based on negative area (NA) and positive area (PA) values are: IP: NA > (66 + 0.66PA) (2) FZRA: NA < (46 + 0.66PA) (3) FZRA/IP: (46 + 0.66PA) ≤ NA ≤ (66 + 0.66PA) (4) IP: NA > (66 + 0.66PA) (2) FZRA: NA < (46 + 0.66PA) (3) FZRA/IP: (46 + 0.66PA) ≤ NA ≤ (66 + 0.66PA) (4) IP: NA > (66 + 0.66PA) (2) FZRA: NA < (46 + 0.66PA) (3) FZRA/IP: (46 + 0.66PA) ≤ NA ≤ (66 + 0.66PA) (4) (2) Several metrics were calculated to characterize each sounding. In addition to surface air temperature, atmospheric thickness between key pressure levels was calculated as an indication of layer temperature. Simple linear interpolation was employed between observed data points to estimate the vertical position of key thresholds (e.g., 0°C air temperature) that fell between observations. Thicknesses were calculated for the 1000–500-, 1000–700-, 850–700-, and 1000–850-hPa layers. a. Warm-nose precipitation events Within the study region, 45 single-site or multi- site (coincidence between two or more ASOS/AWOS locations) WNPEs were identified for the 6-yr period, or an average of 7.5 yr–1 (Table 3). The locations of highest elevation (KBCB) and largest latitude (KIAD) have the greatest number of WNPEs during the 6-yr study period (Table 3). Nearly all of the WNPEs occurred in four of the six winter seasons studied, as few were identified for each of the 2015–2016 and 2016–2017 seasons (Table 3). For the WNPEs at each location and regionally, individual precipitation observations during the event were predominantly a combination of ice pellets and freezing rain, ranging from 44% (KIAD) to 57% (KBCB; Table 3). For all precipitation observations across the region associated with the 45 regional WNPEs, 51% were either ice pellets or freezing rain, 28% were snow, and 21% were rain (Table 3). Of the 45 regional events, 20 were single site while only two involved all five ASOS/AWOS locations (Table 4). Aside from the lengthier five-site events, the mean per-site duration of a WNPE was between 12 and 15 h, with precipitation occurring during 55–70% of the event extent (Table 4). Mean per-site event-total precipitation generally ranged between 9 and 14 mm (0.35 and 0.55 in) with a mean intensity of around 1 mm h–1 (0.04 in h–1; Table 4). While each WNPE inherently involves cold-air damming, approximately 71% (32) of the 45 WNPEs were associated with a low- pressure center/mid-latitude cyclone. These involved either passage to the west of the study area (10), or evolutions of type Miller-A (passage to the east from the southeastern United States; 11) or Miller-B (passage from west-to-east with redevelopment to the east; 11, see Table 4). Seven WNPEs, including the two that involved all five ASOS/AWOS locations (Table 4), were characterized primarily by an anticyclone to the north and classic synoptically driven cold-air damming— Observed and forecast saturated soundings during WNPEs were compared for each of the three sounding locations. Comparisons were made for each of the four model runs of each of the three models. With the research focus on forecasts of the vertical thermal profile rather than error in forecast timing, flexibility in the timing of a saturated forecast sounding (model-generated precipitation) relative to the observed sounding time was instituted. b. Observed versus forecast sounding analysis (6) For the three numerical weather prediction models that are the focus of this study (NAM, NAMnest, and RAP), forecast model sounding data associated with each observed warm-nose sounding were downloaded from the IEM archive site (https://mtarchive.geol. iastate.edu/). The data are Binary Universal Form for the Representation of meteorological data (BUFR) soundings converted to BUFKIT format and represent the native model vertical grid spacing. For each of the three models, data were downloaded for model runs at the observed sounding time (i.e., model initialization) and 12 h before the observed sounding. For the longer- horizon NAM and NAMnest models, data also were downloaded for model runs at 24 and 36 h before the observed sounding. For the shorter-horizon RAP model, data also were downloaded for model runs at 3 and 6 h before the observed sounding. Thus, four model runs were the focus for each of the NAM (0 h, +12 h, +24 h, and +36 h), NAMnest (0 h, +12 h, +24 h, and +36 h) and RAP (0 h, +3 h, +6 h, and +12 h) models. The higher-resolution models and shorter-term forecast horizons were chosen to focus on the winter-storm warning period and the near-term update periods for the higher-resolution models used by the NWS. While the RAP model generates forecasts out to 51 h four times per day, archived BUFR data are only available for the hourly model runs with forecasts out to 21 h, and it is unclear whether forecasters are aware when the blended cycles they are using include the extended RAP cycles. Error assessment focused on atmospheric thickness variables and the characteristics of the cold and warm layers (e.g., depths, positive/negative areas, maximum/ minimum/mean temperatures for a layer). a. Identification of warm-nose precipitation events Multiple variables were recorded or calculated (4) The Bourgouin method is an element of the BUFKIT forecast profile visualization and analysis toolkit (https://training.weather.gov/wdtd/tools/BUFKIT/ index.php, Mahoney and Niziol 1997) used in many NWS forecast offices. 5 ISSN 2325-6184, Vol. 10, No. 1 Ellis et al NWA Journal of Operational Meteorology 4 March 2022 NWA Journal of Operational Meteorology 4 March 2022 (6) (6) a. Warm-nose precipitation events Also listed is the number of events within each category of nSites associated with overrunning (OR), with cyclone passage to the west of the study region (West), with Miller-A or -B cyclone evolution, and with an unclassifiable synoptic pattern (None). Table 4. For the 45 regional WNPE events evident at one or more ASOS/AWOS sites (n Sites), the number of events (n Events), and from all events in each category of n Sites, the per-site mean values of the event length, percent of event hours with precipitation (Percent P), total precipitation, and precipitation intensity. Also listed is the number of events within each category of nSites associated with overrunning (OR), with cyclone passage to the west of the study region (West), with Miller-A or -B cyclone evolution, and with an unclassifiable synoptic pattern (None). y g ( ), y ,i y p p ( ) Mean Event Characteristics Synoptic Pattern Frequency n Sites n Events Length (h) Percent P Total P (mm) P Intensity (mm/hr) OR West Miller-A Miller-B None 1 20 13.4 62.6 11.2 0.9 2 4 3 8 3 2 7 12.2 69.5 14.6 1.5 0 2 4 0 1 3 10 12.3 55.6 8.8 1.0 2 1 3 2 2 4 6 15.2 63.6 13.4 1.2 1 3 1 1 0 5 2 21.5 52.9 12.8 1.1 2 0 0 0 0 Total 45 14.3 60.6 11.7 1.1 7 10 11 11 6 the closest evidence of a surface cyclone being far to the south of the study area—and overlaid by broad southerly isentropic lift, or overrunning. Six WNPEs were associated with a nondescript synoptic pattern, possibly characterized by a weak wave of low pressure or weak frontal passage. These were left unclassified (Table 4). or within 1 h preceding the sounding time reduced the number of soundings for further study to 55 soundings: 19 at KRNK (representing events at KBCB, KCHO, and KROA), 17 at KGSO, and 19 at KIAD (representing events at KCHO and KIAD). Composite soundings for each location depict the warm nose and the veering of low-level winds with height that is symbolic of warm-air advection (Figs. 3a–c). Mean values of sounding metrics at the three locations (Table 5) illustrate the relative strength of the warm nose compared to the surface-based cold layer. a. Warm-nose precipitation events Designated for comparison with an observed sounding was the saturated forecast model sounding nearest the observed sounding time, but with a limit of ≤3 h from the observed sounding. Beyond visual depictions of observed and forecast soundings, mean absolute error [MAE; Eq. (5)] and mean percent error [MPE; Eq. (6)] were calculated to reflect the general magnitude and bias of forecast error. (5) ISSN 2325-6184, Vol. 10, No. 1 6 NWA Journal of Operational Meteorology 4 March 2022 Ellis et al Ellis et al 4 March 2022 Table 3. Number of warm-nose precipitation events, November through April, at each ASOS/AWOS site for the seasons 2013–2014 through 2018–2019. Region values represent the total of single- and multi-location (coincident at two or more locations) events across the study region as a whole. Also included for each location is the mean percentage of event precipitation observations as snow (SN), rain (RA), or either ice pellets (IP) or freezing rain Location Total 2013-14 2014-15 2015-16 2016-17 2017-18 2018-19 SN RA IP/FZRA BCB 27 6 6 1 2 4 8 34 9 57 CHO 13 5 3 1 1 0 3 20 28 52 GSO 19 6 3 2 0 4 4 21 27 52 IAD 24 7 7 1 1 5 3 30 26 44 ROA 18 5 4 1 1 3 4 22 29 49 Region 45 11 11 3 2 10 8 28 21 51 ( ) Table 4. For the 45 regional WNPE events evident at one or more ASOS/AWOS sites (n Sites), the number of events (n Events), and from all events in each category of n Sites, the per-site mean values of the event length, percent of event hours with precipitation (Percent P), total precipitation, and precipitation intensity. Also listed is the number of events within each category of nSites associated with overrunning (OR), with cyclone passage to the west of the study region (West), with Miller-A or -B cyclone evolution, and with an unclassifiable synoptic pattern (None). Table 4. For the 45 regional WNPE events evident at one or more ASOS/AWOS sites (n Sites), the number of events (n Events), and from all events in each category of n Sites, the per-site mean values of the event length, percent of event hours with precipitation (Percent P), total precipitation, and precipitation intensity. ISSN 2325-6184, Vol. 10, No. 1 a. Warm-nose precipitation events At both KGSO and KIAD, the depth of the warm nose is considerably greater than the depth of the cold air beneath (Table 5). The greater depth and/or greater difference from freezing for the warm nose relative to the cold layer at the three locations equates to positive area values (warm layer) that are 1.7 (KRNK) to 2.5 (KGSO, KIAD) times larger than negative-area values (cold layer; Table 5). Mean surface winds are from the For the 101 potential WNPEs across the five ASOS/AWOS locations (Table 3), 88 coincided with ≥1 observed sounding with a distinct warm nose (nine without a warm nose, three missing soundings, and one without a cold surface layer). Some potential WNPEs had multiple soundings within their timeframes, yielding a total of 132 potential soundings for study. However, as soundings at KRNK and KIAD were linked with multiple ASOS/AWOS sites, multi-site events eliminated 37 duplicate soundings to produce a population of 95 soundings for potential analysis. Finally, the requirement of observed precipitation at ISSN 2325-6184, Vol. 10, No. 1 7 NWA Journal of Operational Meteorology 4 March 2022 4 March 2022 Ellis et al Ellis et al Table 5. Mean values from the observed soundings identified for forecast analysis at KRNK (n = 19), KGSO (n = 17), and KIAD (n = 19). In addition to surface air temperature (Ta), metrics for the surface-based cold layer and overlying warm layer are shown, including the negative and positive areas calculated using the Bourgouin method, along with thickness values for key pressure ranges. 1 kt = 0.5144 m s–1. a. Warm-nose precipitation events Layer Metric KRNK KGSO KIAD Surface Surface Ta (°C) –0.9 –0.8 –0.3 Cool Layer Minimum Ta (°C) –3.2 –3.7 –2.9 Mean Layer Ta (°C) –2.0 –2.3 –1.8 Negative Area (J kg–1) 76.2 81.8 60.5 Mean Layer Wind Direction (degrees) 166.1 82.1 126.1 Mean Layer Wind Speed (kt) 13.2 20.3 17.3 Thickness (gpm) 628.5 683.7 701.7 Top Layer Pressure (hPa) 865.2 904.8 915.4 Warm Layer Maximum Ta (°C) 3.7 4.9 4.1 Mean Layer Ta (°C) 2.3 2.9 2.4 Positive Area (J kg–1) 132.9 202.5 149.6 Mean Layer Wind Direction (degrees) 211.2 175.2 164.3 Mean Layer Wind Speed (kt) 35.6 36.1 31.0 Thickness (gpm) 582.5 1395.0 1248.0 Top Layer Pressure (hPa) 746.6 765.1 787.2 Thickness 1000–500 hPa (gpm) 5472.7 5474.8 5457.1 1000–700 hPa (gpm) 2862.8 2867.4 2864.2 850–700 hPa (gpm) 1561.8 1563.5 1560.6 1000–850 hPa (gpm) 1301.1 1303.8 1303.6 in maximum warm-nose temperature within the RAP model. Composite soundings (Fig. 4) for the +12-h runs of the NAM and NAMnest and +3-h cycle of the RAP (model runs prior to, but closest to, the observed sounding time) illustrate the mean accuracy of forecast thermal profiles. Note that differences in composite soundings are somewhat muted when computing mean values on a 25-hPa vertical grid from all soundings; but the relatively coarse vertical grid spacing is necessary to yield a robust computation of mean values at each pressure level. northeast at each location, but the mean wind from the cold-to-warm layers veers from east to south (Figs. 3a–c; Table 5). Atmospheric thickness, reflecting mean temperature, is rather consistent across the three locations for the 1000–500-hPa portion of the atmosphere, and especially so for the partial layers of 1000–700, 850–700, and 1000–850 hPa (Table 5). ISSN 2325-6184, Vol. 10, No. 1 b. Observed versus forecast sounding analysis Forecasts for the NAM and NAMnest are for the model run 12 h before the observed sounding time, while composites for the RAP are for the model run 3 h before the observed sounding time. Also shown are the temperature differences plus/minus one standard deviation (red lines). Figure 4. Composite observed (red) and forecast (blue) air temperature and wind speed/direction through 700 hPa for WNPEs at each location and each model. Forecasts for the NAM and NAMnest are for the model run 12 h before the observed sounding time, while composites for the RAP are for the model run 3 h before the observed sounding time. Figure 4. Composite observed (red) and forecast (blue) air temperature and wind speed/direction through 700 hPa for WNPEs at each location and each model. Forecasts for the NAM and NAMnest are for the model run 12 h before the observed sounding time, while composites for the RAP are for the model run 3 h before the observed sounding time. at KIAD is evident from the surface to about 850 hPa (Fig. 5g), while that for RAP forecasts extends from the surface to about 800 hPa (Fig. 5i). Unlike for any other model-location combination, NAMnest forecasts at KIAD have a mean cool bias between about 875 to 800 hPa, but with the more typical warm bias beneath this layer (Fig. 5h). It is common for the surface-based cold layer to be too thin, and the warm nose to be too thick, across the three models and three locations. In some cases, errors decrease with shorter lead times (e.g., RAP warm-nose depth at KIAD), but in many cases, the biases remain large or even increase at the analysis time (e.g., NAM warm-nose depth at KIAD; Figs. 6–8). For WNPEs at KRNK, the surface-based cold layer is consistently too thin for all the models at all model forecast times (Fig. 6a). The error in RAP model forecasts is generally the largest of the three models, and arguably largest at the time of the observed sounding (0 h; Fig. 6a). For the NAM, the cold layer tends to be too thin at all forecast lead times (Fig. 6a). The forecast warm nose at KRNK mean accuracy of NAMnest forecasts is evident (Fig. 5b). A mean warm bias in RAP forecasts at KRNK is evident between the surface and 850 hPa (Fig. 5c). b. Observed versus forecast sounding analysis For the 55 forecast soundings during WNPEs, each of the models generally simulates the thermal structure of the lower atmosphere well, but with thickness biases for the surface-based cold layer and the overlying warm nose. Because mean-layer temperature is skewed by error in depth, minimum cold-layer temperature and maximum warm-nose temperature are used to evaluate explicit temperature error. Commonly across the three models and three locations, surface air temperature and minimum/maximum air temperatures within the cold/ warm layers are well forecast, with MPEs between 99 and 101% and standard deviations <1% (not shown). A slight warm bias is common, except for a cool bias Repeating the process for generating composite soundings, but focusing on the difference in forecast and observed air temperature (forecast minus observed), provides a clearer illustration of the vertical pattern of model error (Fig. 5). In terms of mean error, there is very good agreement above 800 hPa for each model at each location, while the variability in RAP forecast error (standard deviation of the temperature difference) is larger than that for the NAM or NAMnest (Fig. 5). At KRNK, the warm bias in NAM forecasts is most evident between 900 and 800 hPa (Fig. 5a), and while the same is true within NAMnest forecasts, the better ISSN 2325-6184, Vol. 10, No. 1 8 NWA Journal of Operational Meteorology 4 March 2022 Ellis et al Figure 5. Mean of the difference in forecast and observed air temperature (blue line; forecast minus observed) through 700 hPa for WNPEs at each location and each model. Forecasts for the NAM and NAMnest are for the model run 12 h before the observed sounding time, while composites for the RAP are for the model run 3 h before the observed sounding time. Also shown are the temperature differences plus/minus one standard deviation (red lines). Figure 4. Composite observed (red) and forecast (blue) air temperature and wind speed/direction through 700 hPa for WNPEs at each location and each model. Forecasts for the NAM and NAMnest are for the model run 12 h before the observed sounding time, while composites for the RAP are for the model run 3 h before the observed sounding time. Figure 5. Mean of the difference in forecast and observed air temperature (blue line; forecast minus observed) through 700 hPa for WNPEs at each location and each model. b. Observed versus forecast sounding analysis At KGSO, a warm bias is evident from the surface up to about 875 hPa for NAM forecasts (Fig. 5d) and from the surface up to about 800 hPa for NAMnest forecasts (Fig. 5e), although the mean and variability in error is generally smaller for the NAMnest. A much larger variability in error and a warm bias extending from the surface up to 800 hPa is evident for RAP forecasts at KGSO (Fig. 5f). The patterns of larger variability in error for RAP forecasts and better mean accuracy with NAMnest forecasts also are evident at KIAD (Figs. 5g–i). As at KGSO, the warm bias in NAM forecasts 9 ISSN 2325-6184, Vol. 10, No. 1 Ellis et al NWA Journal of Operational Meteorology 4 March 2022 NWA Journal of Operational Meteorology 4 March 2022 Ellis et al Ellis et al Figure 6. Box plots characterizing the population of forecast minus observed values for (a) cold and (b) warm layer depth (gpm), and (c) negative and (d) positive area (J kg–1) at KRNK for the NAM, NAMnest, and RAP models. The four model runs for the NAM and NAMnest (0 h, +12 h, +24 h, +36 h) and RAP (0 h, +3 h, +6 h, +12 h) models are ordered left-to-right from latest (0 h) to earliest (+36 h or +12 h) on the horizontal axis. Box plot whiskers represent the 10th and 90th percentiles. Figure 7. Same as Fig. 6, but for KGSO. Figure 7. Same as Fig. 6, bu Figure 6. Box plots characterizing the population of forecast minus observed values for (a) cold and (b) warm layer depth (gpm), and (c) negative and (d) positive area (J kg–1) at KRNK for the NAM, NAMnest, and RAP models. The four model runs for the NAM and NAMnest (0 h, +12 h, +24 h, +36 h) and RAP (0 h, +3 h, +6 h, +12 h) models are ordered left-to-right from latest (0 h) to earliest (+36 h or +12 h) on the horizontal axis. Box plot whiskers represent the 10th and 90th percentiles. Figure 7. Same as Fig. 6, but for KGSO. small and a warm nose/positive area that is too thick/ large—are also evident at KGSO. Forecasts of the surface-based cold layer are consistently too thin for all three models, although RAP forecasts improve at the time of the observed sounding (0 h; Fig. 7a). ISSN 2325-6184, Vol. 10, No. 1 b. Observed versus forecast sounding analysis Each model tends to forecast a warm nose that is too thick, but with improvement with model runs for the NAMnest, and improvement at the observed sounding time (0 h) for the NAM and especially for the RAP (Fig. 7b). Negative area forecasts for all three models are too small, but there is some improvement with shorter lead times for the NAM and NAMnest, and modest improvement through model runs for the RAP (Fig. 7c). Forecasts of the positive area are too large for the NAM and NAMnest, and each improves with shorter lead times (Fig. 7d). Similar to KRNK, RAP model forecasts of positive area at KGSO are slightly too small, owing to a cool bias in maximum warm-nose temperature; but unlike at KRNK, variability in model error is generally smaller than for the other models (Fig. 7d). is generally too thick, particularly at +12 h and nearer the observed sounding for the NAM and NAMnest, and for +3- and +6-h forecast soundings for the RAP, which also exhibits high variability in the error of forecast depth (Fig. 6b). Forecast errors for layer depth are realized in forecasts of negative and positive areas. For the negative area, variability in error associated with the NAM improves with model runs to an area that is slightly too small at 0 and 12 h before the observed sounding (Fig. 6c). The NAMnest generally forecasts the negative area better than the NAM, particularly in terms of reduced variability in error, while RAP forecasts of negative area are consistently too small and with a high variability in error (Fig. 6c). For the positive area at KRNK, NAM forecasts are only slightly too large, particularly at 12 h before the observed sounding, while NAMnest forecasts are too small initially and too large closer to the observed sounding time, but with a distinct reduction in error variability (Fig. 6d). In contrast, RAP forecasts of positive area are generally too small (Fig. 6d), owing to a cool bias in maximum warm-layer temperature that supersedes a thick bias in warm-layer depth. In addition to the small bias in the positive area, the error in RAP forecasts of the positive area is highly variable (Fig. 6d). b. Observed versus forecast sounding analysis Unlike at KRNK and KGSO, RAP forecasts of positive area are too large nearer the sounding time, meaning that the warm-layer thick bias (Fig. 8b) outweighs the very slight cool bias in maximum warm-nose temperature. 8d). NAMnest forecasts of positive area are too small early within the model cycle (+36 h), then slightly too large but with a lessening degree of variability in model error (Fig. 8d). RAP model forecasts of positive area at KIAD are generally more accurate than for the NAM and NAMnest, particularly at +6 h and closer to the observed sounding time (Fig. 8d). Unlike at KRNK and KGSO, RAP forecasts of positive area are too large nearer the sounding time, meaning that the warm-layer thick bias (Fig. 8b) outweighs the very slight cool bias in maximum warm-nose temperature. Counts of large +/– biases in the thicknesses of the cold and warm layers illustrate skewness in the forecasts. We define a large model bias as an absolute error magnitude greater than the MAE. Focusing on the forecast model run nearest the observed sounding time for each model (+12 h for NAM and NAMnest, and +3 h for RAP), the thin bias in the surface-based cold layer in each model and the thick bias in the warm nose within the NAM and NAMnest are obvious (Table 6). For the three sounding locations taken together, 96% of the large NAM biases in the depth of surface-based cold layer are too thin, while 95% of large biases in warm- nose depth are too thick (Table 6). While still evident, the contrast is not quite as stark for the NAMnest, with 70% of large biases in the depth of cold air being too thin, and 75% of large biases in the warm nose as too thick (Table 6). For the RAP model, 94% of large biases in surface-based cold-layer depth are too thin, but large biases in warm-nose depth are near evenly split between too thick (58%) and too thin (42%; Table 6). Composite data for observed and forecast wind speed/direction (not explicitly shown, but illustrated in Fig. 4) do not depict clear errors in wind to accompany the temperature error within the models. b. Observed versus forecast sounding analysis is generally too thick, particularly at +12 h and nearer the observed sounding for the NAM and NAMnest, and for +3- and +6-h forecast soundings for the RAP, which also exhibits high variability in the error of forecast depth (Fig. 6b). Forecast errors for layer depth are realized in forecasts of negative and positive areas. For the negative area, variability in error associated with the NAM improves with model runs to an area that is slightly too small at 0 and 12 h before the observed sounding (Fig. 6c). The NAMnest generally forecasts the negative area better than the NAM, particularly in terms of reduced variability in error, while RAP forecasts of negative area are consistently too small and with a high variability in error (Fig. 6c). For the positive area at KRNK, NAM forecasts are only slightly too large, particularly at 12 h before the observed sounding, while NAMnest forecasts are too small initially and too large closer to the observed sounding time, but with a distinct reduction in error variability (Fig. 6d). In contrast, RAP forecasts of positive area are generally too small (Fig. 6d), owing to a cool bias in maximum warm-layer temperature that supersedes a thick bias in warm-layer depth. In addition to the small bias in the positive area, the error in RAP forecasts of the positive area is highly variable (Fig. 6d). Forecast errors at KIAD generally conform to those at KRNK and KGSO, albeit with a more pronounced thin bias within the surface-based cold layer than at the other locations. Forecasts of cold-layer depth are consistently too thin, but variability in forecast error improves approaching the observed sounding time (Fig. 8a). Forecasts of the warm nose are too thick, with some improvement closer to the observed sounding time for the RAP model (Fig. 8b), which more accurately portrays warm-layer depth than cold-layer depth. Forecasts of the negative area at KIAD are consistently too small across all three models (Fig. 8c), while forecast biases in the positive area at KIAD are a bit more variable. NAM forecasts of the positive area are too large at +12 h and closer to the observed sounding time (Fig. The general characteristics of model bias at KRNK—a cold layer/negative area that is too thin/ ISSN 2325-6184, Vol. 10, No. ISSN 2325-6184, Vol. 10, No. 1 b. Observed versus forecast sounding analysis 1 10 NWA Journal of Operational Meteorology 4 March 2022 4 March 2022 Ellis et al Ellis et al Figure 8. Same as Fig. 6, but for KIAD. large (Table 7). As with layer-depth biases, large errors in negative and positive areas within the NAMnest are slightly less skewed than for the NAM, but the contrasts are still very obvious. Of the large errors in negative area forecasts by the NAMnest, 76% are for an area that is too small, and of the large errors in positive area, 79% are for an area that is too large (Table 7). Large RAP model errors in the negative area are highly skewed toward an area that is too small (95% of the cases; Table 7); but large errors within RAP forecasts of positive area are rather evenly split between too large (54%) and too small (46%; Table 7). Comparison of forecast and observed partial atmospheric thicknesses indicate larger errors lower within the cold-layer/warm-nose thermal structure. A larger warm bias within the RAP model at KRNK is evident in atmospheric-thickness biases for the deeper 1000–500-hPa layer and for the lower layers of 1000– 700 and 1000–800 hPa (Fig. 9a). However, the better performance of the RAP model within the warm nose is apparent in the accuracy of 850–700-hPa thickness forecasts at KRNK (Fig. 9a). This also is the case for the RAP model at KGSO (Fig. 9b) and for all three models at KIAD (Fig. 9c), indicating that temperature across essentially the top half of the warm nose is rather well forecast. More problematic for all models at all locations is the warm bias illustrated by thickness forecasts for the lower layers of 1000–700 and 1000– 800 hPa, which translates to pronounced error in the deeper 1000–500-hPa layer for all models at each of the locations (Fig. 9). Figure 8. Same as Fig. 6, but for KIAD. 8d). NAMnest forecasts of positive area are too small early within the model cycle (+36 h), then slightly too large but with a lessening degree of variability in model error (Fig. 8d). RAP model forecasts of positive area at KIAD are generally more accurate than for the NAM and NAMnest, particularly at +6 h and closer to the observed sounding time (Fig. 8d). b. Observed versus forecast sounding analysis Values represent frequency with which the area exceeds the MAE magnitude in the positive (too large) and negative (too small) directions. Table 7. Frequency of large model error in negative and positive areas of the vertical atmospheric sounding at KRNK, KGSO, and KIAD for the 12-h lead-time runs of the NAM and NAMnest models and for the 3-h lead-time run of the RAP model. Values represent frequency with which the area exceeds the MAE magnitude in the positive (too large) and negative (too small) directions. ( g ) g ( ) NAM NAMnest RAP Total Cold Warm Cold Warm Cold Warm Cold Warm KRNK Large 2 7 3 8 0 3 5 18 Small 4 0 4 1 5 2 13 3 KGSO Large 1 6 1 5 0 1 2 12 Small 6 1 5 0 8 3 19 4 KIAD Large 0 5 1 2 1 3 2 10 Small 8 2 7 3 5 1 20 6 Total Large 3 18 5 15 1 7 9 40 Small 18 3 16 4 18 6 52 13 stronger when there is large error in the cold-layer forecast (Fig. 10a) compared to large error in the warm-layer forecast (Fig. 10c). Reinforcing this are the negative differences in observed omega between large error and little error in the cold-layer forecast (Fig. 10b) that are in contrast to the positive differences associated with the warm layer (Fig. 10d). Considering the negative omega values of the observed vertical motion field across the region, the negative differences in omega for the cold layer (Fig. 10b) indicate a more strongly negative omega associated with large error. In other words, the models have a tendency toward a warm bias within the cold layer under stronger synoptic-scale upward motion. The positive differences in observed omega in the warm layer (Fig. 10d) indicate a weaker negative omega associated with large error, indicating that the models have a tendency toward a warm bias in the warm nose under weaker synoptic- area (cold layer) or positive area (warm nose) greater than the MAE. These are distinguished from all other instances, meaning an error in positive or negative area less than the MAE. b. Observed versus forecast sounding analysis However, upon creating synoptic atmospheric composites associated with large warm model bias, it appears that error in the thickness of the surface-based cold layer and the overlying warm nose correspond differently to the strength of observed vertical motion. Using gridded NCEP/NCAR daily reanalysis data (Kalnay et al. 1996), mapped composites of mean observed daily vertical velocity in isobaric coordinates (omega, Pa s–1) at the 850-hPa level were created for instances that coincided with large model errors in cold- and warm- layer thermal characteristics. These were contrasted with composites of vertical velocity coinciding with instances of relatively accurate forecasts of the cold and warm layers. Instances of large model error are defined as before—absolute error in the negative Counts of large biases in negative and positive areas reinforce those for layer depths. Collectively, for the three sounding locations, 86% of large NAM errors in negative area are for an area that is too small, and 86% of large errors in positive area are for an area that is too 11 NWA Journal of Operational Meteorology 4 March 2022 Ellis et al 4 March 2022 Ellis et al Table 6. Frequency of large model error in the depth of the cold and warm layers within the vertical atmospheric sounding at KRNK, KGSO, and KIAD for the 12-h lead-time runs of the NAM and NAMnest models and for the 3-h lead-time run of the RAP model. Values represent frequency with which the depth exceeds the MAE magnitude in the positive (too thick) and negative (too thin) directions. NAM NAMnest RAP Total Cold Warm Cold Warm Cold Warm Cold Warm KRNK Thick 0 8 4 4 0 4 4 16 Thin 8 0 6 1 4 1 18 2 KGSO Thick 0 6 2 6 1 0 3 12 Thin 9 1 8 0 5 2 22 3 KIAD Thick 1 6 2 2 1 3 4 11 Thin 6 0 5 3 7 2 18 5 Total Thick 1 20 8 12 1 7 11 39 Thin 23 1 19 4 16 5 58 10 p ( ) g ( ) Table 7. Frequency of large model error in negative and positive areas of the vertical atmospheric sounding at KRNK, KGSO, and KIAD for the 12-h lead-time runs of the NAM and NAMnest models and for the 3-h lead-time run of the RAP model. ISSN 2325-6184, Vol. 10, No. 1 b. Observed versus forecast sounding analysis Also shown (e) is the difference in observed omega between occurrences of large model error in the warm layer and large model error in the cold layer (map c minus map a). Figure 10. For instances of large warm bias within the cold (a, b) and warm (c, d) atmospheric layers, composite means of observed 850-hPa omega (Pa s–1) (a, c) and the difference from instances with little bias (b, d) collectively for all models regionally. Differences are taken as observed omega during large model error minus small error for the +12-h runs of the NAM and NAMnest models and +3-h model run of the RAP. Also shown (e) is the difference in observed omega between occurrences of large model error in the warm layer and large model error in the cold layer (map c minus map a). Figure 9. Box plots characterizing the population of forecast minus observed values for atmospheric thickness at (a) KRNK, (b) KGSO, and (c) KIAD for the +12-h model runs of the NAM and NAMnest and +3-h run of the RAP model. Thicknesses from 1000–500, 1000–700, 850–700, and 1000–800 hPa are ordered from left-to-right on the horizontal axis. Box plot whiskers represent the 10th and 90th percentiles. scale upward motion. This dichotomy in the strength of observed upward motion corresponding with error in the cold- and warm-layer forecasts is underscored in the composite difference in omega associated with each. When subtracting the composite omega associated with large error in the cold layer from that for the warm layer (Fig. 10e), the positive values indicate the stronger observed upward motion in cases of cold-layer error. than the overrunning pattern, which was associated with more cases of large warm bias in the warm layer (10) than in the cold layer (6; Table 8). Overrunning should generally yield weaker synoptic-scale upward motion (symptomatic of a warm-layer bias) than the Miller-B pattern. That model errors, regionally, tend to correspond with a different strength of observed upward motion depending upon whether the error is in the cold or warm layer reinforces the finding that warm bias in the two layers is usually not coincident. Regionally and across all models, of the 92 instances of large error in a positive area too large (n = 52) and a negative area too small (n = 40; Table 7), only 12 (13%) coincided. b. Observed versus forecast sounding analysis Eliminating duplicate dates (coincident across the three models or three locations) yielded 19 instances of large error in negative area (cold layer), in which the forecast is too small, contrasted with 30 instances of little error in forecast negative area. Similarly, composites relating to the positive area (warm nose) are based on 21 instances of large error, in which the forecast is too large, and 35 instances of little error in forecast positive area. Composite omega fields are characterized by negative values across the study region for large error in both the cold layer (Fig. 10a) and warm nose (Fig. 10c), indicating the expected upward motion (decreasing pressure) associated with the precipitation events studied. Observed upward motion is considerably 12 Ellis et al NWA Journal of Operational Meteorology 4 March 2022 NWA Journal of Operational Meteorology 4 March 2022 Ellis et al Ellis et al Figure 9. Box plots characterizing the population of forecast minus observed values for atmospheric thickness at (a) KRNK, (b) KGSO, and (c) KIAD for the +12-h model runs of the NAM and NAMnest and +3-h run of the RAP model. Thicknesses from 1000–500, 1000–700, 850–700, and 1000–800 hPa are ordered from left-to-right on the horizontal axis. Box plot whiskers represent the 10th and 90th percentiles. Figure 10. For instances of large warm bias within the cold (a, b) and warm (c, d) atmospheric layers, composite means of observed 850-hPa omega (Pa s–1) (a, c) and the difference from instances with little bias (b, d) collectively for all models regionally. Differences are taken as observed omega during large model error minus small error for the +12-h runs of the NAM and NAMnest models and +3-h model run of the RAP. Also shown (e) is the difference in observed omega between occurrences of large model error in the warm layer and large model error in the cold layer (map c minus map a). Figure 10. For instances of large warm bias within the cold (a, b) and warm (c, d) atmospheric layers, composite means of observed 850-hPa omega (Pa s–1) (a, c) and the difference from instances with little bias (b, d) collectively for all models regionally. Differences are taken as observed omega during large model error minus small error for the +12-h runs of the NAM and NAMnest models and +3-h model run of the RAP. ISSN 2325-6184, Vol. 10, No. 1 b. Observed versus forecast sounding analysis In other words, 40 of 52 (77%) large errors in the warm layer did not coincide with a large error in the cold layer, and 28 of 40 (70%) large errors in the cold layer did not coincide with a large error in the warm layer. As isentropic lift is likely an important contributor to the vertical motion associated with the WNPEs studied (warm-air advection over a cold surface layer), the observed depth of the cold surface layer may reinforce the idea of stronger synoptic-scale upward motion with warm bias in the cold layer than with warm bias in the warm layer. The idea is that a deeper cold layer may correspond to stronger isentropic lift, the strength of the flow notwithstanding. As there are layer- depth differences by location, observed cold-layer data are portrayed for the three models collectively (+12-h NAM and NAMnest and +3-h RAP), but segregated by location. Coinciding with a large error in the surface- based cold layer (i.e., too thin) is an observed cold- layer depth that is generally greater than for instances of little error at all three locations (Fig. 11). The greater observed cold-layer depth with warm bias corresponds to the stronger observed upward motion outlined earlier. Conversely, coinciding with instances of a large error in the warm nose (i.e., too thick) is an observed cold- layer depth that is generally less than it is for instances The difference in the strength of upward motion per layer of bias seems to align with the stratification of large model error by synoptic pattern, particularly for the Miller-B type cyclone evolution and for overrunning. The Miller-B pattern was associated with many more cases of a large warm bias in the cold layer (16) than in the warm layer (4; Table 8). The mid-latitude cyclone of the Miller-B pattern should yield stronger synoptic- scale upward motion (symptomatic of a cold-layer bias) 13 NWA Journal of Operational Meteorology 4 March 2022 Ellis et al Ellis et al Table 8. Segregated by synoptic pattern, the frequency of large absolute error in negative area (too warm/shallow cold layer) and positive area (too warm/deep warm layer) for the 12-h lead-time runs of the NAM and NAMnest models and for the 3-h lead-time run of the RAP model at all locations combined (Table 7). b. Observed versus forecast sounding analysis Values are percentages of the total number of large errors of each synoptic pattern, and parenthetical numbers are raw counts. g y p p p Synoptic Pattern Negative Area, Small Bias Positive Area, Large Bias Overrunning 11.5 (6) 25.0 (10) Western L 30.8 (16) 25.0 (10) Miller-A 23.1 (12) 35.0 (14) Miller-B 30.8 (16) 10.0 (4) Unclassified 3.8 (2) 5.0 (2) Total 100 (52) 100 (40) Figure 12. For instances of large warm forecast bias and little forecast bias in cold (a, b) and warm (c, d) atmospheric layers, composite means of the observed 295K isentropic surface elevation (gpm) and wind speed (knots)/directional vector. Elevation data are derived from the 295K elevation at the seven sounding locations for which wind speed and direction are shown. Data correspond to biases within the +12-h runs of the NAM and NAMnest models and +3-h model run of the RAP, collectively, for all models regionally. Figure 11. Box plots characterizing the population of observed surface-based cold-layer depth associated with warm forecast bias and little forecast bias in the cold and warm layers at KRNK, KGSO, and KIAD. Data correspond to biases within the +12-h model runs of the NAM and NAMnest and +3-h run of the RAP model, collectively. Box plot whiskers represent the 10th and 90th percentiles. Figure 11. Box plots characterizing the population of observed surface-based cold-layer depth associated with warm forecast bias and little forecast bias in the cold and warm layers at KRNK, KGSO, and KIAD. Data correspond to biases within the +12-h model runs of the NAM and NAMnest and +3-h run of the RAP model, collectively. Box plot whiskers represent the 10th and 90th percentiles. Figure 11. Box plots characterizing the population of observed surface-based cold-layer depth associated with warm forecast bias and little forecast bias in the cold and warm layers at KRNK, KGSO, and KIAD. Data correspond to biases within the +12-h model runs of the NAM and NAMnest and +3-h run of the RAP model, collectively. Box plot whiskers represent the 10th and 90th percentiles. Figure 12. For instances of large warm forecast bias and little forecast bias in cold (a, b) and warm (c, d) atmospheric layers, composite means of the observed 295K isentropic surface elevation (gpm) and wind speed (knots)/directional vector. Elevation data are derived from the 295K elevation at the seven sounding locations for which wind speed and direction are shown. ISSN 2325-6184, Vol. 10, No. 1 b. Observed versus forecast sounding analysis 12c) compared to little error (Fig. 12d). While not excluding other dynamical processes associated with vertical motion, it appears that the common warm model bias in the cold and warm layers of the WNPEs is related to the degree of isentropic lift occurring across the region. It is intuitive that model forecasts err toward a cold layer that is too thin for those study cases possessing the thicker cold layers, and that they err toward a warm layer that is too thick for those study cases possessing the thinner cold layers. This characterizes the models as too conservative in their forecasts of the thickest and thinnest cold layers for the events studied. But without deeper study of model physics, it is difficult to conclude whether the differences in apparent isentropic lift are an inherent coincidence with model error, or a cause of model error. nose), were both well forecast by the models. However, each of the models exhibited a warm bias that is evident in layer thickness—a cold layer too thin for each of the models, and a warm nose too thick, particularly within NAM and NAMnest forecasts. A well-forecast thermal structure (but with a warm bias) matches the findings of Ikeda et al. (2017) in their study of the HRRR model. Large warm biases in each of the cold and warm layers, in terms of negative and positive areas, infrequently coincided, indicating that model error is not simply a deep-layer warm bias, but rather that it commonly is specific to thermal layer. Differences between forecast and observed atmospheric-layer thickness suggest this as well. While differences in the broader 1000–500-hPa thickness depict the general warm bias within the models, the upper portion of the warm layer, represented by the thickness of the 850– 700-hPa layer, is adequately forecast, particularly for the lower-elevation locations studied. Generally, the warm bias stems from the lower portion of the warm layer and below. While forecast biases within the thermal profile during WNPEs are important, the accuracy with which the resulting precipitation type is forecast is ultimately most important for forecasters. Using the original Bourgouin (2000) method, we estimated precipitation type for the observed and forecast soundings studied, but we re-rendered precipitation type as rain when the surface air temperature was above freezing. b. Observed versus forecast sounding analysis Data correspond to biases within the +12-h runs of the NAM and NAMnest models and +3-h model run of the RAP, collectively, for all models regionally. of little error—most obviously at KRNK, but also at KGSO, while only marginally so at KIAD (Fig. 11). The shallower observed cold-layer depth coinciding with warm bias in the warm nose corresponds to the weaker observed upward motion outlined earlier. models and three study locations (collectively) that were used to create composite omega maps (Fig. 12) were used to create composite maps of the observed 295K height and wind direction/speed (Fig. 12). There is little difference in observed wind direction and speed for the 295K isentropic surface between instances of large error and little error in either layer (Fig. 12). However, the observed isentropic surface across the mid-Atlantic study area is positioned higher in the atmosphere for instances of thin bias in surface-based cold-layer forecasts (Fig. 12a) compared to little error (Fig. 12b). The opposite is the case for the warm nose; models and three study locations (collectively) that were used to create composite omega maps (Fig. 12) were used to create composite maps of the observed 295K height and wind direction/speed (Fig. 12). There is little difference in observed wind direction and speed for the 295K isentropic surface between instances of large error and little error in either layer (Fig. 12). However, the observed isentropic surface across the mid-Atlantic study area is positioned higher in the atmosphere for instances of thin bias in surface-based cold-layer forecasts (Fig. 12a) compared to little error (Fig. 12b). The opposite is the case for the warm nose; In summary, instances of a cold-layer forecast as too shallow generally coincide with observed stronger upward motion over a deeper cold layer, while instances of a warm-nose forecast as too thick generally coincide with observed weaker upward motion over a shallower cold layer. These findings are collectively expressed in synoptic composites of the 295K isentropic surface derived from sounding data from seven sounding sites across and to the south of the study area (Fig. 12). The same warm-bias and little-bias dates for the three ISSN 2325-6184, Vol. 10, No. 1 14 NWA Journal of Operational Meteorology Ellis et al Ellis et al 4 March 2022 4 March 2022 the observed isentropic surface across the mid-Atlantic is positioned lower in the atmosphere for instances of a thick forecast bias (Fig. b. Observed versus forecast sounding analysis Consolidating the results from the three locations yields 52 forecast–observed precipitation-type comparisons for both the NAM and NAMnest models, and 45 for the RAP model. There is agreement in precipitation type for approximately two-thirds of the cases for each model (63.5% for the NAM, 67.3% for the NAMnest, and 64.4% for the RAP). In terms of the nature of bias in precipitation-type forecast, the NAM model is exclusively warm-biased with precipitation type (36.5% of all cases), while the NAMnest and RAP models are nearly so. For the NAMnest model, 28.9% of the 52 cases are warm-biased and 3.8% are cold-biased, while for the RAP model, 26.7% of the 45 cases are warm- biased and 8.9% are cold-biased. Comparison of observed and forecast wind velocity and direction revealed no obvious error for either of the models. However, synoptic-scale composites of observed vertical-velocity fields indicate that the instances of large warm model bias within the cold layer (too thin) tended to coincide with an observed atmosphere characterized by stronger upward motion across the region, while instances of large warm bias within the warm nose (too thick) tended to coincide with weaker observed upward motion across the region. This provides a basis for some speculation of the source of model error, with the degree of isentropic lift representing one possible mechanism by which model errors emerge. The stronger synoptic-scale vertical motion that coincides with the warm bias in the cold surface layer generally occurs when a deeper cold layer and higher isentropic surface are in place. In contrast, the weaker vertical motion that coincides with the warm bias in the elevated warm layer generally occurs when a shallower cold layer and lower isentropic surface are in place across the mid-Atlantic region. ISSN 2325-6184, Vol. 10, No. 1 REFERENCES REFERENCES the degree to which warm air is brought downward from the elevated warm layer and/or the degree of evaporative cooling that is occurring. The latter is an error source suggested by Ikeda et al. (2017) in their study of the performance of the HRRR model. 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https://openalex.org/W4236414643	https://pure.uva.nl/ws/files/54330854/s13068_020_01755_3.pdf	English	null	Using Osmotic Stress to Stabilize Mannitol Production in Synechocystis sp. PCC6803	Research Square (Research Square)	2,020	cc-by	12,347	osmotic stress to stabilize mannitol production in Synechocystis sp. 803 Citation for published version (APA): Wu, W., Du, W., Gallego, R. P., Hellingwerf, K. J., van der Woude, A. D., & Branco Dos Santos, F. (2020). Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803. Biotechnology for Biofuels, 13, Article 117. https://doi.org/10.1186/s13068-020- 01755-3 UvA-DARE (Digital Academic Repository) Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803 Wu, W.; Du, W.; Gallego, R.P.; Hellingwerf, K.J.; van der Woude, A.D.; Branco Dos Santos, F. DOI 10.1186/s13068-020-01755-3 Publication date 2020 Document Version Final published version Published in Biotechnology for Biofuels License CC BY Link to publication Citation for published version (APA): Wu, W., Du, W., Gallego, R. P., Hellingwerf, K. J., van der Woude, A. D., & Branco Dos Santos, F. (2020). Using osmotic stress to stabilize mannitol production in Synechocystis sp PCC6803. Biotechnology for Biofuels, 13, Article 117. https://doi.org/10.1186/s13068-020- 01755-3 UvA-DARE (Digital Academic Repository) Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803 Wu, W.; Du, W.; Gallego, R.P.; Hellingwerf, K.J.; van der Woude, A.D.; Branco Dos Santos, F. DOI 10.1186/s13068-020-01755-3 Publication date 2020 Document Version Final published version Published in Biotechnology for Biofuels License CC BY Link to publication Citation for published version (APA): Wu, W., Du, W., Gallego, R. P., Hellingwerf, K. J., van der Woude, A. D., & Branco Dos Santos, F. (2020). Using osmotic stress to stabilize mannitol production in Synechocystis sp PCC6803. Biotechnology for Biofuels, 13, Article 117. https://doi.org/10.1186/s13068-020- 01755-3 Citation for published version (APA): Wu, W., Du, W., Gallego, R. P., Hellingwerf, K. J., van der Woude, A. D., & Branco Dos Santos, F. (2020). Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803. Biotechnology for Biofuels, 13, Article 117. https://doi.org/10.1186/s13068-020- 01755-3 General rights General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare uva nl) Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Abstract Background: Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p, in short: a ‘mannitol cassette’), proved to be genetically unstable. In this study, we aim to overcome this genetic instability by conceiving a strategy to stabilize mannitol production using Synechocystis sp. PCC6803 as a model cyanobacterium. Results: Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild-type Synechocystis, complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this abil- ity in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions: Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory. Keywords: (D-)Mannitol, Synechocystis sp. PCC6803, Compatible solutes, Production stability, Salt stress Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803 Wenyang Wu1, Wei Du1, Ruth Perez Gallego2,3, Klaas J. Hellingwerf1,2, Aniek D. van der Woude2† and Filipe Branco dos Santos1† Wenyang Wu1, Wei Du1, Ruth Perez Gallego2,3, Klaas J. Hellingwerf1,2, Aniek D. van der Woude2† and Filipe Branco dos Santos1† © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. 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 in a credit line to the data. Disclaimer/Complaints regulations f UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) will be contacted as soon as possible. Download date:24 Oct 2024 Wu et al. Biotechnol Biofuels (2020) 13:117 https://doi.org/10.1186/s13068-020-01755-3 Biotechnology for Biofuels Biotechnology for Biofuels Open Access Background Mannitol is a six-carbon sugar alcohol with multiple bio- logical applications, such as a sweetener and an antioxi- dant. Therefore, mannitol has been widely applied in the food-, pharmaceutical-, and chemical industry, and it is of high commercial value [1]. Recently, cyanobacteria have gained much attention to be developed as photosynthetic cell factories to convert CO2 directly into biochemical *Correspondence: f.brancodossantos@uva.nl †Aniek D. van der Woude and Filipe Branco dos Santos contributed equally to this work 1 Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands Full list of author information is available at the end of the article 1 Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands Full list of author information is available at the end of the article The first report of mannitol production directly from CO2 in the marine cyanobacterium Synechococcus.sp PCC7002 (hereafter, Synechococcus) appeared in 2014 [9]. This production was achieved via heterologous expression of the genes encoding mannitol-1-phosphate- 5-dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p) either as individual proteins [9] or as a fused pro- tein [10], to convert (part of) the endogenous metabolite fructose-6-phosphate into mannitol. This two-step con- version from fructose-6-phosphate to mannitol is in prin- ciple superior to the single-step conversion of fructose to mannitol via mannitol dehydrogenase (mdh). This is due to the fact that in cyanobacteria, fructose-6-phosphate is more abundant comparing to fructose, since fructose- 6-phosphate is one of the main metabolites in the Pen- tose Phosphate Pathway that carries a high metabolic flux under photoautotrophic conditions. Hence, more manni- tol would be expected to be produced via this two-step conversion, though the accumulation of the intermedi- ate mannitol-1-phosphate might be harmful for the cells resulting in genetic instability problems [11]. In the study of Jacobsen and Frigaard, a concentration of mannitol of 1.1 g L−1 was reached after 12 days, with an average productivity of 0.15 g L−1 day−1 [9]. However, this pro- duction system turned out to be genetically unstable, possibly because the heterologous mannitol production pathway directly competes for metabolic intermediates with biomass formation [12], which imposes a fitness burden on the mannitol producing cells. These strains thus become susceptible to suppressor mutations, such as insertions or deletions, that would lower/remove this fitness burden. In the study of Jacobsen and Frigaard [9], the genetically engineered Synechococcus strains suffered from both incomplete genome segregation and from sup- pressor mutations occurring in the mtlD locus, which clearly indicates the genetic instability of mannitol pro- duction in those strains. To implement this idea, the model freshwater cyano- bacterium Synechocystis sp. PCC6803 (hereafter, Syn- echocystis) was chosen over Synechococcus for a few reasons. In addition to being extensively genetically engineered for the production of a variety of biofuels and chemicals [4], Synechocystis is the only cyano- bacterium in which the molecular mechanism of salt stress has been studied in detail [17]. The genes and biosynthetic pathways of its native compatible solutes (i.e., sucrose and glycosyl-glycerol) under salt stress have been clarified [18], which facilitates their targeted deletion and provides a straightforward approach to introduce a ‘mannitol cassette’ (mtlD and m1p). © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. 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 in a credit line to the data. Wu et al. Biotechnol Biofuels (2020) 13:117 Page 2 of 12 non-producing mutant cells, i.e., the production would be stabilized. Before such rationale can be applied to mannitol production in cyanobacteria, we first want to understand the function that mannitol plays inside the cells that are able to produce this compound. According to literature, mannitol can serve as a compatible solute in selected bacteria, presumably because it will protect cells under high-salt- and/or oxidative stress condi- tions [14]. Since cyanobacteria natively produce vari- ous compatible solutes (e.g., trehalose, glycine betaine, sucrose, glucosyl-glycerol, etc.) to accommodate the consequences of salt stress [15, 16], we want to first experimentally validate whether mannitol can be used to replace the cyanobacterial native compatible solutes under salt stress conditions. If so, mannitol production would be expected to be beneficial for cyanobacterial cells that lack the endogenous compatible solutes in their resistance to salt stress. A mannitol production system would hence be expected to be stable in such cyanobacterial mutants under salt stress conditions. compounds of interest. Enabled by genetic engineer- ing, the production of many different compounds [2–8] (e.g., ethanol, lactate, terpenes to name only a few) has already been achieved in different cyanobacterial species. Given the commercial value of mannitol and our cur- rent need for green alternatives, sustainable production of this sweetener by cyanobacteria is therefore becoming increasingly attractive. Engineering mannitol‑producing Synechocystis strains Engineering mannitol‑producing Synechocystis strains It has been reported that mannitol can be syn- thesized from fructose-6 phosphate via the sequential enzymatic reactions catalyzed by CO2 3PG G3P Calvin cycle F6P RuBP ATP ATP NADPH MtlD M1P Mannitol M1p NADH NaCl pressure Synechocystis.sp PCC 6803 ADP-Glucose G3P GG-P GG GgpS GgpP UDP-Glucose F6P Sps S6P Sucrose Spp Glycogen Glycogen PSII PSI Fig. 1 Overview of the engineered biosynthetic pathway to mannitol in the cyanobacterium Synechocystis sp. PCC6803. Solid arrows without wings represent native steps in cell; dashed arrows represent introduced pathway of mannitol synthesis; engineered enzymes are shown in blue; red circles with diagonal red line indicate deleted proteins; red cross-lines indicate metabolites that cannot be synthesized. White double circles represent protection against osmotic pressure by compatible solutes or mannitol in the cells. Grey ovals represent salt stress. 3PG, 3-phosphoglycerate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; M1P, mannitol-1-phosphate; M1p, Mannitol-1-phosphatase (M1Pase; encoded by m1p from E.tenella); MtlD, Mannitol-1-phosphate dehydrogenase (M1PDH; encoded by mtlD from E. coli); GgpS, Glucosyl-glycerol phosphate synthase; GG-P, glucosyl-glycerol-phosphate; GgpP, Glucosylglycerol phosphate phosphatase; GG, glucosyl-glycerol; Sps, Sucrose phosphate synthase; S6P, sucrose-6-phosphate; Spp, Sucrose phosphate phosphatase; RuBP, ribulose-1,5-bisphosphate CO2 3PG G3P Calvin cycle F6P RuBP ATP ATP NADPH MtlD M1P Mannitol M1p NADH NaCl pressure Synechocystis.sp PCC 6803 ADP-Glucose G3P GG-P GG GgpS GgpP UDP-Glucose F6P Sps S6P Sucrose Spp Glycogen Glycogen PSII PSI Fig. 1 Overview of the engineered biosynthetic pathway to mannitol in the cyanobacterium Synechocystis sp. PCC6803. Solid arrows without wings represent native steps in cell; dashed arrows represent introduced pathway of mannitol synthesis; engineered enzymes are shown in bl d i l ith di l d li i di t d l t d t i d li i di t t b lit th t t b th i d Whit d bl Fig. 1 Overview of the engineered biosynthetic pathway to mannitol in the cyanobacterium Synechocystis sp. PCC6803. Solid arrows without wings represent native steps in cell; dashed arrows represent introduced pathway of mannitol synthesis; engineered enzymes are shown in blue; red circles with diagonal red line indicate deleted proteins; red cross-lines indicate metabolites that cannot be synthesized. White double circles represent protection against osmotic pressure by compatible solutes or mannitol in the cells. Grey ovals represent salt stress. 3PG, 3-phosphoglycerate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; M1P, mannitol-1-phosphate; M1p, Mannitol-1-phosphatase (M1Pase; encoded by m1p from E.tenella); MtlD, Mannitol-1-phosphate dehydrogenase (M1PDH; encoded by mtlD from E. Fur- thermore, the freshwater Synechocystis has a much higher sensitivity to salt stress than the marine Syn- echococcus [19, 20]. During high salt acclimation, one would therefore expect more mannitol to be produced in Synechocystis than in Synechococcus, provided salt tolerance increases with cellular mannitol production. Given these reasons, we have selected Synechocystis as the microbial host to be genetically engineered for mannitol production. In this study, we achieved mannitol production in Syn- echocystis via heterologous gene expression of a cassette composed of the codon-optimized mannitol-1-phos- phate-5-dehydrogenase (mtD) from E. coli and manni- tol-1-phosphatase (m1p) from Eimeria tenella. We have further shown that mannitol can indeed function as a compatible solute, to benefit cell growth especially for the Synechocystis mutant strains that have lost the abil- ity to synthesize their endogenous compatible solute(s). Significantly, by adding salt to the growth media, this strategy has been proven capable of stabilizing mannitol By aligning product formation to biomass synthe- sis, growth-coupled production promises to become a useful strategy to stabilize production from CO2 in cyanobacteria [13]. To implement this strategy, prod- uct formation needs to be either beneficial to the cells, or become a mandatory process for biomass synthesis. Under such conditions, Darwinian selection will ensure that the producing cells will not be outcompeted by Wu et al. Biotechnol Biofuels (2020) 13:117 Page 3 of 12 production during prolonged cultivations in these latter Synechocystis strains. production during prolonged cultivations in these latter Synechocystis strains. mannitol-1-phosphatedehydrogenase (mtlD) and manni- tol-1-phosphatase (m1p) (Fig. 1). All our efforts to con- struct plasmids containing a functional mannitol cassette under control of the strong constitutive Ptrc1 promoter (a hybrid between the trp and lac UV5 promoters) have failed in E. coli, presumably due to the toxicity of certain sugar phosphates [11]. For instance, E. coli mutants that lose the function of fructose-1-phosphate dehydrogenase, Results and discussion Engineering mannitol‑producing Synechocystis strains Engineering mannitol‑producing Synechocystis strains coli); GgpS, Glucosyl-glycerol phosphate synthase; GG-P, glucosyl-glycerol-phosphate; GgpP, Glucosylglycerol phosphate phosphatase; GG, glucosyl-glycerol; Sps, Sucrose phosphate synthase; S6P, sucrose-6-phosphate; Spp, Sucrose phosphate phosphatase; RuBP, ribulose-1,5-bisphosphate Wu et al. Biotechnol Biofuels (2020) 13:117 Wu et al. Biotechnol Biofuels (2020) 13:117 Wu et al. Biotechnol Biofuels (2020) 13:117 Page 4 of 12 Page 4 of 12 This mannitol cassette was expressed in the wild type (WT), ∆ggpS (ΔGGPS) and ∆ggpS∆sps (ΔCS) Synecho- cystis backgrounds, resulting in the strains: WT_M, ΔGGPS_M and ΔCS_M, respectively (Fig. 2a, b and Additional file 1: Table S1). Compared with a previ- ous study in Synechococcus [9], this mutated mannitol cassette seems much easier to fully segregate, which occurs for the slr0168 neutral site of Synechocystis in only 5 days under 50 µg mL−1 kanamycin. This could be influenced by the lower expression and/or activity level of MtlD due to the mutation on position 332. Next, we monitored the growth of each mutant in regular BG-11 medium. Growth of the strains with a functional mannitol cassette appeared clearly impaired (Fig. 3a). This is due to the burden of mannitol production, for instance because part of the carbon fixed in photosyn- thesis is not available for biomass formation. Strikingly, impairing the ability to synthesize glycosyl-glycerol, or both sucrose and glycosyl-glycerol, did not lead to any improvement in mannitol production; it even had a negative impact relative to the Synechocystis WT back- ground (Fig. 3b and Additional file 1: Table S1). via mutagenesis, have impaired cell growth due to the intracellular accumulation of fructose-1-phosphate [21]. A similar phenomenon was also observed in Salmonella typhimurium, when mannitol was added to the growth medium, cells that lost the function of mannitol dehydro- genase could not grow because of the intracellular accu- mulation of mannitol-1-phosphate [22]. Hence, instead of using an intact plasmid, a fused and linear DNA frag- ment, consisting of the homologous regions of slr0168 (a non-essential hypothetical protein), the mannitol pro- duction cassette and a kanamycin resistance cassette, was constructed to be integrated via natural transformation at the neutral site slr0168 of the Synechocystis genome [23]. After several attempts, we obtained a few positive colonies, though a methionine was always stubbornly missing at position 332 of mtlD (even though not present in the linear DNA fragment used for the transformation). Engineering mannitol‑producing Synechocystis strains Nonetheless, since even with this mutated mtlD, manni- tol production could still be observed, we decided to con- tinue with this construct for the subsequent experiments. a slr0022 sps slr0023 sll1085 ggpS slr1670 slr0168 slr0168 mtlD m1p CheckSPS_F CheckSPS_R CheckGGPS_F CheckGGPS_R Checkslr0168_F Checkslr0168_R b sps ggpS mannitol cassette mannitol cassette 1 2 3 4 5 6 7 8 9 10 11 12 13 14 5K bp 250 bp 2K bp Kanr Fig. 2 Identification and confirmation genotype by PCR in the Synechocystis genome. a Map of primer-binding sites. Primers CheckSPS_F and CheckSPS_R were used for segregation analysis of the sps locus (PCR products 300 bp in mutant and 2400 bp in WT). Primers CheckGGPS_F and CheckGGPS_R were used to check the ggpS locus (PCR products 2100 bp in mutant and 4800 bp in WT). Primers Checkslr0168_F and Checkslr0168_R were used to check the slr0168 locus (PCR products 5200 bp in mutant and 200 bp in WT). b PCR analyses for genotyping of mutants. The chromosomal DNA is from WT (lanes 2, 6 and 10), WT_M (lanes 3,7 and 11), ΔGGPS_M (lanes 4,8 and 12) and ΔCS_M (lanes 5,9 and 13) of Synechocystis as a template and primers CheckSPS_F and CheckSPS_R specific for selected genes sps (lanes 2 to 5), primers CheckGGPS_F and CheckGGPS_R specific for selected genes ggpS (lanes 6 to 9), and primers Checkslr0168_F and Checkslr0168_R specific for selected genes mannitol cassette (lanes 10 to 13) to verify the genotype in the chromosomal DNA of the mutants. Lanes 1 and 14 ladder (1 kb from Fisher Scientific Company). Kanr, indicates kanamycin resistance cassette b sps ggpS mannitol cassette 1 2 3 4 5 6 7 8 9 10 11 12 13 14 5K bp 250 bp 2K bp b a a mannitol cassette Fig. 2 Identification and confirmation genotype by PCR in the Synechocystis genome. a Map of primer-binding sites. Primers CheckSPS_F and CheckSPS_R were used for segregation analysis of the sps locus (PCR products 300 bp in mutant and 2400 bp in WT). Primers CheckGGPS_F and CheckGGPS_R were used to check the ggpS locus (PCR products 2100 bp in mutant and 4800 bp in WT). Primers Checkslr0168_F and Checkslr0168_R were used to check the slr0168 locus (PCR products 5200 bp in mutant and 200 bp in WT). b PCR analyses for genotyping of mutants. 0 mM salt Fig. 4 The effect of salt concentrations on the growth of Synechocystis. The strains WT, WT_M, ΔGGPS, ΔGGPS_M, ΔCS and ΔCS_M were grown to OD730 = 1.0, after which 5 µl of WT, WT_M, ΔGGPS and ΔGGPS_M were transferred to a plate with a linear gradient of NaCl with a maximum salt concentration of 1 M; in addition, 5 µl of ΔCS and ΔCS_M was grown on a plate with a linear gradient with a maximum salt concentration of 0.5 M. The plates were incubated for 12 days at 30 °C. Red rectangles emphasize differences in salt resistance between corresponding strains (i.e., the same genetic background, but with and without the mannitol cassette) Fig. 4 The effect of salt concentrations on the growth of Synechocystis. The strains WT, WT_M, ΔGGPS, ΔGGPS_M, ΔCS and ΔCS_M were grown to OD730 = 1.0, after which 5 µl of WT, WT_M, ΔGGPS and ΔGGPS_M were transferred to a plate with a linear gradient of NaCl with a maximum salt concentration of 1 M; in addition, 5 µl of ΔCS and ΔCS_M was grown on a plate with a linear gradient with a maximum salt concentration of 0.5 M. The plates were incubated for 12 days at 30 °C. Red rectangles emphasize differences in salt resistance between corresponding strains (i.e., the same genetic background, but with and without the mannitol cassette) Fig. 4 The effect of salt concentrations on the growth of Synechocystis. The strains WT, WT_M, ΔGGPS, ΔGGPS_M, ΔCS and ΔCS_M were grown to OD730 = 1.0, after which 5 µl of WT, WT_M, ΔGGPS and ΔGGPS_M were transferred to a plate with a linear gradient of NaCl with a maximum salt concentration of 1 M; in addition, 5 µl of ΔCS and ΔCS_M was grown on a plate with a linear gradient with a maximum salt concentration of 0.5 M. The plates were incubated for 12 days at 30 °C. Red rectangles emphasize differences in salt resistance between corresponding strains (i.e., the same genetic background, but with and without the mannitol cassette) All the strains carrying a mannitol cassette displayed increased salt tolerance (Fig. 4). In the Synechocystis WT background, sucrose and glycosyl-glycerol are the two main endogenous compatible solutes that are used by the cells to cope with high salt stress. Engineering mannitol‑producing Synechocystis strains The strains WT, WT_M, ΔGGPS, ΔGGPS_M, ΔCS and ΔCS_M were grown to OD730 = 1.0, after which 5 µl of WT, WT_M, ΔGGPS and ΔGGPS_M were transferred to a plate with a linear gradient of NaCl with a maximum salt concentration of 1 M; in addition, 5 µl of ΔCS and ΔCS_M was grown on a plate with a linear gradient with a maximum salt concentration of 0.5 M. The plates were incubated for 12 days at 30 °C. Red rectangles emphasize differences in salt resistance between corresponding strains (i.e., the same genetic background, but with and without the mannitol cassette) 0 3 6 9 12 0 1 2 3 4 5 6 7 Cultivation time (day) OD730 WT WT_M ΔGGPS ΔGGPS_M ΔCS ΔCS_M 0 1 2 3 4 5 0 1 2 3 4 5 6 7 Cultivation time (day) (μM OD730 −1 ) a b Mannitol titer Fig. 3 Growth curve and mannitol production of Synechocystis mutants in normal BG-11 medium. a Growth curve in cultures of WT, WT_M, ΔCS, ΔCS_M, ΔGGPS and ΔGGPS_M b Mannitol accumulation in three different mannitol-producing strains WT_M, ΔCS_M and ΔGGPS_M. Symbols: solid triangles, WT_M; solid squares, ΔGGPS_M; solid circles, ΔCS_M; open triangles, WT; open squares, ΔGGPS; open circles, ΔCS. Values represent the average of at least three biological replicates (error bars represent standard deviation). In a, the broken lines connect the data points of the strains lacking the mannitol cassette 0 3 6 9 12 0 1 2 3 4 5 6 7 Cultivation time (day) OD730 WT WT_M ΔGGPS ΔGGPS_M ΔCS ΔCS_M a 0 1 2 3 4 5 0 1 2 3 4 5 6 7 Cultivation time (day) (μM OD730 −1 ) b Mannitol titer b Fig. 3 Growth curve and mannitol production of Synechocystis mutants in normal BG-11 medium. a Growth curve in cultures of WT, WT_M, ΔCS, ΔCS_M, ΔGGPS and ΔGGPS_M b Mannitol accumulation in three different mannitol-producing strains WT_M, ΔCS_M and ΔGGPS_M. Symbols: solid triangles, WT_M; solid squares, ΔGGPS_M; solid circles, ΔCS_M; open triangles, WT; open squares, ΔGGPS; open circles, ΔCS. Values represent the average of at least three biological replicates (error bars represent standard deviation). In a, the broken lines connect the data points of the strains lacking the mannitol cassette WT_M WT ΔGGPS_M ΔGGPS ΔCS_M ΔCS 0 mM salt 1 M salt 0 mM salt 500 mM salt Fig. Engineering mannitol‑producing Synechocystis strains 4 The effect of salt concentrations on the growth of Synechocystis. The strains WT, WT_M, ΔGGPS, ΔGGPS_M, ΔCS and ΔCS_M were grown to OD730 = 1.0, after which 5 µl of WT, WT_M, ΔGGPS and ΔGGPS_M were transferred to a plate with a linear gradient of NaCl with a maximum salt concentration of 1 M; in addition, 5 µl of ΔCS and ΔCS_M was grown on a plate with a linear gradient with a maximum salt concentration of 0.5 M. The plates were incubated for 12 days at 30 °C. Red rectangles emphasize differences in salt resistance between corresponding strains (i.e., the same genetic background, but with and without the mannitol cassette) Mannitol production confers cells with higher salt tolerance To test whether mannitol can functionally replace the native compatible solutes of Synechocystis to resist high- salt stress, the salt sensitivity of the WT, ΔGGPS and ΔCS mutants, in the absence and presence of the man- nitol cassette, was assayed under a wide range of salt stress conditions via a spot assay. Linear gradient NaCl plates were prepared with a salt gradient from 0 to 1 M for the WT and ΔGGPS strains, and from 0 to 0.5 M for the ΔCS strain. The two salt gradients were used because the ΔCS strain is much more sensitive to increasing salt concentrations than the other two strains. This is to such an extent that the salt tolerance conferred by mannitol production can in fact only be clearly noticed for the ΔCS strain when the concentrations are between 0 and 0.5 M. Next, exponentially growing cultures of all strains were diluted to a concentration of 12,500 cells µL−1 and 5 µL of each strain was spotted multiple times across the salt gradient on the BG-11 plate. The two different salt gradi- ent plates were incubated at 30 °C under a constant mod- erate light intensity of ~ 50 µmol photons m−2 s−1. After about 1 week, the spots became green and the results were then analyzed. 0 mM salt 500 mM salt Engineering mannitol‑producing Synechocystis strains The chromosomal DNA is from WT (lanes 2, 6 and 10), WT_M (lanes 3,7 and 11), ΔGGPS_M (lanes 4,8 and 12) and ΔCS_M (lanes 5,9 and 13) of Synechocystis as a template and primers CheckSPS_F and CheckSPS_R specific for selected genes sps (lanes 2 to 5), primers CheckGGPS_F and CheckGGPS_R specific for selected genes ggpS (lanes 6 to 9), and primers Checkslr0168_F and Checkslr0168_R specific for selected genes mannitol cassette (lanes 10 to 13) to verify the genotype in the chromosomal DNA of the mutants. Lanes 1 and 14 ladder (1 kb from Fisher Scientific Company). Kanr, indicates kanamycin resistance cassette Fig. 2 Identification and confirmation genotype by PCR in the Synechocystis genome. a Map of primer-binding sites. Primers CheckSPS_F and CheckSPS_R were used for segregation analysis of the sps locus (PCR products 300 bp in mutant and 2400 bp in WT). Primers CheckGGPS_F and CheckGGPS_R were used to check the ggpS locus (PCR products 2100 bp in mutant and 4800 bp in WT). Primers Checkslr0168_F and Checkslr0168_R were used to check the slr0168 locus (PCR products 5200 bp in mutant and 200 bp in WT). b PCR analyses for genotyping of mutants. The chromosomal DNA is from WT (lanes 2, 6 and 10), WT_M (lanes 3,7 and 11), ΔGGPS_M (lanes 4,8 and 12) and ΔCS_M (lanes 5,9 and 13) of Synechocystis as a template and primers CheckSPS_F and CheckSPS_R specific for selected genes sps (lanes 2 to 5), primers CheckGGPS_F and CheckGGPS_R specific for selected genes ggpS (lanes 6 to 9), and primers Checkslr0168_F and Checkslr0168_R specific for selected genes mannitol cassette (lanes 10 to 13) to verify the genotype in the chromosomal DNA of the mutants. Lanes 1 and 14 ladder (1 kb from Fisher Scientific Company). Kanr, indicates kanamycin resistance cassette Wu et al. Biotechnol Biofuels (2020) 13:117 Page 5 of 12 Mannitol production confers cells with higher salt tolerance To test whether mannitol can functionally replace the native compatible solutes of Synechocystis to resist high- salt stress, the salt sensitivity of the WT, ΔGGPS and ΔCS mutants, in the absence and presence of the man- nitol cassette, was assayed under a wide range of salt stress conditions via a spot assay. Linear gradient NaCl plates were prepared with a salt gradient from 0 to 1 M for the WT and ΔGGPS strains, and from 0 to 0.5 M for the ΔCS strain. Engineering mannitol‑producing Synechocystis strains The two salt gradients were used because the ΔCS strain is much more sensitive to increasing salt concentrations than the other two strains. This is to such an extent that the salt tolerance conferred by mannitol production can in fact only be clearly noticed for the ΔCS strain when the concentrations are between 0 and 0.5 M. Next, exponentially growing cultures of all strains were diluted to a concentration of 12,500 cells µL−1 and 5 µL of each strain was spotted multiple times across the salt gradient on the BG-11 plate. The two different salt gradi- ent plates were incubated at 30 °C under a constant mod- erate light intensity of ~ 50 µmol photons m−2 s−1. After about 1 week, the spots became green and the results were then analyzed. All the strains carrying a mannitol cassette displayed increased salt tolerance (Fig. 4). In the Synechocystis WT background, sucrose and glycosyl-glycerol are the two main endogenous compatible solutes that are used by the cells to cope with high salt stress. It is quite interesting to see that even with the native compatible solutes pre- either glycosyl-glycerol (in the ΔGGPS strain), or both glycosyl-glycerol and sucrose (in the ΔCS strain), was 0 3 6 9 12 0 1 2 3 4 5 6 7 Cultivation time (day) OD730 WT WT_M ΔGGPS ΔGGPS_M ΔCS ΔCS_M 0 1 2 3 4 5 0 1 2 3 4 5 6 7 Cultivation time (day) (μM OD730 −1 ) a b Mannitol titer Fig. 3 Growth curve and mannitol production of Synechocystis mutants in normal BG-11 medium. a Growth curve in cultures of WT, WT_M, ΔCS, ΔCS_M, ΔGGPS and ΔGGPS_M b Mannitol accumulation in three different mannitol-producing strains WT_M, ΔCS_M and ΔGGPS_M. Symbols: solid triangles, WT_M; solid squares, ΔGGPS_M; solid circles, ΔCS_M; open triangles, WT; open squares, ΔGGPS; open circles, ΔCS. Values represent the average of at least three biological replicates (error bars represent standard deviation). In a, the broken lines connect the data points of the strains lacking the mannitol cassette WT_M WT ΔGGPS_M ΔGGPS ΔCS_M ΔCS 0 mM salt 1 M salt 0 mM salt 500 mM salt Fig. 4 The effect of salt concentrations on the growth of Synechocystis. 0 mM salt It is quite interesting to see that even with the native compatible solutes pre- sent in the cell, mannitol production still confers cells even higher salt tolerance in the WT background (see top panel of Fig. 4). When the biosynthetic capacity for either glycosyl-glycerol (in the ΔGGPS strain), or both glycosyl-glycerol and sucrose (in the ΔCS strain), was deleted, cells displayed a lower salt tolerance relative to the WT strain. This further supports the functionality of the native compatible solutes to resist salt stress. In Wu et al. Biotechnol Biofuels (2020) 13:117 Page 6 of 12 decided to select the NaCl concentration that allows 70% of the maximum growth rate. We decided to leave out the ΔGGPS strain, because of possible contribution of sucrose to the salt tolerance of the cells. Hence, 420 mM and 200 mM NaCl were the salt concentrations chosen to test the WT_M and ΔCS_M strains, respectively. the ΔCS_M mutant, mannitol is the only (known) com- patible solute remaining. Indeed, mannitol production helped the ΔCS_M mutant to cope with higher salt stress (Fig. 4). Accordingly, we anticipated that mannitol pro- duction in Synechocystis can be stabilized via salt stress; yet this still needed to be directly tested. Mannitol production for the WT_M and ΔCS_M strains was monitored with and without salt stress, in prolonged turbidostat cultivations. As shown in Fig. 5b, for both the WT_M and the ΔCS_M strain without salt stress, mannitol production was quickly lost until no mannitol could be measured after only 6 days of cultiva- tion (13.5 generations). This further supports the argu- ment that mannitol production is indeed unstable via common metabolic engineering strategies as previously observed in Synechococcus [9]. For the WT_M strain under salt stress, mannitol production gradually dropped to below the detection limit in 11 days (17.1 genera- tions). This phenomenon is to be expected because native compatible solutes are likely to be preferred to resist salt stress, over the exogenous mannitol. In the ΔCS_M strain, mannitol will be the only compatible solute avail- able to the cells to tolerate salt stress. Therefore, manni- tol production is expected to be stable in this strain. This is in accordance with what we observed, as throughout the entire experiment (i.e., 12 days, 21.6 generations, see Fig. 5b), only a slight drop in mannitol production was observed in the ΔCS_M strain, especially during days 6 and 7. 0 mM salt We speculate that this slight decrease might be due to the fact that cells within the population are selected when mannitol production is fine-tuned to the amounts required by the environment imposed. These results Salt stress stabilized mannitol production during prolonged cultivations To test the (genetic) stability of mannitol production, a suitable NaCl concentration has to be selected. This salt concentration should partially inhibit cell growth, but should still allow cells to replicate at a certain rate, such that reverting cells that may arise, but should not be able to take over the population in a relatively short time. Hence, we decided to first determine the growth rate of all the background strains under different NaCl con- centrations in a 96-well plate growth assay (Fig. 5a and Additional file 1: Figure S1). As depicted in Fig. 5a, the results obtained showed that the growth rate of the WT strain was hardly affected, at least up to 400 mM NaCl, and then its growth rate gradually decreased until the growth was completely arrested at 500 mM NaCl. For the ΔGGPS strain, which lacks the main native compat- ible solute glucosyl-glycerol, the growth rate slightly decreased at 350 mM NaCl and suddenly dropped sharply to zero at 400 mM NaCl, and the same trend is observed for the ΔCS. For this latter strain, the growth rate already decreased at 200 mM NaCl and went to zero at 300 mM NaCl. Based on these results, to check the stability of man- nitol production during prolonged cultivation, we 0.00 0.02 0.04 0.06 0.08 0 100 200 300 400 500 NaCl (mM) Maximum growth rate (h-1) WT ΔGGPS ΔCS 0 3 6 9 12 0 2 4 6 8 10 12 Cultivation time (days) Mannitol titer (µM OD730 −1 ) WT_M -NaCl WT_M +NaCl ΔCS_M -NaCl ΔCS_M +NaCl a b Fig. 5 Maximum growth rate for mutants under different salt concentration and mannitol production changed from mannitol producer under specific salt pressures in Multi-cultivator. a Growth rate of WT, ΔGGPS and ΔCS as a function of salt concentration. Symbols: open triangles, WT; open squares, ΔGGPS; open circles, ΔCS. Values represent the average of biological duplicates (error bars represent standard deviation). b Mannitol production of WT_M and ΔCS_M with and without salt stress in a Multi-cultivator. Symbols: crosses, WT_M grown in the presence of 420 mM NaCl; triangles, WT_M grown without added salt; bars, ΔCS_M grown without added salt; squares, ΔCS_M grown in the presence of 200 mM NaCl. Characterization of mutations that are selected by the phenotypic instability Mannitol production from WT_M and ΔCS_M under no salt condition in the multi-cultivator was completely absent after only 13.5 generations, which corresponds to only 6 days of cultivation. Under 420 mM salt, the WT_M produced decreasing amounts of mannitol until it completely ceased to do so after 17.1 generations in 11 days of cultivation. In sharp contrast, the ΔCS_M under 200 mM salt stress displayed remarkably stable mannitol production, which could still be observed after 12 days cultivation in the multi-cultivator, cor- responding to over 21.6 generations. To clarify the molecular mechanism(s) behind the phenotypic insta- bility of mannitol production, we decided to sequence the mannitol cassette of cultures derived from pro- longed turbidostat cultivation experiments. From each strain and growth condition, 5 single colonies (i.e., 20 single colonies in total) were isolated when the man- nitol productions from their responding culture were completely disrupted and their mannitol-producing cassette was amplified by PCR and sent for sequenc- ing (Additional file 1: Table S2). Indeed, the results obtained showed that for those cultivations in which mannitol production was lost, various mutations could be detected, either leading to a truncated, non-func- tional, MtlD protein, or to impairment of its enzyme activity. All mutations found could be grouped in one of three types: single nucleotide insertions (SNI), sin- gle nucleotide deletions (SND) and single point muta- tions (PM) (see Additional file 1: Table S2 and Fig. 6). Among 14 mutations found in the strains that lost the ability to synthesize mannitol, 13 mutations occurred in mtlD reading frame and one was in its (Ptrc1) pro- moter region. All these mutants of different mannitol producers cultured under various salt conditions were re-cultivated in the shake flasks, and none of them showed the ability of producing mannitol (data not shown). These results indicated that mannitol produc- tion was disrupted due to malfunction of MtlD and that mannitol-1-phosphate accumulation might be harmful to cells, even though the underlying mechanism is still unclear [11]. We know the MtlD protein contains two pfam domains, the mannitol dehydrogenase C-termi- nal domain (pfam08125) and mannitol dehydrogenase Rossmann domain (pfam01232). The Rossmann domain is specific for binding NAD(P)+ and contains the con- served consensus motif G-x-G-x-x-G. Salt stress stabilized mannitol production during prolonged cultivations Values represent the average of at least three biological replicates (error bars represent standard deviation) 0 3 6 9 12 0 2 4 6 8 10 12 Cultivation time (days) Mannitol titer (µM OD730 −1 ) WT_M -NaCl WT_M +NaCl ΔCS_M -NaCl ΔCS_M +NaCl b 0.00 0.02 0.04 0.06 0.08 0 100 200 300 400 500 NaCl (mM) Maximum growth rate (h-1) WT ΔGGPS ΔCS a Fig. 5 Maximum growth rate for mutants under different salt concentration and mannitol production changed from mannitol producer under specific salt pressures in Multi-cultivator. a Growth rate of WT, ΔGGPS and ΔCS as a function of salt concentration. Symbols: open triangles, WT; open squares, ΔGGPS; open circles, ΔCS. Values represent the average of biological duplicates (error bars represent standard deviation). b Mannitol production of WT_M and ΔCS_M with and without salt stress in a Multi-cultivator. Symbols: crosses, WT_M grown in the presence of 420 mM NaCl; triangles, WT_M grown without added salt; bars, ΔCS_M grown without added salt; squares, ΔCS_M grown in the presence of 200 mM NaCl. Values represent the average of at least three biological replicates (error bars represent standard deviation) Wu et al. Biotechnol Biofuels (2020) 13:117 Page 7 of 12 indicate that by exploiting salt stress as a selection pres- sure, one can stabilize mannitol production from CO2 in Synechocystis. substrate fructose-6-phosphate, thus facilitating the catalytic reaction of the enzyme [24]. Our data revealed that three of the mutations were distributed over the Rossmann domain, 9 were in the C-terminal domain, including two identical mutations isolated indepen- dently, and one was in the linker region. It is impor- tant to note that one of the isolates from the ΔCS_M population, cultured under no salt condition, showed no mutation while its culture had stopped producing mannitol within 6 days. This result suggests that though a very small proportion of this population might still have the ability to synthesize mannitol, its ratio of non- mutated over mutated cells must be too low to make detectable amounts of mannitol by enzymatic assay. Characterization of mutations that are selected by the phenotypic instability The C-terminal domain contains conserved residues which bind the y y y In sharp contrast, no mutation in the mannitol cas- sette was found in the ΔCS_M strain during the entire prolonged cultivation under salt stress. This observa- tion corroborates the result that cells also maintained a continuous mannitol producing phenotype under such conditions (Fig. 6a) and leads us to conclude that man- nitol production in Synechocystis can be stabilized by salt stress, in mutants lacking the pathways for synthesis of the two endogenous osmoprotectants. In addition, by purposely weakening the native abilities of cells to tol- erate salt stress [25, 26], this strategy could allow more mannitol to be synthesized to compensate the loss of salt tolerance ability. Such could allow that mannitol produc- tion could be even further sustainably enhanced. Conclusions l d Mannitol production was achieved in a freshwater cyano- bacterium, a derivative of Synechocystis, this time confer- ring the producing strain with increased salt tolerance. With this approach we have tackled the major hurdle to mannitol production in cyanobacteria—genetic instabil- ity—by specifically aligning the production of a target compound with a fitness gain for the producing cells. Given the osmoprotectant properties of mannitol, we tar- geted the native osmotic stress response of Synechocystis, by replacing the synthesis of the endogenous compatible solutes by mannitol production. This strategy has been shown to be very successful, resulting in a production system for mannitol directly from CO2 with increased stability. The principle developed here, i.e., of coupling product formation to increased fitness under mild stress conditions, can potentially be applied to other host/prod- uct combinations as well. In future work it is important to take this principle of product-mediated stress protec- tion already into account during the metabolic engineer- ing design stage, as this engineering is likely to involve the identification and deletion of native stress response genes. Wu et al. Biotechnol Biofuels (2020) 13:117 Page 8 of 12 Wu et al. Biotechnol Biofuels Mannitol producer under salt pressure Mannitol producer WT_M under no salt pressure ΔCS_M under no salt pressure WT_M under 420 mM salt pressure ΔCS_M under 200 mM salt pressure Single nucleotide insertion Single nucleotide deletion Point mutation No mutation mtlD Ptrc1 480 1016 260 775 608 1016 103 1100 506 -40 161 405 934 941 -40 103 1016 1016 161 260 405 480 506 608 775 934 941 1100 A B a b Fig. 6 Characterization of mutations underpinning phenotypic instability surrounding mannitol production under non-stabilizing conditions in Multi-cultivator. a Schematic representation on the analysis of genetic stability of mannitol producers in the Multi-cultivator. The colored arrows in the center refer to the four combinations of a relevant strain (i.e., WT and ΔCS_M) plus cultivation condition (i.e., in the presence and absence of NaCl). Mannitol production capacity of WT_M and ΔCS_M under no salt condition was completely eliminated within only 6 days. WT_M produces decreasing amounts of mannitol, until it ceases to do so, during the first 10 days while under 420 mM salt. The mannitol production from ΔCS_M under 200 mM salt was found to be the most stable, which could still be detected after 12 days. The grey ovals in green color indicate salt pressure. Conclusions l d For further details, see Results and Discussion. b Schematic overview of all mutations observed in the mtlD-part of the mannitol cassette of the mannitol-producing strains. The numbers in the circles refer to the position of a mutation in the promoter region or in the reading frame of MtlD. “A” in the bar above the reading frame of MtlD represents the Rossmann domain, and “B” the c-terminal domain. In MtlD, the two domains are linked via a linker region Mannitol producer under salt pressure Mannitol producer WT_M under no salt pressure ΔCS_M under no salt pressure WT_M under 420 mM salt pressure ΔCS_M under 200 mM salt pressure Single nucleotide insertion Single nucleotide deletion Point mutation No mutation 480 1016 260 775 608 1016 103 1100 506 -40 161 405 934 941 a mtlD Ptrc1 -40 103 1016 1016 161 260 405 480 506 608 775 934 941 1100 A B b b Fig. 6 Characterization of mutations underpinning phenotypic instability surrounding mannitol production under non-stabilizing conditions in Multi-cultivator. a Schematic representation on the analysis of genetic stability of mannitol producers in the Multi-cultivator. The colored arrows in the center refer to the four combinations of a relevant strain (i.e., WT and ΔCS_M) plus cultivation condition (i.e., in the presence and absence of NaCl). Mannitol production capacity of WT_M and ΔCS_M under no salt condition was completely eliminated within only 6 days. WT_M produces decreasing amounts of mannitol, until it ceases to do so, during the first 10 days while under 420 mM salt. The mannitol production from ΔCS_M under 200 mM salt was found to be the most stable, which could still be detected after 12 days. The grey ovals in green color indicate salt pressure. For further details, see Results and Discussion. b Schematic overview of all mutations observed in the mtlD-part of the mannitol cassette of the mannitol-producing strains. The numbers in the circles refer to the position of a mutation in the promoter region or in the reading frame of MtlD. “A” in the bar above the reading frame of MtlD represents the Rossmann domain, and “B” the c-terminal domain. In MtlD, the two domains are linked via a linker region Fig. 6 Characterization of mutations underpinning phenotypic instability surrounding mannitol production under non-stabilizing conditions in Multi-cultivator. a Schematic representation on the analysis of genetic stability of mannitol producers in the Multi-cultivator. Strains and culture conditions Strains of E. coli were grown on Lysogeny Broth (LB) liq- uid medium at 37 °C in a shaking incubator at 200 rpm, or on the solid LB plates with 1.5% agar. Antibiotics were added to LB liquid medium or to solid plates, with the appropriate concentration as follows: kanamycin (50 µg mL−1) or ampicillin (100 µg mL−1), either sepa- rately or in combination. Synechocystis, a glucose-tolerant wild type, obtained from D. Bhaya, University of Stanford, Stanford CA, was cultivated in a modified BG-11 medium enriched with 25 mM PIPPS buffer (pH 8.0) [27] at 30 °C, either in a shaking incubator at 120 rpm, or on solid BG-11 plates, supplemented with 1.5% agar and 0.3% (w/v) sodium thiosulphate. The cells were grown with white light of moderate intensity (~ 50 µmol photons m−2 s−1), except when indicated. To construct Synechocystis mutants, kanamycin or nickel was added separately into BG-11 liq- uid medium or solid plates, with the appropriate concen- tration as follows: kanamycin (50 µg mL−1) and/or nickel (20 µM). The culture density was monitored by determin- ing the optical density at a wavelength of 730 nm (OD730). The mtlD and m1p genes were PCR amplified from pHKHmtlD and pUCm1p, respectively. Initially, we attempted to clone these genes using E. coli as a shut- tle host. To correctly express mtlD in Synechocystis, the weaker promoter PnrsB was used to control the expression level of mtlD in Synechocystis, but repeat- edly failed. This result surprised us, as PnrsB is from the nickel response system (nrs [31]), and is regarded as one of the weakest promoters in this host in the absence of an inducer. This result indicated the difficulty of high expression levels of MtlD, and that this might become a bottleneck for the synthesis of large amounts of manni- tol in Synechocystis. So we decided to bypass the usage of a shuttle host. Instead we chose to fuse these two frag- ments together with the kanamycin resistance gene, and the upstream and downstream homologous regions of slr0168, via overlap PCR. The resulting mannitol cassette plus resistance marker was placed under control of the Ptrc1 promoter. The fused fragment was sequenced to check for the absence of mutations and then used directly for transformation of the chromosome of Synechocystis at the neutral site present in locus slr0168. Conclusions l d The colored arrows in the center refer to the four combinations of a relevant strain (i.e., WT and ΔCS_M) plus cultivation condition (i.e., in the presence and absence of NaCl). Mannitol production capacity of WT_M and ΔCS_M under no salt condition was completely eliminated within only 6 days. WT_M produces decreasing amounts of mannitol, until it ceases to do so, during the first 10 days while under 420 mM salt. The mannitol production from ΔCS_M under 200 mM salt was found to be the most stable, which could still be detected after 12 days. The grey ovals in green color indicate salt pressure. For further details, see Results and Discussion. b Schematic overview of all mutations observed in the mtlD-part of the mannitol cassette of the mannitol-producing strains. The numbers in the circles refer to the position of a mutation in the promoter region or in the reading frame of MtlD. “A” in the bar above the reading frame of MtlD represents the Rossmann domain, and “B” the c-terminal domain. In MtlD, the two domains are linked via a linker region Wu et al. Biotechnol Biofuels (2020) 13:117 Page 9 of 12 each upstream and downstream homologous region of either sps or ggpS was individually PCR-amplified from the genome of Synechocystis, using Herculase II Fusion DNA Polymerase (Agilent Technologies). After gel puri- fication, each set of fragments of an upstream and down- stream homologous region was fused by overlap PCR, and the entire fused fragment was then further amplified by PCR. After the fused fragment was gel purified, an extra adenosine was added to the 3′ ends of these frag- ments and it was then ligated to the pFL-AN-T vector [30], resulting in plasmids pFL-AN2 (Δsps) and pFL-AN4 (ΔggpS), respectively. Because an XbaI restriction site was introduced via the primers during overlap PCR, the selection cassette, if provided with an XbaI site on both ends, can be easily inserted into pFL-AN2 (Δsps) and pFL-AN4 (ΔggpS), resulting in pFL-AN1 (Δsps) and pFL- AN3 (ΔggpS), respectively. Gene synthesis with codon optimizationh The gene sequence of mannitol-1-phosphate-5-dehydro- genase (mtlD) from E. coli (NCBI Reference Sequence: NC_000913.3) and mannitol-1-phosphatase (m1p) from Eimeria tenella (NCBI Reference Sequence: AF032462.1) was taken directly from the NCBI database. Codon opti- mization was performed based on the codon usage table compiled for Synechocystis (https://www.kazusa.or.jp/ codon/). The genes were synthesized by GenScript Bio- tech Corp, and were ligated to the pHKH and pUC57 plasmids, respectively [28] resulting in pHKHmtlD and pUCm1p. Plasmids and Synechocystis mutant construction Plasmids and Synechocystis mutant construction To obtain marker-less deletion strains, the genes encod- ing sucrose phosphate synthase (sps) and glucosyl- glycerol phosphate synthase (ggpS) in the genome of Synechocystis were deleted with a counter-selection approach [29]. For each gene deletion, two plasmids are needed: the first one contains only the upstream and the downstream sequences of the region to be deleted, here- after referred as the homologous regions, while the sec- ond plasmid contains an extra selection cassette flanked by both homologous regions. The selection cassette con- sists of a gene conferring kanamycin resistance to the host, as well as a toxic gene (mazF), under transcriptional control of the tightly regulated nickel-inducible promoter PnrsB. To construct each of the two plasmids, ~ 1 kb of It takes two rounds of natural transformation of Syn- echocystis to achieve a markerless gene deletion of either sps or ggpS. The first round is to fully integrate the selec- tion cassette into the chromosome through homologous recombination, while the second round is to completely remove the selection cassette. The method used for nat- ural transformation was essentially as described previ- ously [32]. In brief, 1 ml Synechocystis cultures grown in a shake flask to an OD730 of ~ 0.4 were harvested and concentrated by centrifugation at 5000 rpm for 5 min to a volume of 200 µL. Then, plasmid was added to the concentrated cells at 10 µg mL−1, followed by 5 h’ incu- bation in moderate white light, in a shaking incubator at 150 rpm. After incubation, cells were spread onto a Wu et al. Biotechnol Biofuels (2020) 13:117 Page 10 of 12 Page 10 of 12 commercial membrane (Pall Corporation, USA) resting on a BG-11 plate without antibiotic pressure. After a 24-h incubation in the 30 °C incubator under constant white light illumination, the membrane was transferred onto a new BG-11 plate with 50 µg mL−1 kanamycin. Single colonies appeared after approximately 12 days. The seg- regation status of mutants was confirmed by PCR, using the appropriate primers. When a mutant was confirmed to be fully segregated, a second round of transformation with a plasmid containing only the upstream and down- stream homologous region was performed. The selec- tion was then based on the resistance to nickel as only the colonies with the selection cassette fully removed can survive on the plates with nickel. Measurements of extracellular mannitol concentrations Measurements of extracellular mannitol concentrations Extracellular mannitol concentrations were determined in the supernatant collected from Synechocystis cultures using a D-Mannitol-L-Arabitol Assay Kit (Megazyme) [9]. Cells from shake-flask cultures were removed by centrifugation at 12,000 rpm for 1 min. Then, 100 µL of the supernatant samples was used for mannitol measure- ment according to manufacturer’s instructions. In this assay, the conversion of the mannitol present in the sam- ple to mannose—catalyzed by mannitol dehydrogenase— is stoichiometrically coupled to the conversion of NAD+ to NADH. This leads to an increase of absorbance at 340 nm that can be measured using a plate reader (BMG FLUOstar OPTIMA Microplate Reader). For mannitol quantification, the assay was calibrated with a standard curve (from 3 to 100 µM mannitol) obtained under the same conditions. Plate assay with a linear NaCl gradienth The method for making linear salt gradient plates has been described previously by [33]. In brief, BG-11 media containing agar, with and without NaCl (either 0.5 M or 1 M), were individually prepared. When making linear salt gradient plates, one side of a square petri dish was lifted and the plate was filled with BG-11 agar with- out NaCl. After the agar solidified, the plate was placed in a horizontal position and BG-11 agar with NaCl was poured on top of the first layer. To test the salt toler- ance of all the mutants, the cells in Synechocystis cultures were first counted using a Casy 1 Model TTC cell coun- ter (Schärfe System GmbH, Reutlingen, Germany) with a 60-µm diameter capillary, and diluted to a total cell number of 12,500 cells µL−1. A 5 µL culture from each mutant, grown with 200 mM salt, was spotted on the linear gradient plates containing a 0 to 0.5 M or a 0 to 1 M NaCl concentration gradient. Visible, green colonies appeared within 1 week. Plasmids and Synechocystis mutant construction The protocol for trans- formation of the DNA fragment containing the man- nitol cassette and the kanamycin resistance fragment integrated at the slr0168 site was similar to the protocol mentioned above. Full segregation of this construct was achieved by propagations in the presence of kanamycin. All the mutants were confirmed by PCR and the prim- ers that were used are listed in Additional file 1: Table S3. The confirmed mutants were routinely stored at −80 °C in BG-11 medium supplemented with 20% (v/v) glycerol. the maximum growth rate under each condition, the first 6 consecutive data points (from time 0 to 10 h) were used by fitting a linear function through the natural logarithm of the OD. The slope of the linear function was computed and designated as the growth rate. A representative set of growth curves in the 96 well plate together with the data points for growth rate calculation for all the strains is presented in Additional file 1: Fig. S1. Growth rate determination under salt stress To measure the growth rate for each strain under salt stress, we monitored the growth of each strain in a 96-well plate under a range of NaCl concentrations, from 0 to 500 mM. A preculture was prepared by inoculating cells from glycerol stocks directly into shake flasks con- taining liquid BG-11 medium and cultivated in the incu- bator with shaking of 120 rpm under continuously white light of moderate intensity (~ 50 µmol photons m−2 s−1). Once the precultures reached OD730 = 1, a total volume of 1 mL of a pre-culture was harvested and inoculated in each well of a 48-well plate, supplemented with 50 mM NaHCO3 plus increasing salt concentrations, ranging from 0 to 500 mM. After 2 days, the final optical density of the cultures in the 48-well plate was measured using a SPECTROstar Nano Microplate Photometer (BMG LABTECH GmbH, Germany) at 730 nm. To initiate experiments with a 96-well plate, pre-cultures acclimated to the corresponding salt stress in the 48-well plate were used for inoculation. This is to prevent the prolonging of the lag-phase of growth, resulting from the addition of salt, such that the same stage of cell growth under each condition can be extracted from the data for growth rate calculation. Pre-cultures were then diluted using 50 mM NaHCO3 and the respective concentration of NaCl in BG-11 to an initial OD of 0.05. Plates were incubated under constant white light illumination, with shaking at 600 rpm. Growth was monitored every 2 h within the 36-h incubation in the plate reader, to reliably calculate Turbidostat cultivationh Once the thresh- old of OD730 was reached (OD730 = 1) cell cultures were automatically diluted by 5% (v/v) with fresh BG-11 medium and the same volume of culture as the volume of medium just added, was discarded; all under control of “pycultivator”. All the cultures in the Multi-Cultivator were exposed to continuous white light with an intensity of 100 µmol photons m−2 s−1 OD−1. The genetic stabil- ity of each strain was assessed by mannitol production. Growth rate was calculated by fitting a linear function through the natural logarithm of the OD730 during each “growth-dilution” cycle. Samples for extracellular man- nitol quantification were periodically taken during the cultivation period. The variation of growth rate and man- nitol productivity throughout the whole experiment was then calculated relative to the initial values obtained at the beginning of each specific experiment. Funding The work of W.W. was supported by the China Scholarship Council. F.B.S. and W.D. were supported by the European Union’s EFRO grant ‘Kansen voor West II’. The Netherlands Organization for Scientific Research (NWO) supported F.B.S. through Solar-2-product Grant 733 000 005 and from the European Union’s Horizon 2020 research and innovation programme under Grant agreement No. 760994 (ENGICOIN project). The funders had no role in study design, data collection and analysis, decision to publish, nor preparation of the manuscript. Availability of data and materials Competing interests Klaas J. Hellingwerf and Aniek van der Woude have active roles as the scientific advisor and lead scientist, respectively, within Photanol B.V., a University of Amsterdam spin-off company aiming at commercializing sustainable applications with cyanobacteria. The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. Sequencing of the mannitol cassette To check the sequence, the mannitol cassette for the occurrence of (a) spontaneous mutation(s), single colo- nies from each independent Multi-Cultivator culture were first isolated. This is achieved by taking 5 µL of culture and re-streaking the cells on BG-11 agar plates. After picking a single colony and inoculating it into liq- uid BG-11 medium, genomic DNA was extracted as pre- viously described [34] and used as a template to amplify the mannitol cassette by PCR, using the high-fidelity Herculase II Fusion DNA Polymerase. The PCR product was then purified using MSB Spin PCRapace (STRATEC Molecular, Germany) and sent for sequencing (Macro- gen) using the primers listed in Additional file 1: Table S3. Received: 13 March 2020 Accepted: 23 June 2020 Authors’ contributions WW has performed most of the strain engineering and cultivation of strains under different regimes supervised by WD and FBS. RPG constructed some of the deletion strains and conducted preliminary characterization assays of their salt tolerance under the supervision of AW. WW and WD analyzed the data obtained supervised by FBS. AW, KJH and FBS conceived the project. WW and WD wrote the first draft of the manuscript assisted by FBS and KJH. All authors read and approved the final manuscript. Author details 1 1 Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands. 2 Photanol B.V, Matrix V, Science Park 406, 1098 XH Amsterdam, The Netherlands. 3 Present Address: NIOZ Royal Netherlands Institute for Sea Research, Department of Marine Microbiology and Biogeochemistry, Utrecht University, P.O. Box 59, Den Burg, Texel, 1790 AB Utrecht, The Netherlands. Turbidostat cultivationh The phenotypic stability of mannitol-producing strains was studied with the turbidostat mode of a Multi-Culti- vator (MC1000-OD, PSI, Czech Republic). In this culti- vation mode, Synechocystis populations can be kept at a fixed biomass density by continuously diluting cultures Wu et al. Biotechnol Biofuels (2020) 13:117 Wu et al. Biotechnol Biofuels (2020) 13:117 Page 11 of 12 Page 11 of 12 with fresh BG-11 medium without antibiotic, while simultaneously taking out an identical volume of cultured cells. Accordingly, cells in a turbidostat are under con- tinuous selection for maximal specific growth rate. For this we used a modified Multi-Cultivator with additional pumps (Reglo ICC, ISMATEC, Germany) and controlled by the “pycultivator” software package [13]. Pre-cultured cells were transferred into 8 independent cylindrical vessels of a multi-cultivator, filled with BG-11 medium with 0, 200 mM or 420 mM NaCl to an initial OD730 of 0.05. OD730 was measured every 5 min. Once the thresh- old of OD730 was reached (OD730 = 1) cell cultures were automatically diluted by 5% (v/v) with fresh BG-11 medium and the same volume of culture as the volume of medium just added, was discarded; all under control of “pycultivator”. All the cultures in the Multi-Cultivator were exposed to continuous white light with an intensity of 100 µmol photons m−2 s−1 OD−1. The genetic stabil- ity of each strain was assessed by mannitol production. Growth rate was calculated by fitting a linear function through the natural logarithm of the OD730 during each “growth-dilution” cycle. Samples for extracellular man- nitol quantification were periodically taken during the cultivation period. The variation of growth rate and man- nitol productivity throughout the whole experiment was then calculated relative to the initial values obtained at the beginning of each specific experiment. with fresh BG-11 medium without antibiotic, while simultaneously taking out an identical volume of cultured cells. Accordingly, cells in a turbidostat are under con- tinuous selection for maximal specific growth rate. For this we used a modified Multi-Cultivator with additional pumps (Reglo ICC, ISMATEC, Germany) and controlled by the “pycultivator” software package [13]. Pre-cultured cells were transferred into 8 independent cylindrical vessels of a multi-cultivator, filled with BG-11 medium with 0, 200 mM or 420 mM NaCl to an initial OD730 of 0.05. OD730 was measured every 5 min. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. References 1. Saha BC, Racine FM. Biotechnological production of mannitol and its applications. Appl Microbiol Biotechnol. 2011;89(4):879–91. 1. Saha BC, Racine FM. Biotechnological production of mannitol and its applications. Appl Microbiol Biotechnol. 2011;89(4):879–91. 1. Saha BC, Racine FM. Biotechnological production of mannitol and its applications. Appl Microbiol Biotechnol. 2011;89(4):879–91. 2. Angermayr SA, Gorchs Rovira A, Hellingwerf KJ. 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Ferenci T, Kornberg HL. The utilization of fructose by Escherichia coli Properties of a mutant defective in fructose 1-phosphate kinase activity. Biochem J. 1973;132(2):341–7. 21. Ferenci T, Kornberg HL. The utilization of fructose by Escherichia coli Properties of a mutant defective in fructose 1-phosphate kinase activity. Biochem J. 1973;132(2):341–7. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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https://openalex.org/W2949682053	https://hal.archives-ouvertes.fr/hal-01445705/document	English	null	Discovery of flavivirus-derived endogenous viral elements in<i>Anopheles</i>mosquito genomes supports the existence of<i>Anopheles</i>-associated insect-specific flaviviruses	Virus evolution	2,017	cc-by	6,377	Discovery of flavivirus-derived endogenous viral elements in Anopheles mosquito genomes supports the existence of Anopheles -associated insect-specific flaviviruses Sebastian Lequime, Louis Lambrechts Sebastian Lequime, Louis Lambrechts To cite this version: Sebastian Lequime, Louis Lambrechts. Discovery of flavivirus-derived endogenous viral elements in Anopheles mosquito genomes supports the existence of Anopheles -associated insect-specific fla- viviruses. Virus Evolution, 2017, 3 (1), 10.1093/ve/vew035. hal-01445705 Distributed under a Creative Commons Attribution 4.0 International License Abstract The Flavivirus genus encompasses several arboviruses of public health signiﬁcance such as dengue, yellow fever, and Zika viruses. It also includes insect-speciﬁc ﬂaviviruses (ISFs) that are only capable of infecting insect hosts. The vast majority of mosquito-infecting ﬂaviviruses have been associated with mosquito species of the Aedes and Culex genera in the Culicinae subfamily, which also includes most arbovirus vectors. Mosquitoes of the Anophelinae subfamily are not considered signiﬁ- cant arbovirus vectors; however, ﬂaviviruses have occasionally been detected in ﬁeld-caught Anopheles specimens. Whether such observations reﬂect occasional spillover or laboratory contamination or whether Anopheles mosquitoes are natural hosts of ﬂaviviruses is unknown. Here, we provide in silico and in vivo evidence of transcriptionally active, ﬂavivirus-derived endoge- nous viral elements (EVEs) in the genome of Anopheles minimus and Anopheles sinensis. Such non-retroviral endogenization of RNA viruses is consistent with a shared evolutionary history between ﬂaviviruses and Anopheles mosquitoes. Phylogenetic analyses of the two newly described EVEs support the existence of a distinct clade of Anopheles-associated ISFs. Key words: Flavivirus; host range; Anopheles sinensis; Anopheles minimus; ISF; EVE. V C The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Sebastian Lequime1,2,3,,† and Louis Lambrechts1,2 1Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, Paris, France, 2Centre National de la Recherche Scientiﬁque, Unite´ de Recherche Associe´e 3012, Paris, France and 3University Pierre et Marie Curie, Cellule Pasteur UPMC, Paris, France Corresponding author: E-mail: sebastian.lequime@gmail.com †http://orcid.org/0000-0002-3140-0651 *Corresponding author: E-mail: sebastian.lequime@gmail.com †http://orcid.org/0000-0002-3140-0651 HAL Id: hal-01445705 https://hal.science/hal-01445705v1 Submitted on 27 Jan 2017 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 Virus Evolution, 2017, 3(1): vew035 doi: 10.1093/ve/vew035 Research article doi: 10.1093/ve/vew035 Research article flaviviruses † and Louis Lambrechts1,2 Discovery of flavivirus-derived endogenous viral elements in Anopheles mosquito genomes supports the existence of Anopheles-associated insect-specific flaviviruses Discovery of flavivirus-derived endogenous viral elements in Anopheles mosquito genomes supports the existence of Anopheles-associated insect-specific flaviviruses 2 \| Virus Evolution, 2017, Vol. 3, No. 1 2 \| Virus Evolution, 2017, Vol. 3, No. 1 Anopheles punctipennis (Bernard et al. 2001; Kulasekera et al. 2001). In Asia, Japanese encephalitis virus was detected in Anopheles sinensis (Feng et al. 2012; Liu et al. 2013). In Europe, Anopheles maculipennis was found positive for WNV (Filipe 1972; Kemenesi et al. 2014) and Usutu virus (Calzolari et al. 2013). Interestingly, some ISFs were also detected in An. sinensis (Zuo et al. 2014; Liang et al. 2015) and Anopheles atroparvus (Aranda et al. 2009). It is unknown whether these detections reflect occa- sional spillover or laboratory contamination, or whether Anopheles mosquitoes are in fact natural hosts of flaviviruses. Katzourakis and Gifford 2010; Chen et al. 2015), which is consis- tent with the ancient evolutionary relationship between Aedes mosquitoes and ISFs. Here, we report the discovery of two flavivirus-derived EVEs in the genomes of An. minimus and An. sinensis mosquitoes. We screened 24 publicly available Anopheles genomes (Holt et al. 2002; Zhou et al. 2014; Logue et al. 2015; Neafsey et al. 2015) for flaviviral sequences, and validated in silico hits both at the DNA and RNA levels in vivo. The two newly described flavivirus- derived EVEs are phylogenetically related to ISFs, and support the existence of a previously unsuspected Anopheles-associated clade of ISFs. p q Endogenous viral elements (EVEs) are chromosomal integra- tions of partial or full viral genetic material into the genome of a host species. Not only retroviruses, whose replication cycle in- cludes incorporation of a DNA form of the RNA viral genome into the host cell genome, but virtually all types of eukaryotic vi- ruses can become endogenous (Feschotte and Gilbert 2012). Non-retroviral EVEs have been documented in the genome of a wide variety of host species, including vertebrates and arthro- pods (Feschotte and Gilbert 2012). Unlike detection of exogenous viruses, subject to possible laboratory or environmental con- tamination, EVEs are likely to reflect a long-lasting evolutionary relationship between an RNA virus and its natural host. This is because endogenization, for a single-stranded RNA virus, re- quires 1, reverse transcription; 2, integration of virus-derived DNA into the genome of germinal host cells; and 3, fixation of the integrated sequence in the host population (Holmes 2011; Aiewsakun and Katzourakis 2015). The low probability of this combination of events makes endogenization exceedingly un- likely to occur unless the viral infection is common in the host population over long evolutionary times. WGS, whole-genome shotgun sequencing. 2.1 In silico survey 2.1.1 Genome screen Twenty-four Anopheles genomes (full list and accession num- bers are provided in Table 1) were screened by tBLASTn (BLASTþ 2.2.28) (Camacho et al. 2009) for the presence of flavivirus-derived EVEs using a collection of 50 full flavivirus polyprotein queries (full list and accession numbers are pro- vided in Supplementary Table S1) representing the currently known diversity of the Flavivirus genus. The sequences of hits whose E-value was < 104 were extracted using an in-house bash shell script. In order to reconstruct putative flavivirus- derived EVEs, BLAST hits were clustered using CD-HIT v.4.6.1 (Li and Godzik 2006) and aligned using MAFFT v7.017 (Katoh et al. 2002). Putative EVEs were extracted and used as query for a re- ciprocal tBLASTn (BLASTþ 2.2.30) against the National Center for Biotechnology Information (NCBI) nucleotide database (E- value < 104). Genetic features of identified EVE were analyzed using the NCBI Conserved Domain Database (Marchler-Bauer et al. 2015). Nucleotide sequence data reported are available in 1. Introduction subfamily, mainly from the Culex and Aedes genera. Anopheles mosquitoes in the Anophelinae subfamily are well known for their role in the transmission of human malaria parasites, but they are not considered important vectors of arboviruses in gen- eral, and of flaviviruses in particular (Clements 2012). The only notable exception of Anopheles-transmitted arbovirus is the alphavirus O’nyong’nyong virus (Brault et al. 2004). Nevertheless, several studies have detected flaviviruses in field- caught Anopheles mosquitoes from different parts of the world. In North America, West Nile virus (WNV) was detected in Flaviviruses are positive-sense, single-stranded RNA viruses that infect a broad range of hosts including vertebrates (e.g. birds, primates) and arthropods (e.g. ticks, mosquitoes). In addi- tion to major arboviruses of public health significance such as dengue, Zika, West Nile and yellow fever viruses, the Flavivirus genus also includes vertebrate-specific (not known vector; NKV) and insect-specific (insect-specific flaviviruses; ISFs) members (Moureau et al. 2015). The majority of mosquito-infecting flavi- viruses have been associated with members of the Culicinae V The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1 1 2 \| Virus Evolution, 2017, Vol. 3, No. 1 For example, flavivirus-derived EVEs have been reported in the genomes of Aedes aegypti (Crochu et al. 2004; Katzourakis and Gifford 2010) and Aedes albopictus (Crochu et al. 2004; Roiz et al. 2009; Chen et al. 2015). These EVEs are phylogenetically related to the clade of Aedes-associated ISFs (Crochu et al. 2004; Roiz et al. 2009; 3. Results The in silico screen of 24 Anopheles genomes identified two flavivirus-derived EVEs, one in the reference genome sequence of An. minimus and one in both genome sequences available for An. sinensis (Table 2). 2.1.3 Transcriptome screen Published RNA sequencing (RNA-seq) data were retrieved from NCBI Sequence Read Archive (Leinonen et al. 2011) and explored for the presence of the previously identified EVE sequences. Only one An. minimus transcriptome sequence read archive was found under accession number SRX265162. Six An. sinensis tran- scriptome sequence read archives were found under accession numbers SRX448985, SRX449003, SRX449006, and SRX277584 for experiments using Illumina sequencing technology, and SRX218691 and SRX218721 for experiments using Roche 454 se- quencing technology. RNA-seq reads were mapped to the EVE nucleotide sequence using Bowtie2 v2.1.0 (Langmead and Salzberg 2012). The alignment file was converted, sorted, and indexed with Samtools v0.1.19 (Li et al. 2009). Coverage was as- sessed using bedtools v2.17.0 (Quinlan and Hall 2010). 2.2.3 EVE transcription level . .3 sc p o eve Mosquitoes were homogenized in pools of five separated by sex or development stage in 300 ml of DPBS during two rounds of 30 s at 5,000 rpm in a mixer mill (Precellys 24). RNA was ex- tracted from mosquito homogenates separated by sex using TRIzol Reagent (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Samples were treated with Turbo DNA-free kit (Life Technologies) and reverse transcribed using random hexamers and M-MLV reverse transcriptase (Invitrogen). Complementary DNA was amplified with 35 cycles of PCR for An. minimus and 40 cycles of PCR for An. sinensis, respectively, using DreamTaq po- lymerase (Thermo Fisher Scientific) and primers located within the EVE sequence (Supplementary Table S2). To verify that RNA samples were free of DNA contamination, two sets of primers spanning exons 3 and 4 of the RPS7 gene of both Anopheles spe- cies (under VectorBase annotation number AMIN008193 and ASIC017918 for An. minimus and An. sinensis, respectively) were designed (Supplementary Table S2). Because the corresponding DNA sequence includes intron 3, DNA contamination is ex- pected to result in a larger PCR product. The length of intron 3 is 252 base pairs (bp) for An. minimus and 295 bp for An. sinensis. 2.2.1 Mosquitoes An. minimus and An. sinensis mosquitoes were obtained through BEI Resources (www.beiresources.org), National Institute of Allergy and Infectious Diseases, National Institutes of Health (An. minimus MINIMUS1, MRA-729; An. sinensis SINENSIS, MRA- 1154). Anopheles minimus and An. sinensis specimens came from the 132nd and 65th generations of laboratory colonization, re- spectively. Eggs were hatched in filtered tap water, reared in 24 34 9 cm plastic trays and fed with fish food (TetraMin, Tetra, Melle, Germany). Adults were maintained in 30 30 30 cm screened cages under controlled insectary conditions (28 6 1 C, 75 6 5% relative humidity, 12:12 h light–dark cycle). They were provided with cotton soaked in a 10% (m/v) sucrose solution ad libitum. An. stephensi nucleic acids, used as a reaction control, were kindly provided by the Genetics and Genomics of Insect Vectors unit, Institut Pasteur, Paris. 2.1.2 Phylogenetic analyses Translated EVE sequences were aligned to the corresponding sections of several flavivirus polyproteins (Supplementary Table S1) with MAFFT v7.017 and phylogenetically uninforma- tive positions were trimmed using TrimAI v.1.3 (Capella- Gutie´rrez et al. 2009) accessed through the webserver Phylemon 2 (Sanchez et al. 2011). The trimmed alignments were used to construct phylogenetic trees with PhyML Best AIC Tree (Sanchez et al. 2011). Best substitution models were Blosum62 þ I þ GþF for An. minimus (regardless of gene-coding domains) and Blosum62 þ I þ G for An. sinensis. 2.1.1 Genome screen Table 1. Anopheles genomes screened in this study. Table 1. Anopheles genomes screened in this study. Species GenBank WGS project Assembly GenBank assembly ID An. albimanus APCK01 AalbS1 GCA_000349125.1 An. arabiensis APCN01 AaraD1 GCA_000349185.1 An. atroparvus AXCP01 AatrE1 GCA_000473505.1 An. christyi APCM01 AchrA1 GCA_000349165.1 An. coluzzii ABKP02 AcolM1 GCA_000150765.1 An. culicifacies A AXCM01 AculA1 GCA_000473375.1 An. darlingi ADMH02 AdarC3 GCA_000211455.3 An. dirus A APCL01 AdirW1 GCA_000349145.1 An. epiroticus APCJ01 AepiE1 GCA_000349105.1 An. farauti AXCN01 AfarF1 GCA_000473445.1 An. funestus APCI01 AfunF1 GCA_000349085.1 An. gambiae AAAB01 AgamP4 GCA_000005575.2 An. koliensis JXXB01 AKwgs3 GCA_000956275.1 An. maculatus B AXCL01 AmacM1 GCA_000473185.1 An. melas ACXO01 AmelC1 GCA_000473525.1 An. merus AXCQ01 AmerM1 GCA_000473845.1 An. minimus A APHL01 AminM1 GCA_000349025.1 An. nili ATLZ01 Anili1 GCA_000439205.1 An. punctulatus JXXA01 APwgs2 GCA_000956255.1 An. quadriannulatus A APCH01 AquaS1 GCA_000349065.1 An. sinensis: Sinensis (Korean strain) AXCK02 AsinS2 GCA_000472065.2 An. sinensis: China (Chinese strain) ATLV01 AsinC2 GCA_000441895.2 An. stephensi: Indian ALPR002 AsteI2 GCA_000300775.2 An. stephensi: SDA-500 APCG01 AsteS1 GCA_000349045.1 Lequime and Lambrechts \| 3 the Third Party Annotation Section of the DDBJ/ENA/GenBank databases under the accession numbers TPA: BK009978- BK009980. 2.2.2 EVE genomic integration Mosquitoes were homogenized in pools of 10 separated by sex in 300 ml of Dulbecco’s phosphate-buffered saline (DPBS) during two rounds of 30 s at 5,000 rpm in a mixer mill (Precellys 24, Bertin Technologies, Montigny le Bretonneux, France). DNA was extracted using All Prep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. EVE pres- ence in genomic DNA was assessed by 35 cycles of PCR using Taq Polymerase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) (Supplementary Table S2). PCR primers were designed to generate an amplicon spanning part of the EVE sequence and a section of the flanking host sequence. Identity of the EVE sequence was confirmed by Sanger sequenc- ing of the PCR product. 2.1.2 Phylogenetic analyses 3.1 An. minimus EVE The An. minimus EVE is 1,881-bp long (627 amino-acid residues) with Nienokoue virus as the closest BLAST hit (44% amino-acid identity). The integrated sequence spans non-structural protein Table 2. Newly described ﬂavivirus-derived EVEs in An. minimus and An. sinensis genomes. Element GenBank accession no. Protein identity Supercontig accession no. Supercontig length (bp) Coordinates Supercontig Viral genomea Start End Start End An. minimus EVE BK009978 NS4-NS5 KB664005.1 2,043 107 1,987 6,567 8,432 An. sinensis EVE (Korean strain) BK009979 NS3 AXCK02024744 2,797 822 1,613 5,214 5,822 An. sinensis EVE (Chinese strain) BK009980 NS3 ATLV01019207.1 7,380 3,578 4,376 5,214 5,822 aViral genomes coordinates are based on the closest tBLASTn hits: Nienokoue virus (GenBank accession no. JQ957875) for the An. minimus EVE and Culex ﬂavivirus (GenBank accession no. JQ308188) for the An. sinensis EVE. Table 2. Newly described ﬂavivirus-derived EVEs in An. minimus and An. sinensis genomes. aViral genomes coordinates are based on the closest tBLASTn hits: Nienokoue virus (GenBank accession no. JQ957875) for the An. minimus EVE and Culex ﬂavivirus (GenBank accession no. JQ308188) for the An. sinensis EVE. 4 \| Virus Evolution, 2017, Vol. 3, No. 1 4A (NS4A), NS4B and NS5 (Fig. 1A) without any stop codon. Conserved-domain search identified the NS5- methyltransferase domain involved in RNA capping and part of the RNA-directed RNA-polymerase domain. Phylogenetically, the An. minimus EVE is sister to the ISF clade (Fig. 1B). The An. minimus genome supercontig where the EVE was detected is 2,043-bp long and consists almost exclusively of the EVE sequence. identity. The An. minimus EVE was found in both male and fe- male genomic DNA, and was transcriptionally expressed for all combinations of sex and development stages tested (Fig. 2B). Evidence for transcriptional activity of the An. minimus EVE was confirmed in published RNA-seq data (Figure S1A). 3.2 An. sinensis EVE Presence of the EVE was verified in vivo by PCR on genomic DNA (Fig. 2A), followed by amplicon sequencing to confirm The An. sinensis EVE was detected in two distinct genome se- quences that are available for this mosquito species, derived A A B ery of a ﬂavivirus-derived EVE in An. minimus. (A) EVE location in a generic Flavivirus genome. Positioning is based on the genome sequen (GenBank accession no. JQ957875). C, capsid protein; E, envelope glycoprotein; M, membrane glycoprotein; NS1, non-structural glycoprotein 1; N rotein 2A; NS2B, non-structural protein 2B; NS3, non-structural protein 3 (protease/helicase); NS4A, non-structural protein 4A; NS4B, non-stru non-structural protein 5 (RNA-dependent RNA polymerase). (B) Phylogenetic relationships of the newly discovered An. minimus ﬂavivirus-derive ﬂaviviruses. Maximum likelihood trees were constructed based on the translated EVE sequence. Clades are color-coded according to known hos B B Figure 1. Discovery of a ﬂavivirus-derived EVE in An. minimus. (A) EVE location in a generic Flavivirus genome. Positioning is based on the genome sequence of Nienokoue virus (GenBank accession no. JQ957875). C, capsid protein; E, envelope glycoprotein; M, membrane glycoprotein; NS1, non-structural glycoprotein 1; NS2A, non-structural protein 2A; NS2B, non-structural protein 2B; NS3, non-structural protein 3 (protease/helicase); NS4A, non-structural protein 4A; NS4B, non-structural protein 4B; NS5, non-structural protein 5 (RNA-dependent RNA polymerase). (B) Phylogenetic relationships of the newly discovered An. minimus ﬂavivirus-derived EVE with exogenous ﬂaviviruses. Maximum likelihood trees were constructed based on the translated EVE sequence. Clades are color-coded according to known host spe- ciﬁcity: green, ISFs; purple, tick-borne arboviruses; black, ‘NKV’ (vertebrate speciﬁc); blue, mosquito-borne arboviruses; red: EVEs. Scale bar indicates the number of substitutions. Node values represent Shimodaira-Hasegawa (SH)-like branch support (only values > 0.8 are shown). Figure 1. Discovery of a ﬂavivirus-derived EVE in An. minimus. (A) EVE location in a generic Flavivirus genome. Positioning is based on the genome sequence of Nienokoue virus (GenBank accession no. JQ957875). C, capsid protein; E, envelope glycoprotein; M, membrane glycoprotein; NS1, non-structural glycoprotein 1; NS2A, non-structural protein 2A; NS2B, non-structural protein 2B; NS3, non-structural protein 3 (protease/helicase); NS4A, non-structural protein 4A; NS4B, non-structural protein 4B; NS5, non-structural protein 5 (RNA-dependent RNA polymerase). (B) Phylogenetic relationships of the newly discovered An. minimus ﬂavivirus-derived EVE with exogenous ﬂaviviruses. Maximum likelihood trees were constructed based on the translated EVE sequence. 3.2 An. sinensis EVE Clades are color-coded according to known host spe- ciﬁcity: green, ISFs; purple, tick-borne arboviruses; black, ‘NKV’ (vertebrate speciﬁc); blue, mosquito-borne arboviruses; red: EVEs. Scale bar indicates the number of substitutions. Node values represent Shimodaira-Hasegawa (SH)-like branch support (only values > 0.8 are shown). A B Figure 2. In vivo detection of the An. minimus ﬂavivirus-derived EVE. (A) EVE detection in genomic DNA. Lane 1: size marker; lane 2: ampliﬁed genomic DNA from a pool of 10 An. minimus adult females; lane 3: ampliﬁed genomic DNA from a pool of 10 An. minimus adult males; lane 4: ampliﬁed genomic DNA from a pool of 10 An. sinensis adult females; lane 5: ampliﬁed genomic DNA from a pool of 10 An. sinensis adult males; lane 6: ampliﬁed DNA from a pool of 10 An. stephensi females; lane 7: no tem- plate control (NTC). (B) EVE detection in total RNA. Lane 1: size marker; lanes 2 and 3: ampliﬁed cDNA from pools of ﬁve adult females; lanes 4 and 5: ampliﬁed cDNA from pools of ﬁve adult males; lanes 6 and 7: ampliﬁed cDNA from pools of ﬁve L4 larvae; lane 8: ampliﬁed DNA from a pool of 10 females; lane 9: ampliﬁed cDNA from a pool of 5 An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of ﬁve adult females as lane 2; lane 11: NTC. First row: EVE; second row: RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA contamination is expected to result in a larger PCR product. Lequime and Lambrechts \| 5 Lequime and Lambrechts \| 5 A B A B Figure 2. In vivo detection of the An. minimus ﬂavivirus-derived EVE. (A) EVE detection in genomic DNA. Lane 1: size marker; lane 2: ampliﬁed genomic DNA from a pool of 10 An. minimus adult females; lane 3: ampliﬁed genomic DNA from a pool of 10 An. minimus adult males; lane 4: ampliﬁed genomic DNA from a pool of 10 An. sinensis adult females; lane 5: ampliﬁed genomic DNA from a pool of 10 An. sinensis adult males; lane 6: ampliﬁed DNA from a pool of 10 An. stephensi females; lane 7: no tem- plate control (NTC). (B) EVE detection in total RNA. 4. Discussion ISFs have attracted substantial interest in recent years after some of them were shown to enhance or suppress the replica- tion of medically important flaviviruses in co-infected mosquito cells (Blitvich and Firth 2015). Over a dozen of ISFs have been formally identified to date, mainly in Aedes and Culex genera of the Culicinae subfamily (Blitvich and Firth 2015). ISFs were also reported in Anopheles mosquitoes of the Anophelinae subfamily (Aranda et al. 2009; Zuo et al. 2014; Liang et al. 2015). However, these Anopheles-associated ISFs are thought to infect a broad range of hosts including several mosquito species, mainly in the Culex genus, and are phylogenetically related to Culex-asso- ciated ISFs. Therefore, it is unclear whether Anopheles mosqui- toes are true natural hosts of flaviviruses. Detection of ISFs in field-caught mosquitoes could result from incidental infection, or from a laboratory artifact. In this study, we discovered flavivirus-derived EVEs in the genomes of two Anopheles species. Phylogenetic analyses indicated that both EVEs are related to ISFs but belong to a clade that is distinct from Aedes-associated and Culex-associated ISFs. The An. sinensis supercontig containing the EVE is 2,797-bp long for the Korean strain and 7,380-bp long for the Chinese strain. Analyses of flanking regions revealed the presence of another EVE in the same orientation, upstream of the flavivirus-derived EVE in both the Korean and the Chinese strains (Supplementary Table S3 and Supplementary Fig. S2). The closest BLAST hit of this addi- tional EVE is Xincheng mosquito virus (43 and 42% amino-acid identity for the Korean and Chinese strains, respectively), an un- classified, negative-sense, single-stranded RNA virus detected in a pool of Chinese mosquitoes including An. sinensis specimens (Supplementary Table S3 and Supplementary Fig. S2). BLAST and conserved-domain search identified a class II Mariner-like trans- posase close to a mariner mos1 element, 1,000 bp downstream of the flavivirus-derived EVE (Supplementary Table S3 and Supplementary Fig. S2). This was only observed for the Chinese strain because the supercontig of the Korean strain did not span the downstream region far enough. Presence of flavivirus-derived EVEs in Anopheles genomes supports the hypothesis that Anopheles mosquitoes are, or were, natural hosts of flaviviruses. Endogenization of non-retroviral RNA viruses is unlikely to occur in the absence of recurrent host-virus interactions over a long evolutionary time scale. Endogenization requires reverse transcription, germ line inte- gration and fixation in the host population, three steps whose Presence of the An. 3.2 An. sinensis EVE Lane 1: size marker; lanes 2 and 3: ampliﬁed cDNA from pools of ﬁve adult females; lanes 4 and 5: ampliﬁed cDNA from pools of ﬁve adult males; lanes 6 and 7: ampliﬁed cDNA from pools of ﬁve L4 larvae; lane 8: ampliﬁed DNA from a pool of 10 females; lane 9: ampliﬁed cDNA from a pool of 5 An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of ﬁve adult females as lane 2; lane 11: NTC. First row: EVE; second row: RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA contamination is expected to result in a larger PCR product. An. sinensis EVE, for all combinations of sex and development stages tested, especially in L4 larvae (Fig. 4B). Low expression ob- served for the An. sinensis EVE is consistent with barely detect- able transcriptional activity in published RNA-seq data (Supplementary Fig. S1B). from a Korean strain and a Chinese strain, respectively. The EVE is 792-bp long (264 amino-acid residues) and 799-bp long (266 amino-acid residues) for the Korean and Chinese strains, respectively (Table 1). The closest BLAST hit is Culex flavivirus (45% amino-acid identity for the Korean strain, 44% amino-acid identity for the Chinese strain). The integrated sequence corre- sponds to the middle part of NS3 (Fig. 3A) and contains six and eight stop codons in the Korean and Chinese strains, respec- tively. Conserved-domain search identified the presence of a P- loop containing the nucleoside triphosphate hydrolase domain found in the NS3 protein of exogenous flaviviruses. Phylogenetically, the An. sinensis EVE is sister to the ISF clade (Fig. 3B). 4. Discussion sinensis EVE was verified in vivo by PCR on genomic DNA from the Korean strain (Fig. 4A), followed by amplicon sequencing to confirm identity. The An. sinensis EVE was found in both male and female genomic DNA, and was transcriptionally expressed, although less abundantly than the 6 \| Virus Evolution, 2017, Vol. 3, No. 1 A B very of a ﬂavivirus-derived EVE in An. sinensis. (A) EVE location in a generic Flavivirus genome. Positioning is based on the genome sequence of Culex ﬂav accession no. JQ308188). C, capsid protein; E, envelope glycoprotein; M, membrane glycoprotein; NS1, non-structural glycoprotein 1; NS2A, non-stru A; NS2B, non-structural protein 2B; NS3, non-structural protein 3 (protease/helicase); NS4A, non-structural protein 4A; NS4B, non-structural protein 4 ural protein 5 (RNA-dependent-RNA polymerase). (B) Phylogenetic relationships of the newly discovered An. sinensis ﬂavivirus-derived EVEs with exog es. Maximum likelihood trees were constructed based on the translated EVE sequence. Clades are color-coded according to known host speciﬁcit ple, tick-borne arboviruses; black, ‘NKV’ (vertebrate speciﬁc); blue, mosquito-borne arboviruses; red, EVEs. Scale bar indicates the number of substit ues represent SH-like branch support (only values > 0.8 are shown). A B Figure 3. Discovery of a ﬂavivirus-derived EVE in An. sinensis. (A) EVE location in a generic Flavivirus genome. Positioning is based on the genome sequence of Culex ﬂavi- virus (GenBank accession no. JQ308188). C, capsid protein; E, envelope glycoprotein; M, membrane glycoprotein; NS1, non-structural glycoprotein 1; NS2A, non-struc- tural protein 2A; NS2B, non-structural protein 2B; NS3, non-structural protein 3 (protease/helicase); NS4A, non-structural protein 4A; NS4B, non-structural protein 4B; NS5, non-structural protein 5 (RNA-dependent-RNA polymerase). (B) Phylogenetic relationships of the newly discovered An. sinensis ﬂavivirus-derived EVEs with exoge- nous ﬂaviviruses. Maximum likelihood trees were constructed based on the translated EVE sequence. Clades are color-coded according to known host speciﬁcity: green, ISFs; purple, tick-borne arboviruses; black, ‘NKV’ (vertebrate speciﬁc); blue, mosquito-borne arboviruses; red, EVEs. Scale bar indicates the number of substitu- tions. Node values represent SH-like branch support (only values > 0.8 are shown). combined probability is exceedingly low (Holmes 2011; Aiewsakun and Katzourakis 2015). The species-wide frequency of the An. minimus EVE is unknown because our in silico and in vivo analyses were based on the same mosquito strain. Presence of the An. sinensis EVE in two mosquito strains from different geographical locations, however, suggests that it could be fixed at the species level. 4. Discussion Thus, our discovery of flavivirus-derived EVEs in Anopheles genomes is consistent with a long-lasting host-virus interaction between flaviviruses and mosquitoes of the Anophelinae subfamily. Although direct identification and characterization of exogenous Anopheles- associated flaviviruses are required to provide definite evidence that Anopheles are natural hosts of flaviviruses, our results pro- vide strong indirect support to this hypothesis. Our finding em- phasizes the need to include a broader range of mosquito genera and species than is typically screened in arbovirus sur- veillance programs. ISFs are thought to be mainly maintained through vertical transmission from an infected female to its offspring (Blitvich and Firth 2015). Vertical transmission is likely to favor co-divergence of pathogens and hosts (Jackson and Charleston 2004), as illus- trated by the existence of Aedes-associated and Culex-associated Lequime and Lambrechts \| 7 Figure 4. In vivo detection of the An. sinensis ﬂavivirus-derived EVE. (A) EVE detection in genomic DNA from the Korean strain of An. sinensis. Lane 1, size marker; lane 2, ampliﬁed genomic DNA from a pool of 10 An. minimus adult females; lane 3, ampliﬁed genomic DNA from a pool of 10 An. minimus adult males; lane 4, ampliﬁed geno- mic DNA from a pool of 10 An. sinensis adult females; lane 5, ampliﬁed genomic DNA from a pool of 10 An. sinensis adult males; lane 6, ampliﬁed DNA from a pool of 10 An. stephensi females; lane 7, NTC. (B) EVE detection in total RNA from the Korean strain of An. sinensis. Lane 1: size marker; lanes 2 and 3, ampliﬁed cDNA from pools of ﬁve adult females; lanes 4 and 5, ampliﬁed cDNA from pools of ﬁve adult males; lanes 6 and 7, ampliﬁed cDNA from pools of ﬁve L4 larvae; lane 8, ampliﬁed DNA from a pool of ten females; lane 9, ampliﬁed cDNA from a pool of ﬁve An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of ﬁve adult females as lane 2; lane 11, NTC. First row, EVE; second row, RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA con- tamination is expected to result in a larger PCR product. Figure 4. In vivo detection of the An. sinensis ﬂavivirus-derived EVE. (A) EVE detection in genomic DNA from the Korean strain of An. sinensis. Lane 1, size marker; lane 2, ampliﬁed genomic DNA from a pool of 10 An. References Aiewsakun, P., and Katzourakis, A. (2015) ‘Endogenous Viruses: Connecting Recent and Ancient Viral Evolution’, Virology, 479– 480, 26–37 Aranda, C. et al. (2009) ‘Detection and Monitoring of Mosquito Flaviviruses in Spain Between 2001 and 2005’, Vector Borne and Zoonotic Diseases, 9: 171–8 Aswad, A., and Katzourakis, A. (2012) ‘Paleovirology and Virally Derived Immunity’, Trends in Ecology and Evolution (Amsterdam), 27: 627–36 Supplementary data Supplementary data are available at Virus Evolution online. Bell-Sakyi, L., and Attoui, H. (2013) ‘Endogenous Tick Viruses and Modulation of Tick-Borne Pathogen Growth’, Frontiers in Cellular and Infection Microbiology, 3: 25 Funding Non-retroviral EVEs are thought to be generated by interac- tion of exogenous viruses with endogenous retro-elements, ei- ther with or without long terminal repeats (Holmes 2011). The short size of the supercontigs containing the An. minimus and An. sinensis EVEs limited our ability to investigate the integra- tion mechanism(s). Another EVE sequence that we identified close to the flavivirus-derived EVE in An. sinensis may point to an EVE hotspot. This work was supported by the French Government’s Investissement d’Avenir program Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases grant ANR-10-LABX-62-IBEID, and the City of Paris Emergence(s) program in Biomedical Research. S.L. was supported by a doc- toral fellowship from University Pierre and Marie Curie. The funders had no role in study design, data collection and inter- pretation, or the decision to submit the work for publication. Sequence conservation of the An. minimus EVE (i.e. absence of stop codon across 1,881 bp) is consistent with a recent inte- gration or a more ancient integration followed by non-neutral evolution. Our observation that the An. minimus EVE is abun- dantly transcribed may reflect a selective advantage for the host (Holmes 2011). 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Lane 1: size marker; lanes 2 and 3, ampliﬁed cDNA from pools of ﬁve adult females; lanes 4 and 5, ampliﬁed cDNA from pools of ﬁve adult males; lanes 6 and 7, ampliﬁed cDNA from pools of ﬁve L4 larvae; lane 8, ampliﬁed DNA from a pool of ten females; lane 9, ampliﬁed cDNA from a pool of ﬁve An. stephensi females; lane 10: DNA contamination control (no reverse transcription) using the same pool of ﬁve adult females as lane 2; lane 11, NTC. First row, EVE; second row, RPS7 (control gene). The RPS7 target DNA sequence includes an intron, so that DNA con- tamination is expected to result in a larger PCR product. with the in silico screen, Davy Jiolle and Elliott Miot for tech- nical assistance, Cle´ment Gilbert for advice and critical reading of an earlier version of the article, and members of the Lambrechts lab for insightful comments and discus- sions. 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https://openalex.org/W4254456297	https://periodicos.ufmg.br/index.php/aletria/article/download/18105/14895	Portuguese	null	A paixão segundo São João: uma retórica intermidiática	Aletria	2,006	cc-by	8,175	A PAIXÃO SEGUNDO SÃO JOÃO u m a r e t ó r i c a i n t e r m i d i á t i c a u m a r e t ó r i c a i n t e r m i d i á t i c a u m a r e t ó r i c a i n t e r m i d i á t i c a u m a r e t ó r i c a i n t e r m i d i á t i c a u m a r e t ó r i c a i n t e r m i d i á t i c a Thiago César Viana Lopes Saltarelli Mestrando em Teoria da Literatura - POSLIT/UFMG Thiago César Viana Lopes Saltarelli Mestrando em Teoria da Literatura - POSLIT/UFMG RRRRR E S U M O E S U M O E S U M O E S U M O E S U M O Este artigo propõe uma leitura da Paixão segundo São João, de Johann Sebastian Bach, como uma obra mixmídia, por meio da análise das relações entre os diversos sistemas de signos que a compõem ou que participam ou influenciam a sua execução. Essa análise, que privilegia as relações entre o sistema musical e o sistema verbal – embora não deixe de considerar outros sistemas de signos – visa também a demonstrar como obras de caráter intersemiótico já existiam em épocas anteriores ao Modernismo. A L E T R I A - jul.-dez. - 2 0 0 6 PPPPP A L A V R A S A L A V R A S A L A V R A S A L A V R A S A L A V R A S ----- C H A V E C H A V E C H A V E C H A V E C H A V E Nos tempos atuais, os chamados estudos interartes vêm, aos poucos, conquistando espaço no meio acadêmico. É óbvio que, desde a Antigüidade, o homem já refletia sobre a relação entre as diversas manifestações artísticas, mas essa reflexão se realizava de forma pouco sistematizada, em ensaios e estudos isolados, pontuais. Ao contrário, o que se tenta fazer nas universidades atualmente é sistematizar esses estudos, desenvolvendo uma metodologia própria e eficaz de análise, com conceitos que, transpostos de um sistema artístico para outro, deixem de ser ambíguos ou metafóricos. Muito do impulso dado a esse tipo de estudo se deve à própria criação artística pós-moderna, que elimina algumas fronteiras antes bem delimitadas entre os diversos sistemas de signos e propõe novos modelos de obras, como, por exemplo, a poesia sonora e o vídeo-poema. Esse paralelismo entre teoria/crítica e produção não é novidade, sendo encontrado no Romantismo alemão, com a colaboração entre filósofos e poetas, senão com o amálgama dessas duas instâncias num filósofo-poeta, como Schiller; no Formalismo Russo, unido à vanguarda cubo-futurista; na atual Crítica Cultural, uma vez que os discursos minoritários começam a ganhar seu espaço de propagação. Estes são apenas alguns exemplos que demonstram como uma determinada produção artística de uma época pode alavancar estudos teóricos e críticos que incorporam a sua perspectiva de mundo e de arte. Assim tem ocorrido no que tange à produção e aos estudos interartes contemporâneos. Há aqui, entretanto, um risco no qual incorrem muitos pesquisadores. Fascinados com as inovações artísticas atuais, muitas vezes proporcionadas pelas também fascinantes inovações tecnológicas da nossa era, esquecem-se de que o passado, se não conheceu a A L E T R I A - jul.-dez. - 2 0 0 6 3 3 4 Disponível em: http://www.letras.ufmg.br/poslit Disponível em: http://www.letras.ufmg.br/poslit consciência crítica e o desenvolvimento teórico que temos hoje com relação aos estudos interartes, produziu obras importantes em que convergiam sistemas de signos diversos. Assim, é importante não se perder essa perspectiva diacrônica do estudo, pois essas obras do passado podem gerar muitas contribuições. No âmbito das relações entre literatura ou texto verbal e música, o período denominado Barroco1 é incrivelmente rico para os estudos interartes, pois a relação música-palavra passa a se constituir numa espécie de coluna mestra da produção de música vocal. PPPPP A L A V R A S A L A V R A S A L A V R A S A L A V R A S A L A V R A S ----- C H A V E C H A V E C H A V E C H A V E C H A V E A adequação entre o texto e a melodia é o que caracteriza o programa estético a partir do século XVII, que se diferencia radicalmente da polifonia da Renascença, em que as palavras se perdem em meio ao turbilhão harmônico, o qual pode chegar a trinta e duas vozes! Nesse intuito é que pretendemos fazer uma análise da Paixão segundo São João, de Johann Sebastian Bach, demonstrando as relações entre os diversos sistemas semióticos que compõem a obra. Poder-se-ia proceder à análise de outra obra do período, mas acreditamos que as Paixões de Bach são exemplares quanto à diversidade de sistemas de signos que evocam. Optamos aqui pela São João devido à sua concisão em relação à São Mateus. 1 Não é nosso objetivo aqui discutir a noção e a validade do conceito de Barroco e tampouco os seus limites temporais. Para os fins de nosso trabalho, consideremos, de maneira genérica, o período entre o século XVII e a primeira metade do século XVIII. 1 Não é nosso objetivo aqui discutir a noção e a validade do conceito de Barroco e tampouco os seus limites temporais. Para os fins de nosso trabalho, consideremos, de maneira genérica, o período entre o século XVII e a primeira metade do século XVIII. 2 MASSIN. História da música ocidental, p. 83. 3 Forma menos rígida a meio caminho do recitativo e da ária. A A A A A QUESTÃO QUESTÃO QUESTÃO QUESTÃO QUESTÃO DO DO DO DO DO GÊNERO GÊNERO GÊNERO GÊNERO GÊNERO As Paixões musicais barrocas são uma espécie do gênero oratório. Este tem sua origem nos dramas litúrgicos medievais, isto é, encenações de passagens da Bíblia nas igrejas, com récitas e cantos. O drama litúrgico foi percorrendo os séculos, sendo retomado e modificado, até que, no século XVI, São Filipe de Néri realizou essas encenações no Oratorio de Santa Maria em Vallicella, em Roma. Daí surgiu o nome que designa o gênero.2 A partir do século XVII, o oratório incorporou os principais elementos inovadores da ópera, que acabara de ser criada, sobretudo a monodia acompanhada por um baixo contínuo. Logo se desenvolveu até a forma consagrada que lhe deram Charpentier, Alessandro Scarlatti e Händel. O que interessa a nosso estudo, entretanto, é perceber que o gênero oratório é constituído normalmente por uma dimensão épica, uma lírica e ainda uma terceira, dramática. No caso da Paixão segundo São João isso pode ser facilmente percebido: a dimensão épica está contida na narração de trechos do Evangelho de João – e eventualmente alguns trechos do de Mateus – pelo evangelista, por meio dos recitativos narrativos. A dimensão dramática consiste, ainda no texto do Evangelho, nas passagens em que os personagens da história bíblica participam diretamente das cenas e tomam a palavra. Em termos textuais, ocorre a transformação do discurso indireto do evangelista no discurso direto dos personagens. Essas passagens são postas em música nos recitativos expressivos e nos coros – estes últimos representando a turba, a multidão de judeus disposta a condenar Jesus. A dimensão lírica é dada em dois momentos de texto poético: nas árias e nos ariosos.3 O compositor O que interessa a nosso estudo, entretanto, é perceber que o gênero oratório é constituído normalmente por uma dimensão épica, uma lírica e ainda uma terceira, dramática. No caso da Paixão segundo São João isso pode ser facilmente percebido: a dimensão épica está contida na narração de trechos do Evangelho de João – e eventualmente alguns trechos do de Mateus – pelo evangelista, por meio dos recitativos narrativos. A dimensão dramática consiste, ainda no texto do Evangelho, nas passagens em que os personagens da história bíblica participam diretamente das cenas e tomam a palavra. Em termos textuais, ocorre a transformação do discurso indireto do evangelista no discurso direto dos personagens. 3 Forma menos rígida a meio caminho do recitativo e da ária. 2 MASSIN. História da música ocidental, p. 83. A A A A A QUESTÃO QUESTÃO QUESTÃO QUESTÃO QUESTÃO DO DO DO DO DO GÊNERO GÊNERO GÊNERO GÊNERO GÊNERO Essas passagens são postas em música nos recitativos expressivos e nos coros – estes últimos representando a turba, a multidão de judeus disposta a condenar Jesus. A dimensão lírica é dada em dois momentos de texto poético: nas árias e nos ariosos.3 O compositor 1 Não é nosso objetivo aqui discutir a noção e a validade do conceito de Barroco e tampouco os seus limites temporais. Para os fins de nosso trabalho, consideremos, de maneira genérica, o período entre o século XVII e a primeira metade do século XVIII. 2 0 0 6 - jul.-dez. - A L E T R I A 3 3 5 Disponível em: http://www.letras.ufmg.br/poslit esboça uma meditação pessoal ou emite um comentário sobre a cena ou a narrativa que acaba de suceder. Nos corais, realiza a prece comunitária de todos os fiéis. Essa noção da prece comunitária será discutida com mais pormenores logo adiante. No momento, é importante ressaltar que coral, aqui, não designa um conjunto de pessoas cantando, mas uma forma musical, geralmente simples e a quatro vozes, na qual estas possuem o mesmo desenho melódico e as frases possuem o mesmo tamanho e estrutura. A maioria dos corais utilizados por Bach, ainda que reelaborados, tanto no nível musical quanto no textual, são bastante conhecidos na Igreja Luterana. Alguns são mesmo de autoria do próprio Lutero. Assim, essas formas são a parte da Paixão oriunda de domínio público, conhecida de todos os fiéis. De qualquer modo, tornamos a ressaltar como a teoria clássica dos gêneros literários, que se afirma muito fortemente no Renascimento pela via de Horácio, Aristóteles e outros escritores da Antigüidade, se presta à classificação do gênero musical oratório, inclusive na sua diferenciação da cantata e da ópera. Considerar as dimensões épica, lírica e dramática no estudo do oratório revela-se uma pertinente chave de leitura, além de já apontar para a característica intermidiática desse gênero tão antigo. 4 HARNONCOURT. O diálogo musical, p. 94. A P A P A P A P A PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO ENQUANTO ENQUANTO ENQUANTO ENQUANTO ENQUANTO RITUAL RITUAL RITUAL RITUAL RITUAL EEEEE AS AS AS AS AS PRÁTICAS PRÁTICAS PRÁTICAS PRÁTICAS PRÁTICAS SOCIAIS SOCIAIS SOCIAIS SOCIAIS SOCIAIS O espectador grego do teatro e o ouvinte seiscentista e setecentista do oratório, respectivamente, nunca consideram o término de uma tragédia ou da Paixão como um fim definitivo, pois ainda estão por vir a comédia ou o oratório de Páscoa para a purgação do horror e da piedade e conseqüente equilíbrio dos sentimentos dos cidadãos ou dos fiéis. Também como o teatro grego, a encenação da Paixão mobilizava toda a comunidade. Esse caráter de ritual comunitário é ressaltado pela opinião de alguns musicólogos de que a congregação dos fiéis participava do canto dos corais, uma vez que estes eram oriundos de domínio público, da tradição da Igreja Luterana. Assim, durante os recitativos, coros e árias, o público apenas assistia à representação da história de Jesus mas, no momento dos corais, levantava-se e entoava a sua prece num ato de comunhão uns com os outros, com os músicos e, obviamente, com Deus. Ainda que o próprio Harnoncourt, no caso específico de Bach, discorde dessa participação da congregação nos corais, alegando que o compositor sofisticava-os em demasia ao reelaborá-los,5 não nos podemos furtar a considerá-los como um símbolo da congregação no contexto da Paixão. Destarte, a obra não só se prestava à representação da narrativa bíblica e à fruição estética, como também, nos termos do teatro grego, possuía uma função política, no sentido da manutenção social da pólis e de suas hierarquias. Essa questão da hierarquia é um ponto capital da sociedade barroca, na qual o Absolutismo e a divisão estamental da sociedade atingiram o seu ápice. Se pudermos extrapolar e pensar na relação entre as hierarquias sociais e as hierarquias dos elementos musicais como uma relação intersemiótica, veremos que a música da época, refletindo a sociedade, também é altamente hierarquizada.6 Harnoncourt afirma que, “na música barroca, tudo é ordenado hierarquicamente, tal como acontecia outrora em todos os outros domínios da vida”. Numa passagem anterior, diz que “esta hierarquia é [...] para a música dos séculos XVII e XVIII, talvez o dado mais fundamental e importante”.7 Em relação à acentuação dos tempos, por exemplo, há notas nobres e vis. Num compasso quaternário, o primeiro tempo é nobre, o segundo é vil, o terceiro é não tão nobre, o quarto é miserável. Perpassando essa hierarquia, há outra, a da acentuação com base na harmonia: uma dissonância, ainda que incida sobre um tempo fraco, deve ser sempre acentuada para causar a sensação de incômodo. A P A P A P A P A PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO ENQUANTO ENQUANTO ENQUANTO ENQUANTO ENQUANTO RITUAL RITUAL RITUAL RITUAL RITUAL EEEEE AS AS AS AS AS PRÁTICAS PRÁTICAS PRÁTICAS PRÁTICAS PRÁTICAS SOCIAIS SOCIAIS SOCIAIS SOCIAIS SOCIAIS Atualmente, a despeito de quaisquer questões mercadológicas que envolvem o universo artístico, é fato que a nossa curiosidade pela arte do passado transforma tudo em espetáculo para ser consumido apenas por fruição estética. Todavia, é importante ter em mente que, para os contemporâneos de determinadas produções artísticas, as obras poderiam não ter esse mesmo valor ou função. É o que acontece com a Paixão segundo São João. Longe de se constituir num espetáculo a ser levado para as salas de concerto – que só no fim da vida de Bach começaram a surgir, de forma muito incipiente – essa obra configurava-se como parte de um ritual a ser realizado na igreja, com a participação da população, de acordo com o calendário litúrgico. Nikolaus Harnoncourt afirma que “as obras sacras de Bach foram compostas para determinadas execuções, em circunstâncias precisas, se não em termos qualitativos, ao menos em termos quantitativos”.4 Assim, as paixões eram executadas na ocasião da Semana Santa, na quinta e na sexta-feira. Estavam inseridas no todo do calendário, entre todas as festividades. Assim, os espectadores da época não compartilhavam da nossa sensação de um fim trágico e lamentoso ao término da obra, pois haveria a sua “continuação” no domingo, com alguma cantata ou oratório de Páscoa, celebrando a ressurreição de Cristo. Aqui se pode estabelecer uma analogia com a idéia dos festivais de Dionísio na Grécia clássica do século V, em que havia os concursos de teatro, e a noção de catarse de Aristóteles. O filósofo afirma que a catarse, em seu processo completo, implica a suscitação do horror e da piedade no espectador e na posterior purgação desses sentimentos. Ora, esse processo como um todo não se completa numa única peça, pois o espectador que sai de uma tragédia não passa pelo processo de purgação do horror e da piedade. Assim, a catarse, na verdade, se completa no decorrer de todo o festival, que inclui sempre uma trilogia 3 3 6 A L E T R I A - jul.-dez. - 2 0 0 6 Disponível em: http://www.letras.ufmg.br/poslit de tragédias seguida de um drama satírico ou de uma comédia. 5 HARNONCOURT. O diálogo musical, p. 199. 6 Não pretendemos aqui defender atodo custo a pertinência dessa relação, mas é interessante observar que após a Revolução Francesa algumas das hierarquias musicais do período anterior deixam de vigorar. 7 HARNONCOURT. O discurso dos sons, p. 41 e 50. A A A A A MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE OUTROS OUTROS OUTROS OUTROS OUTROS SISTEMAS SISTEMAS SISTEMAS SISTEMAS SISTEMAS DE DE DE DE DE SIGNOS SIGNOS SIGNOS SIGNOS SIGNOS NÃO NÃO NÃO NÃO NÃO VERBAIS VERBAIS VERBAIS VERBAIS VERBAIS Em nossa abordagem da Paixão segundo São João e da música barroca dentro dos estudos da intermidialidade, faremos algumas breves considerações sobre a relação da prática musical com outros sistemas semióticos não verbais, tais como a arquitetura e alguns elementos de linguagem visual. Essa última se faz fortemente presente numa sociedade que institui práticas de teatralização e de alegorização da vida e dos fenômenos sacros e profanos. Logo, tudo o que se manifesta visualmente no espaço pode ter um caráter simbólico, desde a estrutura da cidade – com a catedral ou a igreja matriz na praça principal representando o poder espiritual – até as vestimentas das pessoas. Pensando na encenação da Paixão dentro de uma igreja na Sexta-Feira Santa, ela deveria ser associada à cor roxa, um dos símbolos litúrgicos da morte de Jesus. É possível que, nas fachadas principais das igrejas, se estendessem – como ainda hoje em regiões tradicionais – estandartes roxos que simbolizassem o luto pela paixão de Cristo. Assim é que o oratório torna-se um espetáculo de sons e cores, associados pelo receptor da época. Da mesma forma, a estrutura do oratório de Páscoa,8 diferente de uma Paixão, devia ser associada à cor branca, que substitui o roxo para anunciar a ressurreição do Salvador. Com relação à arquitetura, é impensável que, com os recursos acústicos que uma igreja ou catedral possui, o compositor não pensasse nas condições físicas de execução da obra ao compô-la e apresentá-la. Há assim uma estreita relação entre as condições arquitetônicas da igreja e a disposição dos músicos no momento da execução. Além de o coro possuir uma área específica da catedral reservada a ele – e por isso mesmo denominada de coro –, normalmente os instrumentos de corda se posicionavam sob a nave principal, perto do altar, pois, além de serem os componentes fundamentais das orquestras barrocas e tocarem em quase todos os momentos, representavam a condição humana, acompanhando os solistas em árias que transmitiam angústia, melancolia, arrependimento. Ao contrário, alguns instrumentos de sopro, como os trompetes, os cornetti e os sacqueboutes, utilizados especialmente em obras de grande porte, como óperas, oratórios, missas e aberturas, tendiam a ser posicionados nos púlpitos, tanto para que seu som penetrante não encobrisse as cordas, como para representar, em nível topográfico, a glória divina. 8 O que designamos por estrutura pode compreender tanto a constituição das partes do oratório, por exemplo, como os recursos retóricos empregados pelo compositor. É a esse aspecto, o qual comentaremos mais adiante, que gostaríamos de dar ênfase. A P A P A P A P A PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO ENQUANTO ENQUANTO ENQUANTO ENQUANTO ENQUANTO RITUAL RITUAL RITUAL RITUAL RITUAL EEEEE AS AS AS AS AS PRÁTICAS PRÁTICAS PRÁTICAS PRÁTICAS PRÁTICAS SOCIAIS SOCIAIS SOCIAIS SOCIAIS SOCIAIS Ao contrário, a resolução de uma frase num acorde consonante deve ser sempre leve. Uma vez que falamos de harmonia, é impossível deixar de ao menos mencionar a dimensão hierárquica do sistema tonal. Nesse sistema, toda a composição está subordinada aos centros tonais da escala escolhida. Todos os temas se desenvolvem, em termos de harmonia, em torno da tônica e de seus tons vizinhos e, de modo geral, a despeito de quaisquer modulações, o fim da peça musical sempre está subordinado a um retorno à tonalidade inicial. Vale lembrar que é no Barroco que esse sistema tonal se afirma como o grande sistema harmônico da música ocidental, em contraste com o sistema modal da música medieval e renascentista. 3 3 7 2 0 0 6 - jul.-dez. - A L E T R I A Disponível em: http://www.letras.ufmg.br/poslit Disponível em: http://www.letras.ufmg.br/poslit A E A E A E A E A ESTÉTICA STÉTICA STÉTICA STÉTICA STÉTICA DA DA DA DA DA R R R R RECEPÇÃO ECEPÇÃO ECEPÇÃO ECEPÇÃO ECEPÇÃO EEEEE AS AS AS AS AS INTERPRETAÇÕES INTERPRETAÇÕES INTERPRETAÇÕES INTERPRETAÇÕES INTERPRETAÇÕES HISTORICAMENTE HISTORICAMENTE HISTORICAMENTE HISTORICAMENTE HISTORICAMENTE ORIENTADAS ORIENTADAS ORIENTADAS ORIENTADAS ORIENTADAS Em seu livro Literatura e música: modulações pós-coloniais, Solange Ribeiro de Oliveira dedica uma seção à contribuição da Lingüística e dos Estudos Literários para a análise da obra musical. Dentro dessas considerações, reflete sobre o papel da Estética da Recepção nos estudos de musicologia. Essa tendência crítica da análise literária, sob nosso ponto de vista, tem muito a contribuir para esses estudos, sobretudo no que tange às pesquisas da chamada Música Antiga. Diferentemente da literatura, em que o contato do leitor com a obra é imediato,9 a música exige um mediador – o intérprete. Este não deixa de ser um leitor da obra musical, um leitor que ajuda a construir o sentido da obra a cada leitura/execução. Por isso, como advoga a Estética da Recepção, é necessário conhecer a história da obra, pois seu sentido é dado não só por sua estrutura, mas também pelo seu histórico de interpretações. A Paixão segundo São Mateus dirigida por Bach em 1729 não é estritamente a mesma obra que a versão regida por Mendelssohn em 1829, tampouco que uma versão do século XX sob a direção de Karajan ou Mengelberg. Essa perspectiva levou alguns músicos, no final da década de 1950, a empreenderem pesquisas numa tentativa de reconstituição histórica dos instrumentos e das condições de produção e recepção das obras anteriores à Revolução Francesa. Talvez sem saber, esses músicos estavam lidando com a noção de horizonte de expectativas, cara aos teóricos da Estética da Recepção. Essa noção se encaixa perfeitamente com a proposta das interpretações historicamente orientadas: reconstituir o horizonte de expectativas do leitor da época em que a obra musical foi composta. A discussão sobre a validade e autenticidade dessas reconstruções foge aos interesses do presente ensaio,10 mas não conseguimos nos furtar a citar duas passagens de Nikolaus Harnoncourt, um dos pioneiros dessas pesquisas: “O músico de hoje deve se perguntar se não existe realmente outra possibilidade de abordar as obras de Bach de maneira honesta, que não a de interpretá-las no estilo e com os meios do alto Romantismo”.11 É preciso reconhecer que Bach compunha sob uma epistéme diversa daquela vigente no século XIX. A A A A A MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE OUTROS OUTROS OUTROS OUTROS OUTROS SISTEMAS SISTEMAS SISTEMAS SISTEMAS SISTEMAS DE DE DE DE DE SIGNOS SIGNOS SIGNOS SIGNOS SIGNOS NÃO NÃO NÃO NÃO NÃO VERBAIS VERBAIS VERBAIS VERBAIS VERBAIS Esses instrumentos normalmente acompanhavam árias em que se cantava algum feito de Deus ou Sua glória, algum anúncio feito por um anjo ou uma mensagem de júbilo. Outro ponto interessante é a criação de efeitos de eco desejados pelo compositor, que destaca da orquestra os músicos necessários para realizá-lo e põe-nos fora do alcance da visão do público, em algum lugar mais recuado da igreja. Claro que tudo isso que dissemos não eram regras, apenas possibilidades, mas que demonstram satisfatoriamente a relação entre todos esses meios de se transmitir a mensagem da obra. 8 O que designamos por estrutura pode compreender tanto a constituição das partes do oratório, por exemplo, como os recursos retóricos empregados pelo compositor. É a esse aspecto, o qual comentaremos mais adiante, que gostaríamos de dar ênfase. A L E T R I A - jul.-dez. - 2 0 0 6 3 3 8 Disponível em: http://www.letras.ufmg.br/poslit 11 HARNONCOURT. O diálogo musical, p. 93. 12 HARNONCOURT. O diálogo musical, p. 94. 9 Estamos considerando a palavra literatura em seu sentido moderno, sem levar em conta as práticas antigas e medievais de récitas feitas por aedos, bardos e trovadores, quando a prática da leitura silenciosa ainda não havia sido instituída. 10 Conste aqui a nossa simpatia pelas pesquisas no âmbito da Música Antiga e pelas interpretações historicamente orientadas, ditas popularmente “com instrumentos de época”. Acreditamos que os argumentos proferidos contra esse movimento são falhos e destituídos de um verdadeiro conhecimento do universo da Música Antiga, da história e das propostas do movimento. Contudo, como dissemos, essa discussão ultrapassa os limites do presente artigo. 11 HARNONCOURT. O diálogo musical, p. 93. 12 HARNONCOURT. O diálogo musical, p. 94. 10 Conste aqui a nossa simpatia pelas pesquisas no âmbito da Música Antiga e pelas interpretações historicamente orientadas, ditas popularmente “com instrumentos de época”. Acreditamos que os argumentos proferidos contra esse movimento são falhos e destituídos de um verdadeiro conhecimento do universo da Música Antiga, da história e das propostas do movimento. Contudo, como dissemos, essa discussão ultrapassa os limites do presente artigo. 9 Estamos considerando a palavra literatura em seu sentido moderno, sem levar em conta as práticas antigas e medievais de récitas feitas por aedos, bardos e trovadores, quando a prática da leitura silenciosa ainda não havia sido instituída. A E A E A E A E A ESTÉTICA STÉTICA STÉTICA STÉTICA STÉTICA DA DA DA DA DA R R R R RECEPÇÃO ECEPÇÃO ECEPÇÃO ECEPÇÃO ECEPÇÃO EEEEE AS AS AS AS AS INTERPRETAÇÕES INTERPRETAÇÕES INTERPRETAÇÕES INTERPRETAÇÕES INTERPRETAÇÕES HISTORICAMENTE HISTORICAMENTE HISTORICAMENTE HISTORICAMENTE HISTORICAMENTE ORIENTADAS ORIENTADAS ORIENTADAS ORIENTADAS ORIENTADAS “Não se deve, pois, situar Bach na mesma linha dos compositores do século XIX, que escreviam sempre de olho no futuro e raramente para uma efetiva execução”.12 Essa última citação de Harnoncourt mostra a importância da execução, da interpretação na análise das mídias e dos meios de transmissão da mensagem musical. Para concluir, lembremos que o próprio Claus Clüver, um dos principais responsáveis pelos estudos da intermidialidade atualmente, concorda com um determinado resgate histórico das condições de produção e recepção das obras: 9 Estamos considerando a palavra literatura em seu sentido moderno, sem levar em conta as práticas antigas e medievais de récitas feitas por aedos, bardos e trovadores, quando a prática da leitura silenciosa ainda não havia sido instituída. 10 Conste aqui a nossa simpatia pelas pesquisas no âmbito da Música Antiga e pelas interpretações historicamente orientadas, ditas popularmente “com instrumentos de época”. Acreditamos que os argumentos proferidos contra esse movimento são falhos e destituídos de um verdadeiro conhecimento do universo da Música Antiga, da história e das propostas do movimento. Contudo, como dissemos, essa discussão ultrapassa os limites do presente artigo. 2 0 0 6 - jul.-dez. - A L E T R I A 3 3 9 Disponível em: http://www.letras.ufmg.br/poslit incluiremos em nossas investigações históricas a tarefa de reconstrução das preocupações e programas estéticos, dos modos de representação, das convenções estilísticas e estruturais relevantes (ou supostamente relevantes) para o artista, seus modelos negativos ou positivos; e poderemos propor as mesmas tarefas no domínio do público que recebia as obras.13 A A A A A RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO ENTRE ENTRE ENTRE ENTRE ENTRE MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE TEXTO TEXTO TEXTO TEXTO TEXTO NA NA NA NA NA P P P P PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO SEGUNDO SEGUNDO SEGUNDO SEGUNDO SEGUNDO S S S S SÃO ÃO ÃO ÃO ÃO J J J J JOÃO OÃO OÃO OÃO OÃO A reconstrução do horizonte de expectativas do leitor da época barroca não deixa de ser uma tentativa de reconstrução do horizonte de expectativas do intérprete, uma vez que este também é um leitor. Os intérpretes atuais, dessa forma, podem adquirir algum conhecimento sobre como se concebiam as interpretações da música no passado, a partir de estudos de tratados da época. No âmbito da música vocal, o que se sabe cada vez mais é que, no Barroco, a relação entre as palavras e a música passou a ser extremamente valorizada. Ao contrário da estética renascentista, na qual a polifonia alcançou o auge do seu desenvolvimento, o projeto inicial do século XVII, iniciado por artistas italianos que se reuniam em agremiações e academias como a Camerata Fiorentina, era o de compor monodias acompanhadas por um baixo contínuo que ressaltassem o conteúdo do texto cantado. É claro que, como afirma Steven Paul Scher, “na música vocal, o texto literário e a composição musical são ligados de maneira inextricável. Juntos, eles constituem um construto simbiótico que pode ser visto como uma obra de arte completa apenas quando os componentes de ambos estão presentes de modo simultâneo”.14 Mas, no caso do Barroco, isso é ainda mais patente. Em geral, ainda que alguns termos da lingüística, oriundos de um sistema semiótico verbal, possam ser aplicados à música, isso só se verifica sem grandes problemas no nível sintático. Podemos aplicar ao sistema musical as noções de frase, de sincronia/diacronia – relacionadas à harmonia e à melodia, respectivamente –, de langue/parole ou competência/ desempenho,15 mas essas noções dizem respeito à sintaxe musical. Quando se passa à questão semântica, surgem os problemas. Longe de pretendermos discorrer longamente sobre o problema semântico da música, o que nos levaria a uma questão de mímesis e de representação, podemos afirmar, contudo, que a música barroca, devido às práticas de alegorização da sua época, possui códigos de produção muito bem definidos e partilhados pela sociedade, que imprimem à composição musical um sentido para além do subjetivo. Diversos elementos podem se constituir num sistema de significação: a forma musical, a escolha da tonalidade da peça, a instrumentação. Por isso é que se pode falar, pelo menos em se tratando da música barroca, de um sentido musical ou de representação. Esse fenômeno, como demonstrou Saussure no âmbito da linguagem verbal, também é arbitrário, oriundo de convenções. 14 SCHER. Literature and music, p. 226 (em inglês no original). 13 CLÜVER. Estudos interartes, p. 41. 14 SCHER. Literature and music, p. 226 (em inglês no original). 15 As noções de langue/parole ou competência/desempenho podem ser bastante úteis para se pensar a execução e a improvisação. Cada músico, ao improvisar, está exercendo seu desempenho da língua, está criando uma parole individual. Entretanto, nenhuma improvisação é absolutamente livre. Cada um dos tipos de “langue” musical – o jazz, o choro, a música barroca francesa, a música barroca italiana, a música organística da Renascença espanhola, por exemplo – possui seus códigos próprios, cuja competência o músico deve dominar. 15 As noções de langue/parole ou competência/desempenho podem ser bastante úteis para se pensar a execução e a improvisação. Cada músico, ao improvisar, está exercendo seu desempenho da língua, está criando uma parole individual. Entretanto, nenhuma improvisação é absolutamente livre. Cada um dos tipos de “langue” musical – o jazz, o choro, a música barroca francesa, a música barroca italiana, a música organística da Renascença espanhola, por exemplo – possui seus códigos próprios, cuja competência o músico deve dominar. 13 CLÜVER. Estudos interartes, p. 41. A A A A A RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO ENTRE ENTRE ENTRE ENTRE ENTRE MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE TEXTO TEXTO TEXTO TEXTO TEXTO NA NA NA NA NA P P P P PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO SEGUNDO SEGUNDO SEGUNDO SEGUNDO SEGUNDO S S S S SÃO ÃO ÃO ÃO ÃO J J J J JOÃO OÃO OÃO OÃO OÃO Aqui chegamos finalmente 15 As noções de langue/parole ou competência/desempenho podem ser bastante úteis para se pensar a execução e a improvisação. Cada músico, ao improvisar, está exercendo seu desempenho da língua, está criando uma parole individual. Entretanto, nenhuma improvisação é absolutamente livre. Cada um dos tipos de “langue” musical – o jazz, o choro, a música barroca francesa, a música barroca italiana, a música organística da Renascença espanhola, por exemplo – possui seus códigos próprios, cuja competência o músico deve dominar. 3 4 0 A L E T R I A - jul.-dez. - 2 0 0 6 Disponível em: http://www.letras.ufmg.br/poslit à importância da Retórica, enquanto disciplina, para a música barroca. Desde o século XVI, os músicos começaram a estabelecer analogias entre os recursos e figuras do discurso verbal e do musical, tomando como base os tratados de retórica do passado, principalmente o de Aristóteles e o de Quintiliano. Dessa forma, algumas figuras musicais ganharam um sentido próprio, com base em convenções, e mesmo a música puramente instrumental passou a ter um significado em alguma medida objetivo. Isso foi uma das inovações que permitiu a criação da ópera, em que a orquestra16 substitui o coro do teatro grego nos comentários sobre a fala/canto dos atores/cantores. Essa utilização da retórica visa à transmissão de um pathos musical, que, no Barroco, é trazido para um mesmo nível que o logos e o ethos, mais valorizados em períodos anteriores. A presença desse pathos, que pode ser traduzido para o latim affectus, levou ao desenvolvimento do que se convencionou chamar de Affektenlehre, isto é, Doutrina dos Afetos. De acordo com ela, os procedimentos retóricos de composição, unidos à escolha das tonalidades e à instrumentação,17 deveriam despertar no ouvinte os mais diversos affetti dell’anima. No caso da música vocal, esses afetos deveriam se adequar ao texto que estava sendo cantado. Surgem então as noções de musica pathetica e musica poetica, as quais são bastante pertinentes para se classificar a Paixão segundo São João. Essa última expressão é singular em nosso estudo, pois, em seus próprios elementos, contém o princípio da interação entre as artes: uma música que é poética, que se une à poesia e se realiza com recursos poéticos. 16 Vale a pena recordarmos a origem da palavra grega orchestra, que designava justamente a parte do teatro onde o coro fazia suas evoluções. Na ópera, o antigo coro, constituído de atores/cantores/bailarinos, é substituído pelos instrumentistas. Isso faz com que os comentários sobre o texto dos cantores sejam puramente instrumentais. Daí a necessidade de se desenvolver um código de significação para essa música instrumental. Disponível em: http://www.letras.ufmg.br/poslit A A A A A RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO ENTRE ENTRE ENTRE ENTRE ENTRE MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE TEXTO TEXTO TEXTO TEXTO TEXTO NA NA NA NA NA P P P P PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO SEGUNDO SEGUNDO SEGUNDO SEGUNDO SEGUNDO S S S S SÃO ÃO ÃO ÃO ÃO J J J J JOÃO OÃO OÃO OÃO OÃO Uma vez que o presente trabalho não se quer um tratado sobre a história da música, mas um breve ensaio sobre as relações entre o sistema semiótico musical e o sistema semiótico verbal, passemos à análise de algumas passagens da Paixão segundo São João em que essa relação se mostra evidente. No recitativo em que o evangelista diz “Da nahm Pilatus Jesum und geißelte ihn” – “Então, por isso, Pilatos tomou a Jesus e mandou açoitá-lo” (Jo cap. 19, v.1), observamos, na linha do baixo contínuo, o ritmo característico dos açoites, isto é, grupos de colcheias pontuadas seguidas de semicolcheias.18 Na linha do canto, sobre a palavra geißelte, há uma catabasis (sucessão de notas descendentes) completamente cromática, isto é, a descida das notas se faz toda por semitons: 16 Vale a pena recordarmos a origem da palavra grega orchestra, que designava justamente a parte do teatro onde o coro fazia suas evoluções. Na ópera, o antigo coro, constituído de atores/cantores/bailarinos, é substituído pelos instrumentistas. Isso faz com que os comentários sobre o texto dos cantores sejam puramente instrumentais. Daí a necessidade de se desenvolver um código de significação para essa música instrumental. 17 Os afetos aqui mencionados não devem ser confundidos com os sentimentos livres e subjetivos do Romantismo. Na música barroca, cada um desses afetos está condicionado por um código objetivo que envolve, como já dissemos, as figuras de retórica, as tonalidades e a instrumentação. Assim, por exemplo, de acordo com alguns tratados, a tonalidade de ré maior evoca alegria triunfal, enquanto a de mi bemol maior é pungente e dolorida. Da mesma forma, os instrumentos também são ligados a uma significação. Trombones podem remeter a um ambiente marítimo ou aos domínios de Hades, enquanto flautas doces e oboés da caccia evocam ambientes pastoris. 18 Mais uma vez recordamos que esses significados são arbitrários, oriundos de convenções. No caso do ritmo dos açoites, trata-se de algo que vem já desde o início do século XVII, com Frescobaldi. 3 4 1 2 0 0 6 - jul.-dez. - A L E T R I A Fig. 1: Johann Sebastian Bach (1685-1750), Johannes-Passion (1724, versão final 1740). Partitura, p. 75. Fig. 1: Johann Sebastian Bach (1685-1750), Johannes-Passion (1724, versão final 1740). Partitura, p. 75. Essa figura retórica evoca afetos de dor e lamento. Unida ao ritmo dos açoites, torna-se um comentário sobre o sofrimento despertado pelo açoitamento de Jesus. 19 A passagem de São Mateus é a seguinte: “51 E eis que se rasgou o véu do Templo em duas partes dalto abaixo: e tremeu a terra, e partiram-se as pedras. 52 E abriram-se as sepulturas: e muitos corpos de santos, que eram mortos, ressurgiram”. Disponível em: http://www.letras.ufmg.br/poslit A A A A A RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO ENTRE ENTRE ENTRE ENTRE ENTRE MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE TEXTO TEXTO TEXTO TEXTO TEXTO NA NA NA NA NA P P P P PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO SEGUNDO SEGUNDO SEGUNDO SEGUNDO SEGUNDO S S S S SÃO ÃO ÃO ÃO ÃO J J J J JOÃO OÃO OÃO OÃO OÃO Da mesma forma, o recitativo que termina com a frase de Jesus “Es ist vollbracht!” – “Está consumado!” (Jo cap. 19, v.30), também apresenta uma catabasis sobre essa exclamação. O interessante, no entanto, é que o acorde final tocado pelo baixo contínuo pode ser realizado com um intervalo de terça maior, e não menor, como seria esperado. Essa surpresa causada pela alteração da terça é certamente um prenúncio da ressurreição, pois a terça maior evoca alegria e júbilo, enquanto a menor se refere a um afeto mais pungente. Eis o respectivo trecho da partitura: Fig. 2: Johann Sebastian Bach. Johannes-Passion. Partitura, p. 150. Fig. 2: Johann Sebastian Bach. Johannes-Passion. Partitura, p. 150. Um dos pontos mais interessantes para analisarmos os comentários da orquestra ao texto é o arioso para tenor que se inicia pela exclamação “Mein Herz!”. Esse arioso é um comentário sobre o recitativo que o precede, retirado do Evangelho de Mateus (Mt cap. 27, v.51-52)19 e inserido por Bach na Paixão segundo São João. O texto do arioso repete algumas expressões do texto de São Mateus, a saber: “der Vorhang reißt” (o véu se rasga), “der Fels zerfällt” (as rochas se fendem), “die Erde bebt” (a terra treme), “die Gräber spalten” (os túmulos se abrem). Cada um desses fenômenos é ilustrado por um conjunto específico de notas que surge logo após a declamação do texto: 19 A passagem de São Mateus é a seguinte: “51 E eis que se rasgou o véu do Templo em duas partes dalto abaixo: e tremeu a terra, e partiram-se as pedras. 52 E abriram-se as sepulturas: e muitos corpos de santos, que eram mortos, ressurgiram”. 3 4 2 A L E T R I A - jul.-dez. - 2 0 0 6 Disponível em: http://www.letras.ufmg.br/poslit Fig. 3: Johann Sebastian Bach, Johannes-Passion. Partitura, p. 163 e 164. Fig. 3: Johann Sebastian Bach, Johannes-Passion. Partitura, p. 163 e 164. Temos, assim, um grupo de fusas em catabasis representando os véus do templo se rasgando de alto a baixo – por isso o movimento descendente. Em seguida, um ritmo “quadrado”, um tanto marcial, criado por alternância de fusas e semicolcheias, ilustra a pulverização das rochas. Uma bateria de fusas na altura de sol executadas pelos violinos e violas comenta o tremor da terra. Disponível em: http://www.letras.ufmg.br/poslit 20 Considera como cada parte de suas costas manchadas de sangue combinam com as cores do céu. Assim que o maremoto de nosso dilúvio de pecados se retrai, o mais belo arco-íris fica como sinal da graça de Deus (tradução livre de Elisa Freixo). A A A A A RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO ENTRE ENTRE ENTRE ENTRE ENTRE MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE TEXTO TEXTO TEXTO TEXTO TEXTO NA NA NA NA NA P P P P PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO SEGUNDO SEGUNDO SEGUNDO SEGUNDO SEGUNDO S S S S SÃO ÃO ÃO ÃO ÃO J J J J JOÃO OÃO OÃO OÃO OÃO Finalmente, uma nova sucessão de fusas, agora em anabasis (sucessão de notas ascendentes), mimetiza o movimento dos túmulos se abrindo, desde as profundezas da terra até a superfície. Podemos afirmar que nesse arioso o comentário da orquestra ao texto, escrito de maneira exemplar por Bach, chega a ser pictórico, pois 3 4 3 2 0 0 6 - jul.-dez. - A L E T R I A a disposição das notas musicais parece ilustrar os fenômenos narrados no Evangelho de Mateus, tanto pela sugestão do movimento, quanto pela mancha gráfica que se dispõe na partitura. Ainda há dois aspectos que gostaríamos de comentar a respeito da retórica e dos afetos. O primeiro se refere à instrumentação. Na ária que serve de comentário às palavras de Jesus “Es ist vollbracht!”, há duas partes claramente distintas. A primeira delas repete a frase dita por Jesus e fala da noite de luto que se seguiu à crucificação. Aqui, somente uma viola da gamba acompanha o contratenor, representando a dor e a fragilidade da condição humana diante da morte do filho de Deus. A segunda parte promove uma mudança de tom, enaltecendo os acontecimentos, considerando-os uma vitória de Jesus e chamando-lhe herói de Judá. Acompanhando essa mudança de tom, o acompanhamento do canto ganha agora o reforço de dois violinos, que entram na música num ritmo caracterizado por alguns estudiosos como ritmo de trompetes. Representam, assim, a glória do herói de Judá, concebido para se tornar o salvador da humanidade: Fig. 4: Johann Sebastian Bach, Johannes-Passion. Partitura, p. 153. Fig. 4: Johann Sebastian Bach, Johannes-Passion. Partitura, p. 153. O segundo aspecto a ser discutido é o que de mais estrito em termos de literatura se pode apontar, ou seja, o trabalho poético realizado nos textos das árias. Veja-se a ária que se segue: Erwäge, wie sein blutgefärbter Rücken In allen Stücken Dem Himmel gleiche geht, Daran, nachdem die Wasserwogen Von unsrer Sündflut sich verzogen, Der allerschönste Regenbogen Als Gottes Gnadenzeichen steht.20 Erwäge, wie sein blutgefärbter Rücken In allen Stücken Dem Himmel gleiche geht, Daran, nachdem die Wasserwogen Von unsrer Sündflut sich verzogen, Der allerschönste Regenbogen Als Gottes Gnadenzeichen steht.20 20 Considera como cada parte de suas costas manchadas de sangue combinam com as cores do céu. Assim que o maremoto de nosso dilúvio de pecados se retrai, o mais belo arco-íris fica como sinal da graça de Deus (tradução livre de Elisa Freixo). Disponível em: http://www.letras.ufmg.br/poslit A A A A A RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO RELAÇÃO ENTRE ENTRE ENTRE ENTRE ENTRE MÚSICA MÚSICA MÚSICA MÚSICA MÚSICA EEEEE TEXTO TEXTO TEXTO TEXTO TEXTO NA NA NA NA NA P P P P PAIXÃO AIXÃO AIXÃO AIXÃO AIXÃO SEGUNDO SEGUNDO SEGUNDO SEGUNDO SEGUNDO S S S S SÃO ÃO ÃO ÃO ÃO J J J J JOÃO OÃO OÃO OÃO OÃO 3 4 4 A L E T R I A - jul.-dez. - 2 0 0 6 Disponível em: http://www.letras.ufmg.br/poslit O trabalho poético desse texto é exemplar. Em primeiro lugar, destaca-se a metáfora que compara as manchas de sangue nas costas de Jesus ao céu avermelhado pelo ocaso. Podemos também ressaltar a hipérbole presente no maremoto formado pelo dilúvio de pecados. Mas o mais interessante é justamente a palavra que, na língua alemã, designa essa última noção – Sündflut. Ela surge de um trocadilho com a palavra Sintflut, que designa o dilúvio do Antigo Testamento. Assim, por uma semelhança no estrato fônico, troca-se Sintflut por Sündflut, que incorpora duas noções simultaneamente. À idéia do dilúvio, que está presente na palavra Sintflut, acrescenta-se a noção de pecado, Sünde em alemão.21 Logo, através de um jogo sonoro, cria-se uma figura de comparação, ao mesmo tempo metáfora e hipérbole: os nossos pecados são tantos que formam um dilúvio, tão arrasador quanto o castigo enviado por Deus no Antigo Testamento, tanto que gerará um maremoto. Estes são apenas alguns exemplos da riqueza poética da Paixão segundo São João. Essa poesia, unida ao sistema musical e a todas as suas particularidades, ao ambiente de execução da peça e a outras mídias diversas, torna-a um dos mais fascinantes monumentos da arte ocidental, ao mesmo tempo em que ela se configura como uma obra do século XVII na qual se verificam características mixmídias. AA 21 As imagens e metáforas nesses versos, que não são de autoria de Bach, são típicas de hinos do Barroco alemão. É provável que a substituição de Sinflut por Sündflut, ainda muito comum hoje, fosse um chavão na época – o que não diminui sua força. RRRRR É S U M É É S U M É É S U M É É S U M É É S U M É Cet article propose une lecture de la Passion selon Saint Jean, de Johann Sebastian Bach, comme une œuvre de discours mixte. On conduira l’analyse à partir des relations entre les divers systèmes de signes qui la composent ou bien qui participent ou influencent son execution. Cette analyse privilegie les relations entre les systèmes musical et verbal mais sans laisser de considérer d’autres systèmes de signes. Elle a aussi le but de démontrer l’existence d’œuvres de caractère intersémiotique avant le Modernisme. M M M M M O T S O T S O T S O T S O T S ----- C L É S C L É S C L É S C L É S C L É S Relations musico-littéraires, iconisation de la partiture, oratorio, le Baroque M M M M M O T S O T S O T S O T S O T S ----- C L É S C L É S C L É S C L É S C L É S Relations musico-littéraires, iconisation de la partiture, oratorio, le Baroque Disponível em: http://www.letras.ufmg.br/poslit RRRRR E F E R Ê N C I A S E F E R Ê N C I A S E F E R Ê N C I A S E F E R Ê N C I A S E F E R Ê N C I A S BACH, Johann Sebastian. Johannes-Passion. Freiburg: Deutsche Harmonia Mundi, 1990. 2 CDs. BWV 245. Acompanha libretto. BACH, Johann Sebastian. Johannes-Passion. Freiburg: Deutsche Harmonia Mundi, 1990. 2 CDs. BWV 245. Acompanha libretto. BACH, Johann Sebastian. Johannes-Passion. London: Ernst Eulenburg, [s.d.]. 1 partitura. Orquestra e coro. BACH, Johann Sebastian. Johannes-Passion. London: Ernst Eulenburg, [s.d.]. 1 partitura. Orquestra e coro. BARTEL, Dietrich. Musica Poética: Musical-rhetorical figures in German baroque music. Lincoln, NE: University of Nebraska Press, 1997. 21 As imagens e metáforas nesses versos, que não são de autoria de Bach, são típicas de hinos do Barroco alemão. É provável que a substituição de Sinflut por Sündflut, ainda muito comum hoje, fosse um chavão na época – o que não diminui sua força. 3 4 5 2 0 0 6 - jul.-dez. - A L E T R I A Disponível em: http://www.letras.ufmg.br/poslit BÍBLIA sagrada. N. T. O Evangelho segundo São Mateus. Rio de Janeiro: Editora Barsa, 1966. HASIN, Ibaney. O canto dos afetos: um dizer humanista. São Paulo: Perspectiva, 2004. , y f p , 4 CLÜVER, Claus. Estudos interartes: conceitos, termos, objetivos. Literatura e sociedade. Revista de teoria literária e literatura comparada, São Paulo, FFLCH/USP, n. 2, p. 37-55, 1997. HARNONCOURT, Nikolaus. O diálogo musical: Monteverdi, Bach e Mozart. (1984.) Trad. Luiz Paulo Sampaio. Rio de Janeiro: Jorge Zahar, 1993. HARNONCOURT, Nikolaus. O discurso dos sons: caminhos para uma nova compreensão musical. (1982.) Trad. Marcelo Fagerlande. 2. ed. Rio de Janeiro: Jorge Zahar, 1990. HANSEN, João Adolfo. Teatro da memória: monumento barroco e retórica. Revista do IFAC, Ouro Preto, [s.n.t.], p. 40-48. ISER, Wolfgang. Problemas da teoria da literatura atual. In: LIMA, Luiz Costa (Org.). Teoria da literatura em suas fontes. 3. ed. Rio de Janeiro: Civilização Brasileira, 2002, v. 2, p. 927-954. JAUSS, Hans Robert. A história da literatura como provocação à teoria literária. Trad. Sérgio Tellaroli. São Paulo: Ática, 1994. LATERZA FILHO, Moacyr. A ópera dos afetos: para uma intertextualidade das paixões: leitura comparativa das retóricas do Padre Antônio Vieira e do Padre Antonio Vivaldi. 2003. Tese (Doutorado em Letras) – Faculdade de Letras, Pontifícia Universidade Católica de Minas Gerais, Belo Horizonte. MAGNANI, Sérgio. Expressão e comunicação na linguagem da música. Belo Horizonte, Editora da UFMG, 1996. MARAVALL, José Antonio. Novidade, invenção, artifício (papel social do teatro e das festas). In: MARAVALL, José Antonio. A cultura do Barroco: análise de uma estrutura histórica. São Paulo: EDUSP, 1997, p. 353-385. Disponível em: http://www.letras.ufmg.br/poslit RRRRR E F E R Ê N C I A S E F E R Ê N C I A S E F E R Ê N C I A S E F E R Ê N C I A S E F E R Ê N C I A S MASSIN, Jean e Brigitte. História da música ocidental. (1987.) Trad. Ângela Ramalho Viana, Carlos Sussekind e Maria Teresa Resende Costa. Rio de Janeiro: Nova Fronteira, 1997. OLIVEIRA, Solange Ribeiro. Literatura e música: modulações pós-coloniais. São Paulo: Perspectiva, 2002. SANTOS, Luís Otávio. An approach through the analysis of Monteverdi’s Combattimento di Tancredi e Clorinda and Bach’s Erbarme dich from Mathäus-Passion. Trabalho de estudo acadêmico não publicado (mimeo), [199-]. SCHER, Steven Paul. Literature and Music. In: BARRICELLI, Jean-Pierre; GIBALDI, Joseph (Org.). Interrelations of Literature. New York: MLA, 1982, p. 225-250. A L E T R I A - jul.-dez. - 2 0 0 6 3 4 6 Disponível em: http://www.letras.ufmg.br/poslit
https://openalex.org/W4362538769	https://figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_Oncogenic_and_Wild-type_Ras_Play_Divergent_Roles_in_the_Regulation_of_Mitogen-Activated_Protein_Kinase_Signaling/22529156/1/files/39992180.pdf	Latvian	null	Supplementary Figure 6 from Oncogenic and Wild-type Ras Play Divergent Roles in the Regulation of Mitogen-Activated Protein Kinase Signaling	null	2,023	cc-by	294	Supplementary Figure S2. Supplementary Figure S2. p-Akt N-Ras K-Ras ERK Akt Actin p-ERK H-Ras EGF: siRNA: Non- silencing H1 H2 H3 K1 K2 K3 N1 N2 N3 - + - + - + - + - + - + - + T24 HRAS G12V RD NRAS Q61H Akt p-Akt N-Ras ERK p-MEK p-ERK H-Ras EGF: siRNA: Non- silencing N1 N2 K1 K2 K3 H1 H2 H3 - + - + - + - + - + - + p-Akt N-Ras K-Ras ERK Akt Actin p-ERK H-Ras EGF: siRNA: Non- silencing H1 H2 H3 K1 K2 K3 N1 N2 N3 - + - + - + - + - + - + - + T24 HRAS G12V MIA PaCa-2 KRAS G12C p-Akt N-Ras K-Ras ERK Akt p-ERK p-MEK H-Ras EGF: siRNA: Non- silencing K1 K2 N1 N2 N3 H1 H2 H3 - + - + - + - + - + - + RD NRAS Q61H Akt p-Akt N-Ras ERK p-MEK p-ERK H-Ras EGF: siRNA: Non- silencing N1 N2 K1 K2 K3 H1 H2 H3 - + - + - + - + - + - + p-Akt N-Ras K-Ras ERK Akt Actin p-ERK H-Ras EGF: siRNA: Non- silencing H1 H2 H3 K1 K2 K3 N1 N2 N3 - + - + - + - + - + - + - + T24 HRAS G12V MIA PaCa-2 KRAS G12C p-Akt N-Ras K-Ras ERK Akt p-ERK p-MEK H-Ras EGF: siRNA: Non- silencing K1 K2 N1 N2 N3 H1 H2 H3 - + - + - + - + - + - + RD NRAS Q61H Akt p-Akt N-Ras ERK p-MEK p-ERK H-Ras EGF: siRNA: Non- silencing N1 N2 K1 K2 K3 H1 H2 H3 - + - + - + - + - + - +
https://openalex.org/W4327610686	https://www.frontiersin.org/articles/10.3389/fnins.2023.1154637/pdf	English	null	Ethanol-induced cerebellar transcriptomic changes in a postnatal model of fetal alcohol spectrum disorders: Focus on disease onset	Frontiers in neuroscience	2,023	cc-by	13,192	TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fnins.2023.1154637 TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fnins.2023.1154637 TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fnins.2023.1154637 TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fnins.2023.1154637 OPEN ACCESS Drew1,3* 1Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, United States, 2Department of Neuroscience, University of Rochester Medical Center, Rochester, NY, United States, 3Department of Neurology, University of Arkansas for Medical Sciences, Little Rock, AR, United States Fetal alcohol spectrum disorders (FASD) are a group of neurodevelopmental disorders caused by ethanol exposure in utero, which can result in neurocognitive and behavioral impairments, growth defects, and craniofacial anomalies. FASD affects up to 1-5% of school-aged children in the United States, and there is currently no cure. The underlying mechanisms involved in ethanol teratogenesis remain elusive and need greater understanding to develop and implement effective therapies. Using a third trimester human equivalent postnatal mouse model of FASD, we evaluate the transcriptomic changes induced by ethanol exposure in the cerebellum on P5 and P6, after only 1 or 2 days of ethanol exposure, with the goal of shedding light on the transcriptomic changes induced early during the onset and development of FASD. We have highlighted key pathways and cellular functions altered by ethanol exposure, which include pathways related to immune function and cytokine signaling as well as the cell cycle. Additionally, we found that ethanol exposure resulted in an increase in transcripts associated with a neurodegenerative microglia phenotype, and acute- and pan-injury reactive astrocyte phenotypes. Mixed effects on oligodendrocyte lineage cell associated transcripts and cell cycle associated transcripts were observed. These studies help to elucidate the underlying mechanisms that may be involved with the onset of FASD and provide further insights that may aid in identifying novel targets for interventions and therapeutics. COPYRIGHT © 2023 Holloway, Douglas, Rafferty, Majewska, Kane and Drew. 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. OPEN ACCESS OPEN ACCESS EDITED BY Carlos Fernando Valenzuela, The University of New Mexico, United States REVIEWED BY Tou Yia Vue, The University of New Mexico Health Sciences Center, United States J. N. Reynolds, Queen’s University, Canada CORRESPONDENCE Paul D. Drew drewpauld@uams.edu SPECIALTY SECTION This article was submitted to Neurodevelopment, a section of the journal Frontiers in Neuroscience RECEIVED 30 January 2023 ACCEPTED 27 February 2023 PUBLISHED 16 March 2023 CITATION Holloway KN, Douglas JC, Rafferty TM, Majewska AK, Kane CJM and Drew PD (2023) Ethanol-induced cerebellar transcriptomic changes in a postnatal model of fetal alcohol spectrum disorders: Focus on disease onset. Front. Neurosci. 17:1154637. doi: 10.3389/fnins.2023.1154637 OPEN ACCESS EDITED BY Carlos Fernando Valenzuela, The University of New Mexico, United States REVIEWED BY Tou Yia Vue, The University of New Mexico Health Sciences Center, United States J. N. Reynolds, Queen’s University, Canada CORRESPONDENCE Paul D. Drew drewpauld@uams.edu SPECIALTY SECTION This article was submitted to Neurodevelopment, a section of the journal Frontiers in Neuroscience RECEIVED 30 January 2023 ACCEPTED 27 February 2023 PUBLISHED 16 March 2023 CITATION Holloway KN, Douglas JC, Rafferty TM, Majewska AK, Kane CJM and Drew PD (2023) Ethanol-induced cerebellar transcriptomic changes in a postnatal model of fetal alcohol spectrum disorders: Focus on disease onset. Front. Neurosci. 17:1154637. doi: 10.3389/fnins.2023.1154637 OPEN ACCESS EDITED BY Carlos Fernando Valenzuela, The University of New Mexico, United States REVIEWED BY Tou Yia Vue, The University of New Mexico Health Sciences Center, United States J. N. Reynolds, Queen’s University, Canada CORRESPONDENCE Paul D. Drew drewpauld@uams.edu SPECIALTY SECTION This article was submitted to Neurodevelopment, a section of the journal Frontiers in Neuroscience RECEIVED 30 January 2023 ACCEPTED 27 February 2023 PUBLISHED 16 March 2023 CITATION Holloway KN, Douglas JC, Rafferty TM, Majewska AK, Kane CJM and Drew PD (2023) Ethanol-induced cerebellar transcriptomic changes in a postnatal model of fetal alcohol spectrum disorders: Focus on disease onset. Front. Neurosci. 17:1154637. doi: 10.3389/fnins.2023.1154637 Kalee N. Holloway1, James C. Douglas1, Tonya M. Rafferty1, Ania K. Majewska2, Cynthia J. M. Kane1 and Paul D. FASD, microglia, astrocytes, oligodendrocytes, transcriptomics COPYRIGHT © 2023 Holloway, Douglas, Rafferty, Majewska, Kane and Drew. 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. Frontiers in Neuroscience Introduction 1 male and 1 female Control, and 1 male and 1 female Ethanol were randomly selected from each of 3 litters for sequencing on P5 and P6 (N = 6 total litters, 3 male/3 female per treatment group, per timepoint). To determine mean Blood Ethanol Concentration (BEC), 3 separate litters were treated with ethanol as described above and blood was collected from half of each litter on either P4 or P5, 90 minutes after ethanol administration. Brieﬂy, animals were anesthetized using isoﬂurane vapor and trunk blood was collected in heparinized capillary tubes following decapitation. Blood was centrifuged at 4000 RPM for 5 minutes and serum was removed for BEC determination using an Analox AM1 alcohol analyzer (Analox Technologies USA, Atlanta, GA) and companion Alcohol Reagent Kit (Analox #GMRD-113) according to manufacturer speciﬁcations. P4 mean BEC was 324.6 mg/dL ± 7.2 mg/dL SEM, (n = 7 male, 4 female) and P5 mean BEC was 333.7 mg/dL ± 12.6 mg/dL SEM, (n = 7 male, 5 female). Fetal ethanol exposure can induce neuropathology in multiple brain regions, including the cerebellum. Human studies have demonstrated cerebellar susceptibility to ethanol exposure during development, including diminished cerebellar volume and white matter abnormalities, which likely contribute to impaired motor coordination, and learning and memory deﬁcits often found in individuals with FASD (Riley and McGee, 2005; Lebel et al., 2008; Norman et al., 2009). Rodent models of FASD have been valuable in studying fetal ethanol exposure. In mice for instance, the ﬁrst two postnatal weeks coincide with the third trimester of human gestation (Clancy et al., 2001), a critical period for cerebellar development. During this time the cerebellum undergoes a period of secondary neurogenesis, cell migration, and synaptogenesis. This period also exhibits oligodendrocyte maturation and myelination (Rice and Barone, 2000; Camarillo and Miranda, 2007; Wilhelm and Guizzetti, 2016). Rodent studies using third trimester equivalent ethanol exposure paradigms have shown ethanol-induced cerebellar alterations associated with activation of both astrocytes and microglia. The activation states of these cells can aﬀect their overall function, including changes in expression of pro-inﬂammatory molecules, suggesting ethanol-induced cerebellar neuroinﬂammation could be associated with FASD (Kane et al., 2011; Drew et al., 2015; Topper et al., 2015). Introduction Fetal Alcohol Spectrum Disorders (FASD) are a leading cause of preventable developmental abnormalities around the world, and result in a range of clinical outcomes that can include craniofacial anomalies, neurological malformations, cognitive and behavioral impairment, and growth defects (Hoyme et al., 2016). FASD are not only Frontiers in Neuroscience 01 frontiersin.org Holloway et al. 10.3389/fnins.2023.1154637 devastating to aﬀected individuals, but also have signiﬁcant economic impact globally (Greenmyer et al., 2018; Sokol, 2018). The global prevalence of FASD varies by region, with a mean of 0.8 percent (Lange et al., 2017). The prevalence has been estimated to be as high as 1-5 percent of school age children in the United States (May et al., 2018). There is no cure for FASD, and elucidation of the underlying mechanisms which regulate development of these disorders is needed to generate eﬀective therapies. animals. All animal use protocols were reviewed and approved by the UAMS Institutional Animal Care and Use Committee. Individually housed pregnant dams were kept on a 10:14 hour light:dark cycle in static cages on an open-air rack and were checked twice daily for birth of pups, with postnatal day 0 (P0) being designated as the day of birth. Cages were changed weekly or as needed. Dams were allowed unlimited access to food and water for the duration of the experiments. Experimental litters contained 4-8 neonates that were distributed among treatment groups, Ethanol (E) or vehicle Control (C) and were separated according to sex as evenly as possible for each individual litter. Handled- only, untreated Control animals were not included in this study, based on no diﬀerence for analogous endpoints from previous studies (Kane et al., 2011; Drew et al., 2015). On P4-5, Ethanol treated animals were administered 4 g/kg/day of ethanol in 20% intralipid (Fresenius Kabi, Uppsala, Sweden) while Control animals received 20% intralipid in which ethanol was substituted with an equal volume of water. Ethanol and water were administered via intragastric gavage. On P5 or P6, 24 hours after the last ethanol treatment on P4 or P5, respectively, animals were anesthetized using isoﬂurane vapor and transcardially perfused with phosphate- buﬀered saline containing 5 U/mL heparin. The brain was removed, the cerebellum was microdissected, ﬂash-frozen in liquid nitrogen, and stored at −80◦C until used for RNA isolation and subsequent sequencing. Introduction In recent years, RNA sequencing (RNAseq) has become more readily accessible and cost eﬃcient making it a highly eﬀective tool to assess ethanol-induced transcriptomic changes in the CNS (Farris and Mayﬁeld, 2014; Berres et al., 2017; Erickson et al., 2019; Pinson et al., 2021). Using our neonatal model of FASD, in which mice were treated with ethanol on postnatal days (P) 4-9 and tissues harvested on P10, we recently reported that ethanol stimulated transcriptomic changes associated with cell cycle and microglia regulation, and oligodendrocyte lineages in the cerebellum (Pinson et al., 2021). In the current studies, we evaluated ethanol-induced transcriptomic changes in the cerebellum on postnatal days 5 and 6 in this FASD model. These ﬁndings may provide further insight into the underlying mechanisms associated with the early onset of FASD as well as identifying potential targets for clinical interventions and therapeutics. Isolation of RNA, RNA-seq library preparation, and sequencing Frozen cerebellar tissues were rapidly thawed and homogenized in Qiazol with 0.5 mm glass beads (Qiagen #13116-50) in a PowerLyzer 24 homogenizer (Qiagen #13155) for 30s at 3500 rpm. Total RNA was isolated using an miRNeasy Mini kit (Qiagen #217084) and DNA was removed with on-column DNase1 digestion (Qiagen #79254) following manufacturer protocol (Qiagen, Valencia, CA). RNA quantity was evaluated using the Qubit 3.0 ﬂuorometer with the Qubit Broad-Range RNA Assay kit (Thermo Fisher Scientiﬁc, Waltham, MA). RNA quality was assessed using the Agilent Fragment Analyzer with the Standard Sensitivity RNA Gel Kit (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using the Illumina TruSeq mRNA Frontiers in Neuroscience frontiersin.org Bioinformatic analysis Each of these phenotype speciﬁc lists was cross-referenced to the transcripts signiﬁcantly dysregulated by Ethanol (adj. p < 0.05) when compared to Control in our data set for both P5 and P6 and was tested for statistical signiﬁcance. To accomplish this, R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal within a given phenotype list. These z-scores were then averaged across transcripts for each individual animal. Control versus Ethanol groups were then tested for statistically signiﬁcant variance in R by two-tailed, student’s t-test, and graphical results were generated. Raw RNA-sequence data [NCBI gene expression omnibus (GEO) series succession number GSE226532 (Edgar et al., 2002)] was analyzed to identify signiﬁcant diﬀerences in mRNA gene expression and global biological pathways associated with alterations of cerebellar genes between Control and Ethanol treatment groups. Using the Nextﬂow RNAseq pipeline, nf- core/rnaseq (version 3.4) available at DOI: 10.5281/zenodo. 1400710, RNAseq reads were quality-checked, trimmed, and aligned, with the resulting gene counts transformed to Log2 counts per million (CPM) and lowly expressed genes were ﬁltered out (Liao et al., 2014). Libraries were normalized by trimmed mean of M-values (Robinson and Oshlack, 2010). To calculate diﬀerential gene expression, the Limma R package was used (Ritchie et al., 2015). Genes with an adjusted p-value (adj. p < 0.05) were considered statistically signiﬁcant and Log2 fold change values were calculated for Ethanol compared to Control. Our previous studies demonstrated that ethanol altered the expression of molecules associated with various stages of the cell cycle in our FASD model in which animals were treated with ethanol from P4-P9 and cerebellar RNA isolated on P10 (Pinson et al., 2021). In the current study, we performed similar analysis to determine if ethanol altered cell cycle progression during the onset of FASD in this model. Thus, from the Mouse Genome Database Gene Ontology Browser (Bult et al., 2019), we extracted gene lists associated with positive and negative regulation of both G1-S phase transition and G2-M phase transition. Average z-score analysis was conducted in a manner consistent with the glial cell types above. Heatmaps, principal component analysis (PCA), and volcano plots were generated in R from the processed diﬀerential gene expression data. Speciﬁcally, the EnhancedVolcano package was used to generate the volcano plots (Blighe et al., 2022). 1 https://digitalinsights.qiagen.com/IPA Animals C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME; stock #000664) and housed in the federally approved Division of Laboratory Animal Medicine facility at the University of Arkansas for Medical Sciences (UAMS) where an in- house breeding colony was established to produce experimental 02 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. for example, were subcategorized into transcripts related to homeostasis or neurodegenerative diseases (Keren-Shaul et al., 2017; Krasemann et al., 2017; Butovsky and Weiner, 2018; Tatsuyuki Matsudaira, 2022) (Supplementary Table 1B). Astrocyte associated genes were subcategorized into transcripts related to acute injury, chronic injury, or pan-injury, with pan- injury including genes associated with both acute and chronic reactive astrogliosis (Das et al., 2020) (Supplementary Table 2). Oligodendrocyte lineage cell associated genes were subcategorized into transcripts related to oligodendrocyte precursor cells (OPCs), committed oligodendrocyte precursor cells (COPs), newly-formed oligodendrocytes (NFOL), myelin forming oligodendrocytes (MFOL), and mature oligodendrocytes (MOL) (Zeisel et al., 2015, 2018; Artegiani et al., 2017) (Supplementary Table 3). Library Prep Kit with TruSeq unique dual-indexed adapters (Illumina, San Diego, CA). Libraries were quantiﬁed with the Qubit 1X dsDNA High-Sensitivity NGS Gel Kit (Thermo Fisher), and additionally characterized for functionality with the KAPA Library Quantiﬁcation Kit (Roche, Basel, Switzerland) and for fragment size using the Agilent Fragment Analyzer with the High-Sensitivity NGS Gel Kit (Agilent). According to manufacturer’s speciﬁcation for clustering, library molarities were calculated followed by dilution and denaturation. Control and Ethanol-exposed animals were clustered on a high-output NextSeq 500 ﬂow cell and paired- end sequenced with 150-cycle SBS kit for 2 × 75 reads (Illumina). Frontiers in Neuroscience Bioinformatic analysis Utilizing the “Core Expression Analysis” in the QIAGEN Ingenuity Pathway Analysis (IPA) software (QIAGEN Inc.)1, pathway and network analysis were conducted. In IPA, the analysis parameter setting for “species” was set to “mouse” and the “tissues and cell lines” parameter was set to “brain”. The gene cut oﬀcriteria was set to an adj. p < 0.05. Once all analysis parameters were set, the analysis was run. Cerebellar differential gene expression following the onset of ethanol exposure in the third trimester The tabular descriptions of the diseases and functions categories, including annotations, p-value, and associated transcripts that correlate with these diseases and biological function categories are listed in Supplementary Tables 4A for P5 and 4B for P6. Pathway and cellular function analysis of genes dysregulated by ethanol at P5 and P6 IPA was utilized to determine speciﬁc pathways and cellular functions associated with genes signiﬁcantly (adj. p < 0.05) dysregulated by ethanol. Results of the top canonical pathway categories altered by ethanol exposure common to P5 and P6, included neurotransmitters and other nervous system signaling, cytokine signaling, cellular immune response, intracellular and second messenger signaling, degradation/utilization/assimilation, humoral immune response, nuclear receptor signaling, ingenuity toxicity list pathways, organismal growth and development, cellular stress and injury, cell cycle regulation, disease-speciﬁc pathways, cellular growth and development, and cancer (Figures 2A, B). The top altered diseases and biological function categories of genes dysregulated by ethanol conserved between P5 and P6 included cell death and survival, neurological disease, organismal Cerebellar differential gene expression following the onset of ethanol exposure in the third trimester Publicly available single-cell RNA seq (scRNA-seq) resources were used to investigate the speciﬁc cell types and cellular processes that may be altered by ethanol exposure in the cerebellum in our dataset. We and others have used this analysis technique to deduce cell composition of bulk RNAseq tissue previously (Jew et al., 2020; Pinson et al., 2021). Subsequently, we compiled a list of 822 microglia associated genes (Supplementary Table 1A), 309 astrocyte associated genes (Supplementary Table 2), and 799 oligodendrocyte lineage associated genes (Supplementary Table 3) utilizing this approach (Zeisel et al., 2015; Artegiani et al., 2017; Sousa et al., 2018; Jurga et al., 2020; Ochocka and Kaminska, 2021). We evaluated gene expression proﬁles at P5 and P6, 24 and 48 hours, respectively, after ethanol exposure began. First, a principal component analysis (PCA) was performed on male and female Control and Ethanol treated animals to evaluate distinct diﬀerences between the two datasets at P5 and P6 (Figures 1A, B). The ﬁrst and second principal components encapsulate gene expression patterns that diﬀerentiate Control versus Ethanol treated animals. The PCA analysis suggested there were minimum sex diﬀerences in the ethanol regulation of gene expression, thus we combined sexes for the remainder of the analysis. Secondly, hierarchical clustering analysis using Pearson correlation was conducted on those genes that were identiﬁed as signiﬁcantly dysregulated by ethanol (adj. p < 0.05) at P5 and P6 (Figures 1C, D). Volcano plot analysis identiﬁed 2,440 genes that were signiﬁcantly dysregulated (adj. p < 0.05 and log2FC ± 0.5) Cell type speciﬁc gene lists were further characterized by transcripts associated with speciﬁc phenotypes for each of the glial cell populations. Microglia associated genes, 03 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. at P5 and 1,348 genes at P6. Of the 2,440 genes at P5, 1,419 were upregulated and 1,021 were downregulated (Figure 1E). Of the 1,348 genes at P6, 840 were upregulated and 508 were downregulated (Figure 1F). injury and abnormalities, cell-to-cell signaling and interaction, nervous system development and function, cellular growth and proliferation, tissue development, cellular assembly and organization, cellular movement, immune cell traﬃcking, cellular compromise, cellular function and maintenance, free radical scavenging, psychological disorders, organismal development, lipid metabolism, metabolic disease, molecular transport, small molecule biochemistry, cell morphology, developmental disorder, embryonic development, inﬂammatory response, and organ morphology (Tables 1 and 2). Frontiers in Neuroscience Ethanol induced alterations in microglia phenotypic states at P5 and P6 p < 0.05 and analysis settings were set to “brain” in IPA. n = 3 males and 3 females per treatment group E or C. FIGURE 2 Top canonical pathways altered in the brain by ethanol exposure at P5 and P6. Qiagen Ingenuity Pathway Analysis (IPA) software was employed to assess the top canonical pathways altered by ethanol at P5 (A) and P6 (B). All analyses were restricted to genes with an adj. p < 0.05 and analysis settings were set to “brain” in IPA. n = 3 males and 3 females per treatment group E or C. Ethanol induced alterations in microglia phenotypic states at P5 and P6 In the current study, we compared the list of 822 microglia associated genes (described in the Bioinformatic Analysis subsection of the Materials and Methods) to the list of genes dysregulated by ethanol (adj. p < 0.05) at P5 and P6. We identiﬁed 175 microglia associated genes at P5 (Supplementary Table 5A) FIGURE 1 Ethanol-induced differential gene expression in the cerebellum. Principle component analysis (PCA) of the variance of genes in the cerebellum between ethanol (E) and control (C) at P5 (A) and P6 (B). A heatmap and hierarchical clustering dendrogram of the of relative gene expression across samples for the signiﬁcantly (adj. p < 0.05) altered genes for P5 (C) and P6 (D). A volcano plot displaying fold change versus adjusted p-value of all detected genes in the cerebellum at P5 and P6. 2,440 of 19,595 total identiﬁed transcripts at P5 (E) and 1,348 of 19,595 total identiﬁed transcripts at P6 (F) displayed an adjusted p < 0.05 and Log2 fold change ≥0.5 or ≤–0.5, shown in red. PCA, heatmaps, and volcano plots (Enhanced Volcano package) were generated using R statistical software. n = 3 males and 3 females per treatment group E or C. FIGURE 1 Ethanol-induced differential gene expression in the cerebellum. Principle component analysis (PCA) of the variance of genes in the cerebellum between ethanol (E) and control (C) at P5 (A) and P6 (B). A heatmap and hierarchical clustering dendrogram of the of relative gene expression across samples for the signiﬁcantly (adj. p < 0.05) altered genes for P5 (C) and P6 (D). A volcano plot displaying fold change versus adjusted p-value of all detected genes in the cerebellum at P5 and P6. 2,440 of 19,595 total identiﬁed transcripts at P5 (E) and 1,348 of 19,595 total identiﬁed transcripts at P6 (F) displayed an adjusted p < 0.05 and Log2 fold change ≥0.5 or ≤–0.5, shown in red. PCA, heatmaps, and volcano plots (Enhanced Volcano package) were generated using R statistical software. n = 3 males and 3 females per treatment group E or C. 04 Frontiers in Neuroscience frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 2 Top canonical pathways altered in the brain by ethanol exposure at P5 and P6. Qiagen Ingenuity Pathway Analysis (IPA) software was employed to assess the top canonical pathways altered by ethanol at P5 (A) and P6 (B). All analyses were restricted to genes with an adj. Frontiers in Neuroscience Ethanol induced astrocyte phenotypic switch following neonatal ethanol exposure and 105 microglia associated genes at P6 (Supplementary Table 5B) that were signiﬁcantly dysregulated by ethanol. We further categorized these 175 and 105 dysregulated microglia associated genes as being either typical of a homeostatic or a neurodegenerative phenotype (Supplementary Tables 5C, D), as previously deﬁned (Supplementary Table 1B; Keren-Shaul et al., 2017; Krasemann et al., 2017; Butovsky and Weiner, 2018; Sousa et al., 2018; Tatsuyuki Matsudaira, 2022). Heatmaps illustrating relative gene expression across transcripts for signiﬁcantly altered (p < 0.05) homeostatic and neurodegenerative disease microglial associated genes are depicted in Figures 3A, C for P5 and 3B and 3D for P6. A student’s t-test comparing the average z-scores across all relevant genes indicated that ethanol exposure did not signiﬁcantly alter expression of homeostatic transcripts at P5, p = 0.7464 (Figure 4A) or P6, p = 0.0817 (Figure 4B), though P6 appears to approach signiﬁcance. Examination of the average z-scores across all neurodegenerative disease associated microglia genes by student’s t-test showed ethanol induced a signiﬁcant upregulation of these genes at both P5, p = 2.967e-06 (Figure 4C) and P6, p = 3.069e-05 (Figure 4D). When comparing the list of 309 astrocyte associated genes (described in the Bioinformatic Analysis subsection of the Materials and Methods) to our list of genes dysregulated by ethanol (adj. p < 0.05) at P5 and P6, we identiﬁed 58 astrocyte associated genes at P5 and 33 genes at P6. We further characterized these genes as belonging to an acute injury, chronic neurodegenerative diseases, or pan-injury phenotype (Supplementary Tables 6A, B; Das et al., 2020). Heatmaps illustrating relative gene expression across transcripts for signiﬁcantly altered (p < 0.05) acute injury, pan-injury, and chronic neurodegenerative diseases associated genes are depicted in Figures 5A, C, E for P5 and Figures 5B, D, F for P6. A student’s t-test comparing the average z-scores of all relevant astrocyte associated genes indicated that ethanol induced a signiﬁcant increase in astrocyte associated acute injury transcripts at both P5, p = 2.3e-05 (Figure 6A) and P6, p = 0.0376 (Figure 6B), and pan-injury transcripts at both P5, p = 1.465e-07 (Figure 6C) 05 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. TABLE 2 P6-Top diseases and biological functions altered by ethanol exposure. TABLE 1 P5-Top diseases and biological functions altered by ethanol exposure. Ethanol induced astrocyte phenotypic switch following neonatal ethanol exposure (Figure 6E), but ethanol signiﬁcantly increased expression of these transcripts at P6, p = 0.0073 (Figure 6F). Ethanol induced astrocyte phenotypic switch following neonatal ethanol exposure Category P-value range Cell death and survival 4.7E-04–4.46E-02 Neurological disease 4.7E-04–4.93E-02 Organismal injury and abnormalities 4.7E-04–4.93E-02 Cardiovascular disease 1.18E-03–4.02E-02 Cell–to–cell signaling and interaction 1.18E-03–4.02E-02 Nervous system development and function 1.18E-03–4.97E-02 Cellular growth and proliferation 2.81E-03–4.97E-02 Tissue development 2.81E-03–4.97E-02 Cellular assembly and organization 4.61E-03–4.97E-02 Cardiovascular system development and function 4.73E-03–4.17E-02 Cellular movement 4.73E-03–2.61E-02 Hematological system development and function 4.73E-03–2.61E-02 Immune cell traﬃcking 4.73E-03–2.61E-02 Organ development 4.73E-03–4.73E-03 Tissue morphology 9.76E-03–4.97E-02 Cellular compromise 1.35E-02–2.52E-02 Cellular function and maintenance 1.35E-02–4.97E-02 Free radical scavenging 1.35E-02–1.35E-02 Psychological disorders 1.35E-02–4.93E-02 Organismal development 1.39E-02–4.97E-02 Carbohydrate metabolism 1.92E-02–1.92E-02 Lipid metabolism 1.92E-02–2.61E-02 Metabolic disease 1.92E-02–1.92E-02 Molecular transport 1.92E-02–2.61E-02 Small molecule biochemistry 1.92E-02–2.61E-02 Cell morphology 2.52E-02–4.97E-02 Cellular development 2.52E-02–4.97E-02 Developmental disorder 2.52E-02–2.52E-02 Embryonic development 2.52E-02–4.97E-02 Inﬂammatory response 2.52E-02–2.52E-02 Organ morphology 3.42E-02–4.02E-02 All analyses were restricted to genes with an adj. p < 0.05 and analysis settings were set to “brain” in IPA. n = 3 males and 3 females per treatment group E or C. Category P-value range Cellular development 5.63E-05–4.91E-02 Nervous system development and function 5.63E-05–4.91E-02 Tissue development 5.63E-05–4.91E-02 Tissue morphology 7.94E-05–3.92E-02 Cellular growth and proliferation 7.32E-04–4.91E-02 Neurological disease 7.36E-04–4.87E-02 Organismal injury and abnormalities 7.36E-04–4.87E-02 Psychological disorders 7.36E-04–2.81E-02 Cell morphology 1.01E-03–3.85E-02 Cellular assembly and organization 1.01E-03–4.63E-02 Cellular function and maintenance 1.01E-03–4.63E-02 Organismal development 1.01E-03–4.86E-02 Cell death and survival 1.64E-03–3.34E-02 Embryonic development 1.64E-03–3.88E-02 Organ morphology 2.67E-03–3.81E-02 Behavior 4.39E-03–4.86E-02 Cancer 5.09E-03–4.87E-02 Free radical scavenging 7.05E-03–7.05E-03 Cardiovascular disease 7.5E-03–1.93E-02 Cell–mediated immune response 9.11E-03–1.93E-02 Cellular movement 9.11E-03–3.44E-02 Hematological system development and function 9.11E-03–3.88E-02 Immune cell traﬃcking 9.11E-03–2.23E-02 Lipid metabolism 9.11E-03–4.87E-02 Molecular transport 9.11E-03–4.87E-02 Small molecule biochemistry 9.11E-03–4.87E-02 Endocrine system disorders 9.57E-03–9.57E-03 Gastrointestinal disease 9.57E-03–9.57E-03 Metabolic disease 9.57E-03–3.17E-02 Organ development 1.62E-02–3.88E-02 Cardiovascular system development and function 1.93E-02–4.86E-02 Cell–to–cell signaling and interaction 1.93E-02–3.85E-02 Developmental disorder 1.93E-02–1.93E-02 Hereditary disorder 1.93E-02–1.93E-02 Inﬂammatory response 1.93E-02–1.93E-02 Organismal functions 1.93E-02–1.93E-02 Endocrine system development and function 2.15E-02–2.97E-02 Cellular compromise 2.23E-02–2.23E-02 Amino acid metabolism 3.85E-02–3.85E-02 All analyses were restricted to genes with an adj. p < 0.05 and analysis settings were set to “brain” in IPA. n = 3 males and 3 females per treatment group E or C. All analyses were restricted to genes with an adj. p < 0.05 and analysis settings were set to “brain” in IPA. n = 3 males and 3 females per treatment group E or C. (Figure 6E), but ethanol signiﬁcantly increased expression of these transcripts at P6, p = 0.0073 (Figure 6F). Frontiers in Neuroscience Oligodendrocyte lineage cells have mixed effects under ethanol exposure at P5 and P6 All analyses were restricted to genes with an adj. p < 0.05 and analysis settings were set to “brain” in IPA. n = 3 males and 3 females per treatment group E or C. Comparing our list of genes dysregulated by ethanol (adj. p < 0.05) to the extracted list of oligodendrocyte lineage genes, at P5 we identiﬁed 65 OPCs, 14 COPs, 0 NFOL, 45 MFOL, and 1 MOL associated genes (Supplementary Table 7A), and at P6 we identiﬁed 32 OPCs, 3 COPs, 0 NFOLs, 24 MFOLs, and 0 MOL and P6, p = 0.0006 (Figure 6D). Astrocyte associated chronic neurodegenerative diseases transcripts showed no signiﬁcant diﬀerence between Control and Ethanol groups for P5, p = 0.4234 frontiersin.org 06 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 3 Heatmap and hierarchical clustering of microglia associated genes at P5 and P6. R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized microglia associated genes as detailed in Methods. Microglia homeostatic associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. Microglia neurodegenerative associated gene expression is depicted in panel (C) for P5 and panel (D) for P6. FIGURE 3 Heatmap and hierarchical clustering of microglia associated genes at P5 and P6. R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized microglia associated genes as detailed in Methods. Microglia homeostatic associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. Microglia neurodegenerative associated gene expression is depicted in panel (C) for P5 and panel (D) for P6. with ethanol from P4-9 and cerebellar RNA isolated at P10 followed by RNASeq analysis, that ethanol increased the expression of molecules associated with the S and G2M phases of the cell cycle. In addition, our current IPA analysis (Figures 2A, B) suggests that cell cycle associated pathways may be altered by ethanol. Thus, we sought to determine if ethanol altered the expression of transcripts involved in cell cycle progression as early as P5 and P6. Using the Mouse Genome Database Gene Ontology Browser (Bult et al., 2019), genes associated with G1-S phase transition and G2-M phase transition cell cycle phases were extracted. Oligodendrocyte lineage cells have mixed effects under ethanol exposure at P5 and P6 Heatmaps illustrating relative gene expression across transcripts for signiﬁcantly altered (p < 0.05) positive and negative regulation of G1-S transition associated genes and positive and negative regulation of G2-M transition associated genes are depicted in Figures 9A, C, E, G for P5 and Figures 9B, D, F, H for P6. For the positive regulation of G1-S phase transition, there was no signiﬁcant diﬀerence between Control and Ethanol treated groups at P5, p = 0.6024 (Figure 10A); however, ethanol did induce a signiﬁcant increase in those genes associated with this phase at P6, p = 0.0027 (Figure 10B). Ethanol induced a signiﬁcant increase in those genes associated with the negative regulation of G1-S phase transition at both P5, p = 3.004e-05 (Figure 10C), and P6, p = 0.0006 associated genes (Supplementary Table 7B). Heatmaps illustrating relative gene expression across transcripts for signiﬁcantly altered (p < 0.05) OPC, COP, and MFOL associated genes are depicted in Figures 7A, C, E for P5 and Figures 7B, D, F for P6. Student’s t-test comparing the average z-scores across relevant genes showed that ethanol induced a signiﬁcant upregulation of OPC associated transcripts at P5, p = 0.0219 (Figure 8A), with no signiﬁcant eﬀect on OPC associated transcripts at P6, p = 0.1887 (Figure 8B). In terms of COP associated genes, ethanol induced a signiﬁcant upregulation at P5, p = 0.0219 (Figure 8C) but no eﬀect at P6, p = 0.2924 (Figure 8D). Lastly, ethanol induced a signiﬁcant upregulation of MFOL associated genes at P5, p = 0.0009 (Figure 8E), but a signiﬁcant downregulation of MFOL associated genes at P6, p = 0.0340 (Figure 8F). NFOL and MOL were not analyzed. Frontiers in Neuroscience frontiersin.org Alteration of cell cycle progression following ethanol exposure Lastly, looking at the negative regulation of G2-M phase transition, ethanol induced a signiﬁcant increase in those genes associated with this phase at both P5, p = 0.0002 (Figure 10G), and P6, p = 3.042e-05 (Figure 10H). ethanol at early times following exposure to begin to assess transcriptomic changes that may contribute to initial development of FASD. Pathway analysis suggested that ethanol may alter immune related pathways at P5 and P6 soon after initial ethanol exposure. We previously demonstrated that ethanol induced microglial activation, production of pro-inﬂammatory cytokines and chemokines, as well as neuron cell loss in animals treated with ethanol from P4-P9 and tissue harvested at P10 (Kane et al., 2011, 2021; Drew et al., 2015; Pinson et al., 2021). Others have used similar FASD models and have also observed ethanol induced neuroinﬂammation (Topper et al., 2015; Zhang et al., 2018). Thus, the current studies suggest that ethanol-induced neuroinﬂammation occurs rapidly following ethanol exposure and may contribute to the initial neuropathology associated with FASD. In an attempt to deﬁne possible mechanisms by which ethanol induces early immune activation in the current studies, we identiﬁed immune related transcripts whose expression was strongly altered by ethanol in our transcriptomic data. At P5 and Frontiers in Neuroscience Alteration of cell cycle progression following ethanol exposure We previously determined, using a third trimester human equivalent mouse model of FASD in which animals were treated 07 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 4 Microglia associated genes altered by ethanol exposure at P5 and P6 in the cerebellum. Microglia associated genes were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Control versus Ethanol groups were then tested for statistically signiﬁcant variance in R by two-tailed, student’s t-test, and graphical results were generated. Quantiﬁcation by average z-score of homeostatic microglia associated genes at P5 (A) and P6 (B) and neurodegenerative microglia associated genes at P5 (C) and P6 (D). n = 3 males and 3 females per treatment group E or C; p < 0.001. Microglia associated genes altered by ethanol exposure at P5 and P6 in the cerebellum. Microglia associated genes were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Control versus Ethanol groups were then tested for statistically signiﬁcant variance in R by two-tailed, student’s t-test, and graphical results were generated. Quantiﬁcation by average z-score of homeostatic microglia associated genes at P5 (A) and P6 (B) and neurodegenerative microglia associated genes at P5 (C) and P6 (D). n = 3 males and 3 females per treatment group E or C; p < 0.001. (Figure 10D). Looking at the positive regulation of G2-M phase transition, ethanol induced a signiﬁcant downregulation of genes associated with this phase at P5, p = 0.0021 (Figure 10E), with no signiﬁcant changes between our Control and Ethanol groups at P6, p = 0.9278 (Figure 10F). Lastly, looking at the negative regulation of G2-M phase transition, ethanol induced a signiﬁcant increase in those genes associated with this phase at both P5, p = 0.0002 (Figure 10G), and P6, p = 3.042e-05 (Figure 10H). (Figure 10D). Looking at the positive regulation of G2-M phase transition, ethanol induced a signiﬁcant downregulation of genes associated with this phase at P5, p = 0.0021 (Figure 10E), with no signiﬁcant changes between our Control and Ethanol groups at P6, p = 0.9278 (Figure 10F). Discussion These transcription factors regulate the expression of pro-inﬂammatory cytokines that amplify the immune response and are also linked to addictive behaviors (Crews et al., 2017). AP1 consists as a dimer of Fos and Jun proteins, and it is interesting the ethanol increased the expression of Fos and Jun transcripts at P5 (Supplementary Table 5A). Furthermore, CCL2 and CCL3, target genes of NF-κB, are induced by ethanol and are key mediators of CNS inﬂammation and alcohol drinking behavior (Blednov et al., 2005; He and Crews, 2008). We previously demonstrated that ethanol induced the expression of CCL2 in animal models of FASD (Drew et al., 2015) as well as adult models of alcohol use disorder (Kane et al., 2014). Complement receptor C3AR1 expression is induced by ethanol P6, top upregulated transcripts (Supplementary Tables 5A, B) included SPP1, CCL3, C5AR1, C3AR1, MSR1, and CD14. At P5, but not P6, CCL2 transcript levels were highly increased by ethanol. SPP1, which is also termed osteopontin, is a secretory molecule expressed by a variety of immune cells which has functions including immunomodulation, chemotaxis, and cell adhesion (Lin et al., 2022). SPP1 plays a role in alcohol liver disease (Apte et al., 2005; Seth et al., 2006; Lebel et al., 2008; Das et al., 2022). In the presence of ethanol, SPP1 binds to integrins and CD44 and activates transcription factors AP-1 and NF-κB (Das et al., 2005; Bellahcène et al., 2008). These transcription factors regulate the expression of pro-inﬂammatory cytokines that amplify the immune response and are also linked to addictive behaviors (Crews et al., 2017). AP1 consists as a dimer of Fos and Jun proteins, and it is interesting the ethanol increased the expression of Fos and Jun transcripts at P5 (Supplementary Table 5A). Furthermore, CCL2 and CCL3, target genes of NF-κB, are induced by ethanol and are key mediators of CNS inﬂammation and alcohol drinking behavior (Blednov et al., 2005; He and Crews, 2008). We previously demonstrated that ethanol induced the expression of CCL2 in animal models of FASD (Drew et al., 2015) as well as adult models of alcohol use disorder (Kane et al., 2014). Complement receptor C3AR1 expression is induced by ethanol resulting in altered phagocytosis by microglia (Kalinin et al., 2018). Previous studies also indicated that C5AR1 is involved in alcohol- induced inﬂammation (Blednov et al., 2005; He and Crews, 2008). Frontiers in Neuroscience Discussion The current study was designed to evaluate the eﬀects of ethanol on transcriptomic proﬁles in the cerebellum of early postnatal mice, which approximates the third trimester of gestation in humans. This is a period of secondary neurogenesis, and ethanol exposure at this time can result in FASD. The proposed studies were also designed to evaluate the eﬀects of 08 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 5 Heatmap and hierarchical clustering of astrocyte associated genes at P5 and P6. R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized astrocyte associated genes as detailed in Methods. Astrocyte acute injury astrocyte associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. Astrocyte pan-injury astrocyte associated gene expression is depicted in panel (C) for P5 and panel (D) for P6. Astrocyte chronic neurodegenerative diseases astrocyte gene expression is depicted in panel (E) for P5 and (F) for P6. FIGURE 5 Heatmap and hierarchical clustering of astrocyte associated genes at P5 and P6. R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized astrocyte associated genes as detailed in Methods. Astrocyte acute injury astrocyte associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. Astrocyte pan-injury astrocyte associated gene expression is depicted in panel (C) for P5 and panel (D) for P6. Astrocyte chronic neurodegenerative diseases astrocyte gene expression is depicted in panel (E) for P5 and (F) for P6. P6, top upregulated transcripts (Supplementary Tables 5A, B) included SPP1, CCL3, C5AR1, C3AR1, MSR1, and CD14. At P5, but not P6, CCL2 transcript levels were highly increased by ethanol. SPP1, which is also termed osteopontin, is a secretory molecule expressed by a variety of immune cells which has functions including immunomodulation, chemotaxis, and cell adhesion (Lin et al., 2022). SPP1 plays a role in alcohol liver disease (Apte et al., 2005; Seth et al., 2006; Lebel et al., 2008; Das et al., 2022). In the presence of ethanol, SPP1 binds to integrins and CD44 and activates transcription factors AP-1 and NF-κB (Das et al., 2005; Bellahcène et al., 2008). frontiersin.org Discussion Collectively, these results suggest potential mechanisms by which ethanol-induced neuroinﬂammation may contribute to the early onset of neuropathology associated with FASD. Microglia play a role in several developmental homeostatic functions, including synapse development, plasticity, and maintaining the health of neurons, which are altered in FASD (Drew and Kane, 2014). Microglia are the primary resident immune cell in the CNS and become activated in response to a variety of stimuli (Lynch et al., 2010). Ethanol exposure has previously been demonstrated to result in microglial activation and production of pro-inﬂammatory cytokines and chemokines that may contribute to the neuropathology associated with FASD (Kane et al., 2011; Drew et al., 2015). Activated microglia are responsible for aiding in immune functions including phagocytosis, antigen presentation, and generation of inﬂammatory cytokines and chemokines (Ransohoﬀand Perry, 2009; Saijo and Glass, 2011; Ransohoﬀand Brown, 2012). Our IPA analysis indicated that exposure to ethanol resulted in microgliosis of the brain at both P5 and P6. Microgliosis occurs during pathogenic insults to the CNS (Li and Zhang, 2016). Traditionally, microglia 09 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 6 Astrocyte associated genes altered by ethanol exposure at P5 and P6 in the cerebellum. Astrocyte associated genes were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Quantiﬁcation by average z-score of acute injury astrocyte associated genes at P5 (A) and P6 (B), pan-injury astrocyte associated genes at P5 (C) and P6 (D), and chronic neurodegenerative diseases associated genes at P5 (E) and P6 (F). n = 3 males and 3 females per treatment group E or C; p < 0.05, p < 0.01, p < 0.001. FIGURE 6 FIGURE 6 Astrocyte associated genes altered by ethanol exposure at P5 and P6 in the cerebellum. Astrocyte associated genes were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Quantiﬁcation by average z-score of acute injury astrocyte associated genes at P5 (A) and P6 (B), pan-injury astrocyte associated genes at P5 (C) and P6 (D), and chronic neurodegenerative diseases associated genes at P5 (E) and P6 (F). Frontiers in Neuroscience frontiersin.org Discussion n = 3 males and 3 females per treatment group E or C; p < 0.05, p < 0.01, p < 0.001. activation has been separated into either an M1 pro-inﬂammatory phenotype or an M2 anti-inﬂammatory phenotype (Franco and Fernández-Suárez, 2015; Tang and Le, 2016). However, recent literature suggests that microglia phenotypes do not ﬁt into this simple binary system (Franco and Fernández-Suárez, 2015). Because microglia phenotypes are complex, several microglial phenotypes corresponding to diﬀerent diseases and physiological states have been described. However, two microglial gene expression proﬁles appear across multiple studies - homeostatic and neurodegenerative disease associated. Homeostatic microglia are believed to aid in synaptic plasticity and synaptogenesis, neurogenesis, and immune cell recruitment (Butovsky and Weiner, 2018). The neurodegenerative disease related microglia phenotype results from insult to the CNS, and microglia lose their homeostatic signature and gain a chronic inﬂammatory signature (Holtman et al., 2015; Moore et al., 2015; Paolicelli et al., 2022). Although there are a variety of neurodegenerative diseases, assessment of microglia phenotype during these disease states have identiﬁed a common neurodegenerative disease related microglia phenotype (Naj et al., 2014; Moore et al., 2015; Keren- Shaul et al., 2017; Butovsky and Weiner, 2018). When examining microglia phenotypic states in the current study, ethanol induced a phenotypic switch at both P5 and P6 in the cerebellum, resulting in upregulation of neurodegenerative disease associated transcripts. phenotypic switch at both P5 and P6 in the cerebellum, resulting in upregulation of neurodegenerative disease associated transcripts. The expression of homeostatic associated genes was not altered at P5 or P6 in the current studies. However, three of the top four most strongly ethanol-downregulated microglial associated molecules at P5 are considered homeostatic molecules (Supplementary Tables 1B, 5A). Additionally, microglia homeostatic associated molecules at P6 trended toward signiﬁcance. This might suggest ethanol decreases the expression of at least a subset of homeostatic microglial associated transcripts in the current study which could in turn change the phenotype of microglia and alter speciﬁc developmental functions. activation has been separated into either an M1 pro-inﬂammatory phenotype or an M2 anti-inﬂammatory phenotype (Franco and Fernández-Suárez, 2015; Tang and Le, 2016). However, recent literature suggests that microglia phenotypes do not ﬁt into this simple binary system (Franco and Fernández-Suárez, 2015). Because microglia phenotypes are complex, several microglial phenotypes corresponding to diﬀerent diseases and physiological states have been described. Discussion MFOL associated gene expression is depicted in panel (E) for P5 and panel (F) for P6. associated with FASD (Guizzetti et al., 2014; Wilhelm and Guizzetti, 2016). Astrocytes may become reactive in response to various stimuli, resulting in astrogliosis/astrocytosis. During astrogliosis/astrocytosis, astrocytes undergo a phenotypic change which has historically been referred to result in an A1 reactive phenotype characterized as being neurotoxic or an A2 reactive phenotype described as being neuroprotective (Zamanian et al., 2012; Liddelow et al., 2017). However, classifying reactive astrocytes into these two categories does not appear to be adequate. A recent meta-analysis of mouse transcriptomic studies aimed to better categorize astrocyte reactive states (Das et al., 2020). The nomenclature used in this study classiﬁed reactive astrocytes as having three diﬀerent phenotypes; acute injury, chronic neurodegenerative diseases, or pan-injury which has characteristics of both acute injury and chronic neurodegenerative diseases phenotypes (Das et al., 2020). In the current study, at P5, our IPA analysis indicated that ethanol treatment resulted in alterations related to the development of astrocytes, formation of astrocyte precursor cells, and quantity of astrocytes, and at P6, our IPA analysis revealed that ethanol treatment stimulated astrocytosis and gliosis of astrocytes in the cerebellum. These results suggest that 24 h following initial ethanol exposure, astrocyte development and quantity is altered, and after 48 h of ethanol exposure, those astrocytes that are present are becoming reactive. We also revealed that at both P5 and P6, ethanol stimulated an acute injury and pan-injury reactive astrocyte phenotype, with a chronic neurodegenerative diseases phenotype also being seen at P6, but not P5. LPS is known to induce an immune response in the CNS, and was demonstrated to trigger an acute injury astrocyte phenotype (Das et al., 2020). Like LPS, ethanol is believed to trigger immune responses, at least in part, through activation of TLR4 signaling pathways (Blanco et al., 2005; Floreani et al., 2010; Alfonso-Loeches et al., 2014). Therefore, in this model of FASD, it is possible that ethanol stimulates an acute or pan-injury reactive phenotypic state at both P5 and P6, possibly through activation of TLR4. It will be important in the future to deﬁne the mechanisms by which ethanol alters astrocyte phenotype and immune responses and how this may contribute to ethanol-induced neuropathology associated with FASD. Oligodendrocytes generate the myelin sheath which wraps axons to promote the conduction of electrical impulses (Baumann and Pham-Dinh, 2001). Discussion However, two microglial gene expression proﬁles appear across multiple studies - homeostatic and neurodegenerative disease associated. Homeostatic microglia are believed to aid in synaptic plasticity and synaptogenesis, neurogenesis, and immune cell recruitment (Butovsky and Weiner, 2018). The neurodegenerative disease related microglia phenotype results from insult to the CNS, and microglia lose their homeostatic signature and gain a chronic inﬂammatory signature (Holtman et al., 2015; Moore et al., 2015; Paolicelli et al., 2022). Although there are a variety of neurodegenerative diseases, assessment of microglia phenotype during these disease states have identiﬁed a common neurodegenerative disease related microglia phenotype (Naj et al., 2014; Moore et al., 2015; Keren- Shaul et al., 2017; Butovsky and Weiner, 2018). When examining microglia phenotypic states in the current study, ethanol induced a Astrocytes, like microglia, play a role in immune responses in the CNS and produce cytokines and chemokines, nitric oxide, and reactive oxygen species (Ransohoﬀand Brown, 2012). Astrocytes also play roles in maintaining the blood brain barrier and neurotransmitter levels, along with regulating energy balance and modulating synaptic plasticity (Santello et al., 2019). They also have a signiﬁcant immune function, mediating both pro- inﬂammatory and anti-inﬂammatory activities in response to CNS insult (Dong and Benveniste, 2001). Astrocyte production of immune mediators is suspected to contribute to neuropathology 10 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 7 Heatmap and hierarchical clustering of oligodendrocyte lineage associated genes at P5 and P6. R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized oligodendrocyte lineage associated genes as detailed in Methods. OPC associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. COP associated gene expression is depicted in panel (C) for P5 and panel (D) for P6. MFOL associated gene expression is depicted in panel (E) for P5 and panel (F) for P6. Heatmap and hierarchical clustering of oligodendrocyte lineage associated genes at P5 and P6. R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized oligodendrocyte lineage associated genes as detailed in Methods. OPC associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. COP associated gene expression is depicted in panel (C) for P5 and panel (D) for P6. Frontiers in Neuroscience Discussion Prior to myelination, oligodendrocytes undergo a series of diﬀerentiation steps, beginning as OPCs and terminating as mature myelinating oligodendrocytes 11 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 8 Oligodendrocyte lineage associated altered genes by ethanol exposure at P5 and P6 in the cerebellum. Oligodendrocyte lineage associated genes were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Quantiﬁcation by average z-score of OPC associated genes at P5 (A) and P6 (B), COP associated genes at P5 (C) and P6 (D), and MFOL associated genes at P5 (E) and P6 (F). n = 3 males and 3 females per treatment group E or C; p < 0.05, **p < 0.001. FIGURE 8 Oligodendrocyte lineage associated altered genes by ethanol exposure at P5 and P6 in the cerebellum. Oligodendrocyte lineage associated genes were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Quantiﬁcation by average z-score of OPC associated genes at P5 (A) and P6 (B), COP associated genes at P5 (C) and P6 (D), and MFOL associated genes at P5 (E) and P6 (F). n = 3 males and 3 females per treatment group E or C; p < 0.05, *p < 0.001. (Bradl and Lassmann, 2010; El Waly et al., 2014). Studies of children and adolescents with FASD have demonstrated white matter abnormalities, suggesting that ethanol has a long- lasting impact on myelination (Wilhelm and Guizzetti, 2016). During development, OPCs migrate from their origin to their functional site where they diﬀerentiate into mature myelinating oligodendrocytes. In rodents, myelin formation occurs abundantly during the ﬁrst two postnatal weeks; however, OPC diﬀerentiation into mature myelinating oligodendrocytes can occur throughout life (El Waly et al., 2014). Ethanol eﬀects on myelination in animal models of FASD have begun to be investigated. Studies have demonstrated that postnatal ethanol exposure in rats resulted in myelin deﬁcits and aberrant eye-blink conditioning, which is a cerebellum-dependent learning task (Rufer et al., 2012). Using a similar postnatal model of FASD, ethanol was demonstrated to decrease both proliferating OPCs and mature oligodendrocytes in the corpus callosum. Interestingly, the eﬀects of ethanol on OPCs depended on the ontogenetic origin of these cells. Frontiers in Neuroscience frontiersin.org Discussion R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized cell cycle associated genes as detailed in Methods. Positive regulation of G1-S transition associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. Negative regulation of G1-S transition associated gene expression is depicted in panel (C) for P5 and (D) for P6. Positive regulation of G2-M transition associated gene expression is depicted in panel (E) for P5 and (F) for P6. Negative regulation of G2-M transition associated genes is depicted in panel (G) for P5 and panel (H) for P6. postnatal development in rodents, cells in the external germinal layer of the cerebellum undergo vast proliferation to generate a substantial pool of cerebellar granule progenitors, which eventually form cerebellar neurons (Miale and Sidman, 1961; Altman, 1997; Li et al., 2002). Additionally, cerebellar interneurons are born and migrate to their ﬁnal destination to form synaptic connections with Purkinje cells (Schilling et al., 2008; Leto and Rossi, 2012). Our data suggest that early third trimester equivalent ethanol exposure halts cell cycle progression in both the synthesis and mitosis phases, preventing cells from replicating. The accumulation of cells in these phases could result in a potential decrease in the pool of cerebellar granule cells that will eventually form mature neurons while also limiting the generation of interneurons. Having deﬁcits in both granule cells and interneurons of the cerebellum could contribute to the reduced cerebellar volume and aberrant motor function and cognitive deﬁcits seen in individuals with FASD. However, further studies are needed to evaluate this possibility. to continue proliferation in order to maintain a consistent pool (Hughes et al., 2013). At P6, ethanol did not alter the expression of transcripts associated with early-stage oligodendrocytes but decreased the expression of transcripts associated with mature oligodendrocytes. The decrease in myelin forming oligodendrocyte transcripts seen at P6 could result in decreased myelination observed in FASD. These results suggest that after two days of consecutive ethanol exposure one begins to see a depletion in myelinating oligodendrocytes. Developmental alcohol exposure is known to aﬀect cell cycle regulation and apoptosis related events (Anthony et al., 2008). Discussion Furthermore, although OPC and oligodendrocyte numbers recovered by adulthood, the myelin microstructure remained aberrant as determined by diﬀusion tensor imaging (Newville et al., 2017). Myelin was also aberrant in third trimester models of FASD in sheep (Dalitz et al., 2008) and oligodendrocyte apoptosis was abundant in a third trimester FASD model in macaques (Creeley et al., 2013). We have recently demonstrated that ethanol dysregulated transcripts associated with OPCs, pre-myelinating oligodendrocytes, and mature oligodendrocytes in a postnatal mouse model of FASD in which animals were treated with ethanol from P4-9 and cerebellum isolated at P10 (Niedzwiedz-Massey et al., 2021a). Using the same model, we demonstrated that ethanol decreased the expression of transcripts associated with OPCs and mature oligodendrocytes in the hippocampus (Niedzwiedz- Massey et al., 2021b). In the current study, we evaluated the eﬀects of ethanol on immature oligodendrocyte lineage cells and mature myelinating oligodendrocytes at P5 and P6. Interestingly, at P5, ethanol induced a signiﬁcant increase in transcripts associated with both immature oligodendrocyte lineage cells and myelinating oligodendrocytes. This increase in the expression of oligodendrocyte related transcripts, particularly those associated with OPCs may result from an initial compensatory response to the toxic eﬀects of ethanol. OPCs are highly proliferative during this stage of development and continue to proliferate until a balanced number of OPCs is reached (Hughes et al., 2013). If this balance is disrupted, perhaps as a response to ethanol, OPCs are triggered Frontiers in Neuroscience 12 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 9 Heatmap and hierarchical clustering of cell cyle associated genes at P5 and P6. R statistical software was utilized to construct heatmaps and hierarchical clustering dendrogram of relative gene expression across samples for signiﬁcantly altered (adj. p < 0.05) and categorized cell cycle associated genes as detailed in Methods. Positive regulation of G1-S transition associated gene expression is depicted in panel (A) for P5 and panel (B) for P6. Negative regulation of G1-S transition associated gene expression is depicted in panel (C) for P5 and (D) for P6. Positive regulation of G2-M transition associated gene expression is depicted in panel (E) for P5 and (F) for P6. Negative regulation of G2-M transition associated genes is depicted in panel (G) for P5 and panel (H) for P6. FIGURE 9 Heatmap and hierarchical clustering of cell cyle associated genes at P5 and P6. Frontiers in Neuroscience frontiersin.org FIGURE 10 GU 0 Ethanol-induced alterations in cell cycle regulation at P5 and P6 in the cerebellum. Gene list associated with positive and negative regulation of G1-S transition and positive and negative regulation of G2-M transition were extracted from the Mouse Genome Database. Genes associated with each cell cycle stage in our dataset at P5 and P6 were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Quantiﬁcation by average z-score of positive regulation of G1-S transition associated genes at P5 (A) and P6 (B), negative regulation of G1-S transition associated genes at P5 (C) and P6 (D), positive regulation of G2-M transition associated genes at P5 (E) and P6 (F), and negative regulation of G2-M transition associated genes at P5 (G) and P6 (H). n = 3 males and 3 females per treatment group E or C; p < 0.01, *p < 0.001. changes need to be evaluated at the level of protein expression. Increasing the sample size would also add conﬁdence that the observed results will be experimentally reproducible. The levels of alcohol used in the current studies are also relatively high. Future studies are needed to determine transcriptomic changes in mice treated with more moderate levels of ethanol. It also should be acknowledged that some of the transcriptomic changes observed may not result solely or speciﬁcally due to ethanol but could result from a more general acute stress response to high doses of ethanol. Future studies are also needed to determine which transcripts and pathways altered by ethanol in these studies may contribute to the pathogenesis of FASD and thus represent potential targets for FASD therapy. We also note that the expression of more transcripts was observed at P5 after a single dose of ethanol than at P6 after two doses of ethanol. Additional studies are needed to determine the potential relevance of these temporal changes in transcriptomic proﬁles to FASD. along with an increase in transcripts associated with acute and pan-injury reactive astrocyte phenotypes at both P5 and P6 and additionally a chronic neurodegenerative disease astrocyte phenotype at P6 but not P5. Lastly, ethanol induced diﬀering eﬀects in the expression of genes associated with immature oligodendrocyte lineage cells and myelinating oligodendrocytes. FIGURE 10 These studies may begin to unravel the eﬀects of ethanol during the onset of FASD. Data availability statement The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number: GSE226532 (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE226532). In conclusion, the current studies demonstrate that ethanol has profound eﬀects on the transcriptomic proﬁle in the developing cerebellum, early following initial ethanol exposure which may be critical in the development of FASD. IPA analysis indicated that ethanol likely alters pathways involved in immune signaling and cell cycle. With regard to glia, ethanol induced an increase in transcripts related to a neurodegenerative microglia phenotype Frontiers in Neuroscience Discussion At P5, we saw no signiﬁcant eﬀect of ethanol on transcripts associated with the positive regulation of G1-S transition, and an increase in transcripts involved in negative regulation of this cell cycle phase. Ethanol also decreased the expression of positive regulators and increased the expression of negative regulators of the G2-M phase at P5. Collectively, these data suggest that ethanol negatively impacts G1-S and G2-M transitions at P5. At P6, ethanol increased the expression of transcripts involved in both positive and negative regulation of the G1-S transition. Ethanol did not alter the expression of positive regulators but increased the expression of negative regulators of the G2-M transition at P6. Collectively, these data show no clear eﬀect of ethanol on G1-S transition and a decrease in G2-M transition at P6. During early The current study demonstrated that ethanol altered the transcriptomic proﬁle in the cerebellum in a postnatal model of FASD. However, there are several limitations in the experimental design which should be considered when interpreting these data. For example, it should be acknowledged that alterations in transcript expression in the current RNASeq analysis will need to be conﬁrmed by RT-PCR analysis. Furthermore, these transcript 13 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. FIGURE 10 Ethanol-induced alterations in cell cycle regulation at P5 and P6 in the cerebellum. Gene list associated with positive and negative regulation of G1-S transition and positive and negative regulation of G2-M transition were extracted from the Mouse Genome Database. Genes associated with each cell cycle stage in our dataset at P5 and P6 were extracted as detailed in Methods. R statistical software was utilized to generate individual z-scores for each transcript of interest and each experimental animal. These z-scores were then averaged across transcripts for each individual animal. Quantiﬁcation by average z-score of positive regulation of G1-S transition associated genes at P5 (A) and P6 (B), negative regulation of G1-S transition associated genes at P5 (C) and P6 (D), positive regulation of G2-M transition associated genes at P5 (E) and P6 (F), and negative regulation of G2-M transition associated genes at P5 (G) and P6 (H). n = 3 males and 3 females per treatment group E or C; p < 0.01, ***p < 0.001. GURE 10 References Bradl, M., and Lassmann, H. (2010). 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Ethics statement This animal study was reviewed and approved by University of Arkansas for Medical Sciences, Institutional Animal Care and Use Committee (IACUC). 14 frontiersin.org 10.3389/fnins.2023.1154637 Holloway et al. Funding This work was supported by grants from the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, RO1 AA024695, RO1 AA026665, and RO1 AA027111. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins.2023. 1154637/full#supplementary-material RNA sequencing was performed by the UAMS Genomics Core which is supported by the Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences. Author contributions 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. PD, CK, and AM: conceptualization. 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https://openalex.org/W4238267373	https://tidsskrift.dk/passage/article/download/1463/1341	Danish	null	null	Passage	2,006	cc-by	8,875	A n n e - M a r i e M a i A n n e - M a r i e M a i Indeni – udenfor Om Kirsten Thorups forfatterskab i litteraturhistorieskrivningen Om Kirsten Thorups forfatterskab i litteraturhistorieskrivningen Det vrimler med danske litteraturhistorier! I det første tiår af det 21. århundrede er der en håndfuld nye danske litteraturhistorier på gaden eller på vej. I en tid hvor genren både nationalt og internationalt har været sendt til tælling i litteraturviden- skabens teoretiske boksering, har lysten til at gøre forsøget med at skrive littera- turhistorie grebet både yngre og ældre litteraturforskere. “En national litteratur- histories formål er og var at nationalisere børn og bønder”1 – lød det advarende fra den polemiske litterat Hans Hauge allerede i 1988. Værre og mindre politisk og videnskabeligt korrekt kunne det næppe blive i 1980´erne, hvor mange af nutidens akademikere og litteraturhistorieskrivere var under uddannelse. Hauge så i stedet en postnational flersproget litteraturhistorie for sig, lykkeligt befriet fra nationalstaten og først og fremmest for alle institutter for nordisk sprog og litteratur! En postnational litteraturhistorie burde vise, at Danmark er et euro- pæisk land med mange indvandrere og en fortid som et multikulturelt imperium. Mens Hauge var modstander af en national litteraturhistorie, men altså for en post- national ditto, havde andre, internationale teoretiske trendsættere længe været kritikere af genren som sådan.2 Traditionel litteraturhistorieskrivning har ikke ret meget at gøre med litteratur, mente allerede en af dekonstruktionskritikkens fou nding fathers, Paul de Man. Med teoretisk konsekvens erklærede de Man i 1971, at tekstanalyse i højere grad kunne betragtes som litteraturhistorie eftersom historisk viden ikke baserer sig på empiriske fakta, men på skrevne tekster, hvad enten de maskerer sig som krige eller revolutioner.3 Den vældige trang til litteraturhistorieskrivning, der trods alskens teoretiske kvababbelser har grebet mere eller mindre poststrukturalistiske og postnationale litterater på dansk grund, finder sted parallelt med den lange kamp om kanon, som Bertel Haa rders kanonudvalg skød i gang i 1994. I 1994 gik kanondebatten i sig selv, da Bertel Haa rder gik af. Men da de borgerlige støttet af Dansk Folkeparti kom til magten i 2001, fik kanonkampen sin anden luft, og den nåede et nyt højdepunkt med undervisningsministeriets offentliggørelse af den officielle, lovpligtige danske 5 kanon i 2004. I 2006 kom der ekstra krudt i kanonen, da kulturministeren afslørede sin kæmpekanon, der skulle kunne ramme alle kunstarter til alle tider. I litteraturen er og var meget på spil: nye og ældre forfatteres overlevelse eller glemsel i en fælles erindring om dansksproget litteratur sammen med mange lit- teratur- og forlagspolitiske interesser. Indeni – udenfor Hvem ville komme hurtigst frem til trykke- maskinen og i øvrigt score kassen på undervisningsbøger til den nye kanon? Og er det linien fra Holberg til Brandes og Rifbjerg, der er hovedsporet i dansk litteratur, hvorom kanon og erindring skal struktureres? Hvor efterlader et sådant hovedspor Leonora Christina, Schack von Staffeldt, Søren Kierkegaa rd og H. C. Andersen? Hvad sker der med samtidslitteraturen? Bortdømmer den vilde kanonjagt og den nye litteraturhistorieskrivning de forfatterskaber, som kritikken og den danske mo- dernismekonstruktion har haft svært ved at tumle? Her er blandt andet den litte- raturhistoriske skæbne for Kirsten Thorups forfatterskab interessant at studere og også forsøge at reformulere. Kirsten Thorup er hverken med på Ulla Tørnæs’ eller Brian Mikkelsens kanon. Hun er helt sat uden for døren såvel i det ene som det andet ministerium. Tørnæs- kanonen slutter som bekendt med forfatterskaber med debut før 1965. Her har man på forhånd bekvemt sikret sig mod overhovedet at skulle tage stilling til opbruddet fra modernismen. Kanon begynder med folkeviserne og Holberg og slutter med Rif- bjerg, så alt kan blive ved at være, som det plejer. Hvornår kanon skal ændres, er nemlig blevet en politisk beslutning, der kun kan træffes af en minister eller et fler- tal i Folketinget. Læserpublikum, kritikere, lærere, undervisere og forskere er som andre grimme smagsdommere helt sat ud af det nye politiske spil om ændringer af det klippefaste litteraturpensum i undervisningen. Brian Mikkelsens kanon tager så småt hul på opbruddet fra modernismen. Både i form af forfatterskaber og tekster fra tiden efter 1965. Man nøjes dog på forfattersiden forsigtigt med at inkludere Henrik Nordbrandt, der debuterede i 1966, i lyrikantologien. Men på tekstsiden går man gerne frem i tid. Den nyeste tekst, der er med på Mikkelsen-kanonen, er Inger Christensens sonetkrans, Sommerfugledalen, fra 1991. I lyrikantologien har Erik A. Nielsen virkelig fået sat fede streger under sin forkærlighed for den symbolistiske linie i dansk lyrik fra Claussen til Bjørnvig, Inger Christensen og udvalgte sider af Højholt. Henry fra Højholts Turbo (1968) ville aldrig få lov at komme ind i dette landskab for slet ikke at tale om de omvandrende ører, Auricula (2001). Der bliver i det hele taget ingen plads til prosa eller lyrik, der går i mere eksperimenterende retninger på tværs af genrer. Thorups tidlige bøger fra slutningen af 1960´erne, der konstant krydser grænsen mellem lyrik og prosa ville forstyrre det nydelige genre- billede. Indeni – udenfor Eftersom der ikke er blik for det 20. århundredes romankunst og slet ikke for hverken den eksistentielt-narrative eller den konstruktivistiske roman,4 er der selvfølgelig heller ikke plads til Kirsten Thorup. På kanten Hvordan står så Kirsten Thorups forfatterskab i litteraturhistorieskrivningen? Tin- genes tilstand minder mest om Brorsons 1700-tals-salme, “Jeg gaa er i fare, hvor jeg gaa er”. Forfatterskabet balancerer nemlig trods læseropbakning og Thorups store 6 gennembrud i den litterære kritik med Himmel og helvede (1982) på yderkanten af den litteraturhistoriske tænkning som en blandingsform, litteraturhistorieskriverne ikke rigtig kan få hold på. Skrækscenariet for forfatterskabet finder vi i Søren Gra- versens og Steen Hvorslev Mogensens Saxo & Co. (2003), der er tænkt til brug i de gymnasiale uddannelser, på HF og VUC. Hos Saxo & Co. er Thorup end ikke nævnt. Konstruktionen af den nyere litteraturhistorie bygges over den velkendte vekseldrift mellem modernisme og realisme, og de centrale figurer blandt “de første postmoder- nister”, der kommer lige efter konfrontationsmodernisterne, er Svend Åge Madsen og Dan Turèll. I afsnittet “Eksistentialisme og postmodernisme 1945-2000” er i øvrigt ikke en eneste kvinde blandt de forfattere, der får et særskilt afsnit. Hverken Dorrit Willumsen, Kirsten Thorup eller Vibeke Grønfeldt bliver omtalt. Inger Christensens forfatterskab får lige en litteraturhistorisk “rundbarbering” over to gange tre linier. Fremstillingen bæres af den ulyksalige modstilling af modernisme og realisme, som gør det af med så meget god og nybrydende litteratur i litteraturhistorieskriv- ningen. Man finder også modstillingen i fuldt flor i Johannes Fibigers og Gerd Lütkens Litteraturens veje, anden udgave 2003, hvor Kirsten Thorup og Dorrit Wil- lumsen placeres som “psykologisk realisme” i afsnittet “1970´erne: Fra Woodstock til Vietnam”. Det hedder her om Kirsten Thorup og Willumsen: “De skriver ikke lit- teratur som diskussionsindlæg i kønskampen, og de har begge et udgangspunkt i en modernistisk tradition, der dog med årene omformes til en bredere realisme. Begge har de sans for at skildre psykologisk indviklede spil, at få personer til at leve indefra og ikke gennem den ydre karakteristik. Deres realisme kan betragtes som en psykologisk realisme, og de mestrer begge en fin portrætteringskunst.”5 Ærger- ligt nok har vi stadig modstillingen, “realisme” og “modernisme” på slæb i det nye årtusind. I Hovedsporet. Dansk litteraturs historie (2005) forsøger man sig med et opbrud fra modernismen, inspireret af som det hedder “nyere forsknings ihærdige fremgravning af den eksperimenterende 70´er-digtning”,6 men en sammenhæng og linie i nybruddet bliver det svært at få fat i, når Hovedsporets forfattere under overskriften “Med andre øjne” udnævner Kirsten Thorup til at være en realistisk forfatter, Suzanne Brøgger til en genrehybrid bekender og Bent Vinn Nielsen til en indignationsforfatter. På kanten Det er ikke sært, at der her kun kan blive plads til at name droppe Vibeke Grønfeldt og Henrik Bjelke under den hjemmestrikkede betegnelse socialmodernister! Hvis en forfatter hverken kan kaldes renlivet modernist eller ditto realist havner forfatterskabet nemt i en farlig gråzone. Man kan sige, at den udskrivning af litteraturhistorien, som Saxo & Co effektu- erer over for Thorup, truer i flere af de øvrige fremstillinger. Mens Dan Turèll begyn- der at blive kanoniseret som en tidlig postmodernist med sin forvandling fra “svær modernistisk forfatter til mediekæledægge”,7 glider Kirsten Thorup ned i rækkerne blandt 1960´ernes nye forfattere. Hovednavnene i Hovedsporet er: Hans-Jørgen Nielsen, Inger Christensen, Svend Åge Madsen og Jens Smærup Sørensen, mens det i Litteraturens veje entydigt er Hans-Jørgen Nielsen, “generationens seismograf”, som han kaldes. Hvad skal man stille op over for disse konstruktioner af den ny- ere litteratur og over for to kanoner, der i udtalt grad centrerer sig om 1800-tallets moderne gennembrud og vælger at glemme det meste af det 20. århundrede? Kan Thorup blive en hovedperson i den nyere danske litteraturhistorie? Og hvordan ser 7 litteraturhistorien i givet fald ud? Fortsættelse følger i næste afsnit af den litteratur- historiske soap! Fra kritik til litteraturhistorie Der er sikkert flere grunde til, at man som læser af litteraturhistorierne sidder med livet i hænderne, når det gælder den noget rutschende placering af Thorups forfat- terskab. En årsag til miséren kunne ligge i kritikkens bedømmelse af forfatterskabet. Kritikken er i nutiden litteraturhistorieskrivningens fødekæde. En forfatter, der får forrang i den litterære kritik, har en god chance for litteraturhistorisk og kanonisk overlevelse. Thorup har ofte oplevet en storslået modtagelse og er blevet prisbeløn- net som en mesterlig fortæller. Men der har også været mislyde og paradokser, som kritikken aldrig rigtigt har kunnet bide i sig. De lod sig blandt andet høre i forbin- delse med romanen Elskede ukendte (1994). Hverken den kulturradikalt eller den modernistisk sindede kritik var udelt begejstret for denne vilde fortælling, som blev kaldt alt fra et forlist projekt til monotoni. Den mest rosende bemærkning lød som følger: “en masochistisk fornøjelse, som det står enhver frit for at unddrage sig” (John Christian Jørgensen i Politiken, 17.3.1994). Bonsai (2000) blev genstand for et regulært og ganske umotiveret overfald for være “hæmningsløst selvbiografisk” af Erik Skyum-Nielsen i Information. Midt i historien Det er selvfølgelig ganske umuligt at give et fyldestgørende historisk billede af sam- tidslitteraturen. Den udspiller sig og forandrer sig jo for øjnene af læseren. Vi er midt i en historisk kontekst, og Kirsten Thorup har forhåbentlig stadig mange bøger for sig. Romanen Førkrigstid er netop udkommet her i efteråret 2006. Men det er dog snart 40 år siden hun debuterede, og linier i den nyere litteratur kan optegnes. Litteraturhistorieskrivningen har faktisk altid nøje forholdt sig til sin samtids littera- tur i sit forsøg på overhovedet at skabe forbindelse mellem “litteratur” og “historie”, selv om vidt forskellige historiske perioder har anset forbindelsen for umulig. Et nyt eksempel på en formulering af modsætningen mellem litteratur og historie finder vi på dansk grund i Torben Brostrøms præsentation af bind III i Kanon i dansk (2006), hvor det bliver understreget, at det er en af modernismens erfaringer, at kunsten altid begynder forfra og altid undersiger alle vedtagne dogmer, æstetiske, religiøse og politiske. Den udsletter dermed konstant sin egen forhistorie – alt er og alt bliver modernisme. Dylans faderlige omkvæd, “Forever young”, fra 1974 kunne således være modernismens afskedsreplik til historien. Måske er det denne udmattende sø- gen efter aktualitet og samtid, der får Jørgen Leth til i et af sine digte i samlingen Det gør ikke noget (2006) at skrive: “Jeg er træt af den gærede sure modernisme.”9 Litteraturhistorieskrivningen har forsøgt sig med et umuligt projekt om at kom- me tilbage til, hvordan det egentlig var, dengang i fortiden, men den har også i høj grad søgt sin motivering og legitimitet i samtidslitteraturen. Der har ofte tegnet sig en art krydsstilling i litteraturhistorieskrivningens tænkning af forholdet mellem fortid og samtid. Litteraturhistorieskrivning er blevet motiveret som et forsøg på at forbedre nutidens smag ved at studere fortiden eller som et forsøg på at skabe grundlag for menneskelig identitetsdannelse og frigørelse. Hvis man forsøger at bruge Thorups værker som en nøgle til en fortolkning og konstruktion af en historisk kontekstualisering af de seneste 40 års litteratur, er det som omtalt ganske oplagt at andre værker og forfatterskaber end de kanoniserede rykker ind i det litteraturhistoriske landskab. Ser man Thorup som en hovedperson i litteraturen siden slutningen af 1960´erne, fokuserer fortællingen om litteraturen langt mere på både eksperimenterende og populær litteratur end traditionelt. På kanten Skyum-Nielsen påtvang således hårdhændet Thorup en selvbiografisk kontrakt, som teksten selv ikke indbød til.8 Marie Tetzlaff var forsigtigt forbeholden over for Thorups persontegning i en ellers rosende an- meldelse: “Der er altid nogle mærkelige modsigelser i Kirsten Thorups personteg- ning, og man skal ikke være for smålig med hensyn til hendes hovedpersons udvik- ling fra den næsten debilt naive, men dygtige studerende Nina til den klarsynede, kendte forfatter, der håndplukkes af en fremstormende svensk filminstruktør som manuskriptforfatter på hans livs film” (Politiken, 26.10.2000). Kritikken retter sig karakteristisk nok ofte mod form og formelle forhold. Det gælder også romanen Ingenmandsland (2003), der midt i mange roser og den store læserkåring i forbindelse med BG Banks litteraturpris blev kritiseret for både at spænde formen for hårdt i en halsbrækkende kompositionsform og være for lidt eksperimenterende. Kritikken vil gerne klappe ad Thorups temaer om identitet, kærlighed, køn, galskab og alderdom, men der er og bliver “noget” omkring formen, som flere kritikere ikke rigtig har kunnet komme overens med. Romanernes måde at være romaner på gør vejen fra litteraturkritisk hyldest til de litteraturhistoriske overskrifter vanskelig. Hvis man skal ind med de store typer og selvstændig omtale i den gældende litteraturhistorieskrivning som prosaist, ser det ud til, at man skal placere sig i en psykologisk realisme med indbygget kritik af småborgerlig kultur som Rifbjergs Den kroniske uskyld og Hans-Jørgen Nielsens Fodboldenglen. Man kan dog også praktisere en underholdende fantastik som Svend Åge Madsen. Derfor forsøger mange litteraturhistorikere så ihærdigt at putte Thorups forfatterskab ned i den meget store kasse: psykologisk realisme. Når det gælder Kirsten Thorup er min pointe imidlertid den, at det netop er for- mens dristighed, der burde placere Thorup som en hovedperson i den litteraturhi- 8 8 storiske fortælling. De konstruktioner omkring realisme og modernisme, som såvel mange kritikere som et flertal af litteraturhistorikere tænker i, trænger til en kritisk revision, så der kan skabes bedre kritiske og litteraturhistoriske formidlingsformer af forfatterskaber som Kirsten Thorups. p a s s a g e \| 5 6 \| v i n t e r 2 0 0 6 p a s s a g e \| 5 6 \| v i n t e r 2 0 0 6 Kroppen som medie Midt i historien Først og fremmest tydeliggør Thorup som historisk hovedperson opbruddet fra moder- nismen og det formelle gennembruds begyndelse.10 Modernismen fortsætter netop ikke for evigt, men kulminerer i første halvdel af 1960’erne i Rifbjergs Konfronta tion (1960), Ørnsbos Digte (1960), Cecil Bødkers noveller Øjet (1961), Jørgen Son- nes Krese (1963), Uffe Harders Positioner (1964) og Villy Sørensens Formynderfor tællinger (1964). Fra midten af 1960´erne formuleres en række andre litterære positioner end modernismens, idet æstetiske skift især i Per Højholts og Inger Chri- stensens forfatterskaber finder sted samtidig med at en stribe unge forfattere på 9 tværs af genrer begynder at tematisere litteraturens karakter af i fænomenologisk forstand at være et æstetisk mellemværende mellem den læsende, den skrivende og verden. Denne tematisering spidsformuleres i en række vidt forskellige og for- grenede litterære positioner og programmer – fra dokumentarisme, bekendelses- litteratur, beatdigtning, minimalisme, systemdigtning og nyrealisme til magisk og fantastisk litteratur.11 Det formelle gennembrud er endvidere kendetegnet ved en åbning i forhold til fortidens litteratur og samtidens populærlitteratur og -kultur. Ingen former og temaer er historisk utidige eller upassende efter opbruddet fra modernismen. Det formelle gennembrud skaber netop en samtidighed mellem litterære strømninger af forskelligt historisk ophav. Det er karakteristisk, at flere af de ældre forfattere fak- tisk senere fået en langt mere direkte adkomst til samtiden end tidligere. I dag viser striben af genlæsninger af efterkrigstidens forfattere, at også litteraturforskningen og kritikken er ved at opdage samtidigheden. Rifbjerg kan slippe ud af modernis- me-konstruktionens “ungdomsfængsel” og den evindelige Kroniske uskyld (1958) og læses som forfatter til senere hovedværker som Arkivet (1967), Livsfrisen (1979), Billedet (1998) og Huset (2000). Peter Seebergs forfatterskab genlæses som et af samtidens betydeligste og fokus kan flyttes fra de første bøger til Seebergs senere readymades og mesterværket, romanen Ved havet (1978).12 Kroppen som medie Thorups tidlige værker, især digtene Indeni – udenfor (1967), Love from Trieste (1969) og I dag er det Daisy (1971) skriver sig ind i opbruddet fra modernismen og det formelle gennembruds problematik. I et af de første digte fra Indeni – udenfor13 lyder det: Digtet er hverken verdens eller samlingens allerbedste, men det har alligevel en poetisk styrke og interesse i sin formulering af forholdet mellem jeg’et, det indre, det ydre og digtets udspring. Digtets vigtige og skelsættende vers er vers 8 af de i alt 12: “strander på min krop”. Herefter nævnes jeg’et ikke længere, og der kan i vers 12 sættes et bogstaveligt punktum for den friktionsfri bevægelse mellem ydre 1 0 og indre illustreret ved digtets mange tankestreger. Mens jeg’ets indre er spejlfrit, og altså ikke konfronterer jeg’et med en stribe ubevidste skygger, frygtindgydende mudderpramme og fødende og skidende maskiner som i modernismens spejlka- binet, er det ydre heller ikke modernistisk truende eller kuende og synet af “intet” standser lige så lidt som de symbolistiske “de luftige vækster” jeg’et. Bevægelsen gennem luft er så gnidningsfri, at den også minder om en bevægelse gennem vand. Men bogstavrimene i ordene “standses” og “strander” skaber en cæsur i digtets be- vægelse, som gør kroppen nærværende og får digtet til at bevæge sig fra sit sjælelige og fysiske lufthav til jordens og kroppens element og billedligt og lydligt strande på et rim. Jeg’et forsvinder i kroppen, “går ind”, men resultatet er hverken rekreation (“at hvile”) eller poetisk skønsang (“at synge”), men det digt, som står på siden som et paradoksalt “uafgrænset indelukke”, som læseren kan opleve igen og igen, og hvorfra den skrivende igen og igen formulerer sig. Selv om digtet står tidligt i sam- lingen giver det alligevel en form for poetologisk svar på en række af de digteriske og mentale problemstillinger, de øvrige digte kredser om. Mens jeg’et svinger mel- lem almagt og afmagt i sin omgang med tid og rum, kommer den skrivende til sig selv i digtet, der billedligt danner et “uafgrænset indelukke”. Den digteriske skrift etablerer en usikker, men dog følelig balance og samtidighed i relationen krop, be- vidsthed og verden, mellem “indeni – udenfor”. Digt og krop hører sammen og kan sammenlignes. Man kan her parallelisere Thorups poetiske udsagn med fænomenologiske fore- stillinger om krop og perception, om det levende mellemværende mellem subjekt og objekt. Kroppen som medie Det er netop som et mellemværende, digtene kommer til og bevæger sig tværs over grænsen mellem indeni og udenfor. Den modernistiske modstilling af bevidst og ubevidst, af krop og psyke, af det indre og det ydre, ja selve splittelses- tænkningen afløses af poetiske formuleringer af den skrivendes handlen og ageren i et mellemværende med verden og med læseren. Bevidsthed er at være hos tingene ved hjælp af en krop, som Maurice Merleau-Ponty forklarer14 i en bestemmelse, der passer godt på en filosofisk problematik i Thorups forfatterskab. Men digtsamlin- gens og forfatterskabets tematik kan også forbindes med det kulturhistoriske sig- nalement af mennesket som Hans-Jørgen Nielsen når frem til i sit essay “Det aper- spektiviske rum” fra Nielsen og den hvide verden: “Mennesket eksisterer ikke mere i et lukket, vertikalt værdihierarki, der bygger på over- og underordning i et sindrigt kassesystem, men i en horisontalt åben verden, der bygger på sideordning.”15 Digt- samlingen skrives ud fra den erfaring, at der, som Nielsen pointerer, ikke findes en formel, et system, der på perspektivisk vis dækker hele virkeligheden og alle sammenhænge. Der findes ikke et, men mange gyldige billeder af virkeligheden i samtidens aperspektiviske rum. Thorup selv understreger, at hendes kunstneriske problematik og inspiration bliver til med den internationale nybrydende og tvær æstetiske kunst fra midten af 1960´erne. Hendes tale til Det danske Akademi i år 2000 markerer på flere måder forudsætningerne for forfatterskabet: S“ Samtidig med at en sofistikeret formbevidsthed udviklede ‘systemdigtningen’ og ‘konkre- tismen’, og der opstod en dynamisk dialog mellem folkelighed og avantgarde. Det var i 60’erne min æstetiske lidenskab blev vakt. Ikke som et mål i sig selv, men som vejen ad “ 1 1 hvilken. Æstetikken som det felt, hvor læseren, beskueren lukkes ind i historien. Møde- stedet mellem afsender og modtager. Jeg bliver aldrig færdig med at udforske romanens formsprog og dens æstetiske muligheder. Jeg bliver aldrig færdig med at eksperimentere. I 60’erne havde jeg nogle kunstneriske oplevelser som gjorde et uudsletteligt indtryk på mig, næsten som en åbenbaring. Nogle radikale kunstneriske greb som i dag er gamle travere, men for mig dengang var det noget aldrig før set, som åbnede op for en Pandoras æske. Den polske instruktør Grotowsky gæstede polyteknisk læreanstalt med sit stykke ‘The Constant Prince’. Det var teater opført som overværede man et helligt ritual under- lagt en slags kirurgisk præcision. Det var første gang jeg så kroppen blive brugt som me- die. Kroppen som medie Og den følelse af at fra nu af er alt muligt inden for kunsten, sidder endnu i mig. Andy Warhols undergrundsfilm ‘Chelsea Girls’ og ‘Empire State Building’, den måde han brugte tiden på (også tilskuerens), at han konkretiserede tiden som en del af kunstværket, gav mig den samme følelse af, at der ikke fandtes nogen grænser eller forbud i den skabende proces, kun kunstnerisk nødvendighed.16 Især tre bestemmelser er vigtige her: dialogen mellem folkelighed og avantgarde, den nye spidsformulerede relation mellem afsender og modtager i det æstetiske felt eller fænomenologiske mellemværende og konkretiseringen af tiden og begrebet om kroppen som medie. p a s s a g e \| 5 6 \| v i n t e r 2 0 0 6 Det er også bemærkelsesværdigt, at det er dramatik og film, som Thorup be- skriver som forudsætningen for sit forfatterskab. Hun har selv både skrevet film- manuskripter og skuespiltekster. De står ikke voldsomt markant i forfatterskabets helhed, men de to genrer er stærkt til stede i romanværkernes syntetiske måde at danne genre på.17 I Ingenmandsland er det meget lange rent dialogiske dramatek- ster for to stemmer, og i romanerne følger en form for psykologisk-motiverende og generaliserende regibemærkninger ofte karakterernes replikker i selve teksten. “– Det der er galt med dig er, at du ikke egner dig til at være voksen. Du hører til i barndommens rige. Moren holdt hans blik fast. Som ung havde hun været en livlig og tiltrækkende kvinde” – lyder med den karakteristiske teknik en replik mel- lem Rene og hans mor i romanen Elskede ukendte (1994).18 Det er kendetegnende for Thorup, at man kan henføre det generaliserende regi til en lidt uvis zone mel- lem moderen og den implicitte fortæller. Der bliver ikke tale om et klart optrukket synsvinkelskift. Replikken kan faktisk placeres både inden i og uden for moderens bevidsthed. Det er både eksempler på, at den generaliserende karakteristik må pla- ceres hos en implicit fortæller, der har en større viden end karakteren, og at gene- raliseringen tilskrives karakteren som i passagerne af jeg-fortælling i Den yderste grænse: “Vi følger dig jo i tankerne, lille Jonna, sagde Betty. Jeg hadede hendes ‘lille Jonna’”.19 Der skabes i Thorups romanværker en art kommunikativt og fysisk flow mellem menneskelige eksistenser, hvor kommunikation og ikke-kommunikation på krop- pens, sprogets og bevidsthedens planer tematiseres og udvikles i fortællingen. Over for en moderne og modernistisk analyse af modsætninger mellem det bevidste og det ubevidste, arbejder Thorup med en modstilling mellem karakterens “indeni” og “udenfor” og med gensidige og skiftende tiltrækning og frastødning imellem men- nesker. Den biseksuelle Jonni er delt imellem sit “indeni” og sit “udenfor”, og det 1 2 lykkes ham hverken at “strande på kroppen” i mødet med Maria eller med den tyske Herbert. Karakterernes forandring er ofte knyttet til en symbiose, der opstår og bringes til ophør, og tilfældigheder spinder deres historier sindrigt ind i hinanden. Symbioser forbinder på godt og ondt en række af personerne i Himmel og helvede, og det kom- plicerer historierne, at symbiosen så langt fra er gensidig. Maria søger den symbio- tiske relation med Jonni, mens Johns kærlighed til Maria er uigengældt symbiotisk. p a s s a g e \| 5 6 \| v i n t e r 2 0 0 6 Maria tænker om forholdet: “Deres ægteskab var Johns drøm, og hun havde ladet som om hun var gået ind i den. Hun havde en anden drøm inden i sig, som havde hindret hende i at opfylde Johns.”20 Forholdet til John skaber en “uvirkelighedsfø- lelse” hos Maria: “Fornemmelsen af at jeg ikke eksisterer, men er en spejling af dig, at jeg er din opfindelse.”21 Maria på sin side ender i en symbiose med musikken og violinen, som John fra første færd så som sin modstander. Karakterernes navne er ofte variationer over de bibelske navne Johannes og Anna. Johannes kan både opfat- tes som Johannes Døberen, hvis dåb af Jesus indleder hans lidelseshistorie, og evan- gelisten Johannes, der i et tilbageblik fortæller hans lidelseshistorie. Anna er jomfru Marias mor, som på sin side låner navn til karakteren Maria. Navnevariationerne peger på, at det er flowet mellem eksistenser og deres symbioser, der sammen med karakterens individualitet interesserer forfatteren. Den mest centrale karakter, Jon- na, får en navnekombination af de to betydningsbærende navne, Johannes og Anna. Hendes status som jeg-fortæller lægger endnu en dimension til tematikken omkring “indeni-udenfor”. Jonna-jeg’ets uventede forsvinden og lige så uventede opdukken i romanernes forløb peger på fortællerens tematiske og narrative delagtighed i pro- blematikken omkring “indeni-udenfor”. Jonna stammer fra en kulturel og mental periferi, et Danmark, der er ved at forsvinde i takt med efterkrigstidens moderni- sering. Hun lever således både på grænsen til det moderne liv og på grænsen til det postmoderne liv, som hun kommer i kontakt med i storbyen. Fra dette mentale sted i et nyt aperspektivisk livsrum kan hun både falde ind i og ud af romanens univers og lade sin fortælling opsluges af andre bevidstheder end sin egen. Hun er ikke en alvidende fortæller, men en fortællermulighed og ligner måske deri sine læsere. Man kan endvidere bemærke, at mentale forelskelser er næsten mere hyppige end de kødelige og fysiske hos Thorup, samtidig med at de tydeliggør, at kommuni- kation er både fysisk og verbal. Replikkerne har fysiske undertekster, og den men- tale og seksuelle orientering følges ofte ikke ad. Når den ene karakter føler en virke- ligheds- og eksistensoplevelse, føler den anden tomhed og fravær. Historien om den biseksuelle Jonni og musikeren Maria fra Himmel og Helvede er her forfatterskabets arketype. p a s s a g e \| 5 6 \| v i n t e r 2 0 0 6 I ultimative situationer som fødsel og død placeres karaktererne i forhold til ele- mentære fysiske, åndelige og sjælelige begreber om “de sultende”, “de udødelige” og “de elskende”. Utopi, dystopi og sammenhæng Utopi, dystopi og sammenhæng Der er oplagt en utopisk tematik i forfatterskabet. Hver af romanerne har deres ud- øvere af utopiske drømme, der i deres forsøg på at realisere utopien aktiverer farlige 1 3 og destruktive sider i sig selv og i omverdenen. Fred i Himmel og helvede udøver ud fra sine hippie-utopier sin grusomme magt, mens Asger i Den yderste grænse ud fra sin politiske utopi om social lighed og frihed ender i vanvid og selvdestruktion. I Elskede ukendte når Karl ud i en vild religiøs fundamentalisme, og i Bonsai fantaserer Stefan i sin dødskamp om en avantgardekunstnerisk utopi, hvor kunst og liv begynder at indgå helt nye forbindelser med hinanden på tankstationerne og rastepladserne. Der er faktisk et helt register af utopier hos Thorup: den kulturelle, den politiske, den religiøse, den erotiske og den kunstneriske. Romanerne viser, hvorledes de alle, idet de søges ført ud i livet, ender som dystopier. Maria og Anna flygter fra den vanvittige Fred, der må formodes at fortsætte med at plage livet af sin kone i en afkrog af ver- den. Asger slutter sin mission om den nye økonomiske verden med sit selvmord. Karl indretter en art interneringslejr til sin utopis ofre, mens Stefan trækker sin eks-kone og datter ind i et ingenmandsland, hvor hverken død eller liv opleves som realiteter. Begrebet ingenmandsland forbindes gentagne gange i romanerne med de realisere- de utopier og deres transformation til dystopier.22 Og netop Ingenmandsland (2003) bliver titlen på Thorups roman om den senile Carl, som holdes indespærret på et plejehjem, der helt lever op til den kontroversielle danske standard. Livsaldrene in- deholder således også deres problematik omkring utopi og dystopi. Barndommen kan både være den bedste og den værste af alle verdener hos Tho- rup, mens alderdommen fører mennesket ind i ingenmandslandet. Moster Marie fra Jonna-serien er dog et eksempel på en karakter, der faktisk oplever opfyldelse i forbindelse med sin død. Hun opdager tilværelsens mønster og gennemfører en slags omvendt syndefald og en tilbagevenden til en forskelsløs tilstand. ‘‘Vi har alle planter og dyr i os lige fra de største til de mindste. Vi kan ikke falde ud af møn- stret,”23 tænker hun. Moster Maries dødsscene indledes med, at hun, der har haft manufakturforret- ning, forsøger at tegne tilværelsens mønster op på et papir som en fin knipling. Utopi, dystopi og sammenhæng Der- næst følger et afgørende gennembrud: “Hun drejede hovedet mod vinduet og så at der ingen forskel var på ude og inde. Hun slog et par streger på papiret for at notere sig denne befriende kendsgerning.”24 Hun beder om et æble og bevæger sig videre gennem et “overdådigt solskin” som på et gyldent alter, videre til en figuration som en falden, sort engel og ned i en hvid, frossen verden på havets bund, og hun udån- der med den barnlige remse på læberne: “‘Marie, Marie Marolle flyv op til Vorherre og bed om godt vejr i morgen,’ sagde hun med høj og tydelig stemme som om hun sad ved et omstillingsbord.”25 Referencerne til syndefaldsmyten og til Andersens eventyr, “Snedronningen” og “Den lille havfrue” er tydelige, og Maries forløsning i barneremsen tilsætter en biedermeier-reference, der tager patos ud af skildringen, men samtidig sikrer, at fortællingen bevarer sin billedkraft. Den usædvanlige samling af billedfragmenter gør hvert af elementerne nærværende for læseren og danner et modstykke til den utopiske erfaring hos andre af Thorups karakterer. Man kan perspektivere de mange utopier, ideen om mønstret og ingenmands- landet i forfatterskabet til Kirstens Thorups bestemmelse af kunstens opgave og funktion i hendes tale til Det danske Akademi i år 2000. Hun erklærer her: 1 4 1 4 Det er en kunstnerisk udfordring at skabe fortællinger i en samtid, der er usammenhæn- gende og også forekommer meningsløs i al sin oppustede rigdom i vores del af verden. En udfordring at skabe sammenhæng ud af en verden opsplittet i et kaos af fragmenter og skabe en betydning ud af det tilsyneladende meningsløse. Tilsyneladende – for bag overfloden findes en endnu større og endnu smukkere verden, som er den kunsten ud- springer af. Og det at tage sin tids udfordringer op er netop den trang og den lyst, der driver kunstværket.26 “ Kunsten udspringer af forestillingen om en større og smukkere verden ikke bag syndfloden, men bag overfloden, lyder det. Kunstens utopi har således med skøn- hed og socialitet at gøre, siger Thorup. Selv om der knytter sig en både forståelse og ømhed til utopisterne hos Kirsten Thorup, dementeres utopierne gang på gang, og utopisterne ender, som vi har set det, med at udøve tvang og vold over for deres medmennesker. Den kunstneriske “trang og lyst” i forhold til det utopiske har således i lige så høj grad sine skygge- som sine skønhedssider. Utopi, dystopi og sammenhæng Thorup er imidlertid også inde på en anden bestemmelse i forbindelse med kunstens opgave: at skabe sammenhæng ud af en verden i et kaos af fragmenter og skabe en betydning ud af “det tilsyneladende meningsløse”. Denne ambition er ikke nødvendigvis utopisk, og det er netop begre- bet om den sammenhæng, som Moster Marie erfarer, der i forfatterskabet danner et modstykke til utopien. De vilde håb om en bedre verden brister og hærger som dystopier, men der kan – muligvis – formuleres en sammenhæng i kaos og menings- løshed. Sammenhængen kan forstås som den enkelte karakters tolkende fortælling om sin livssituation og livsbane: her træder den eksistentielt-narrative dimension frem i romanerne, og den erfaring, der er central for tolkningen af sammenhængen er erfaringen af “indeni – udenfor”. Moster Marie oplever ophævelsen af modsæt- ningen mellem “indeni – udenfor”, mens Jonnas usandsynligheder som jeg-fortæl- ler – hendes opdukken midt i tredjepersonsfortællingen og hendes umotiverede forsvinden – peger på hendes stadige konflikt mellem “indeni” og “udenfor”: enten er hun fordybet “indeni” i sin indre verden, eller også mister hun sig selv “udenfor” i den ydre verdens mangfoldigheder, dvs. romanernes persongalleri. Sammenhængen udvikles og undersøges på individets niveau, og de mange tilfældigheder viser, at individers fortællinger faktisk kan og vil blive spundet ind i hinanden netop af det moderne, fortællende menneske, for hvem tilfældet altid både er en mulighed og en fare, når tomrummet efter den traditionelle landsbykul- tur skal udfyldes. Jonnas og hendes slægtninges liv udspiller sig netop i en kulturel forandring og et opbrud, hvor forskellige livsrum og tidsaldre er blevet samtidige. Sammenhængen opsøges således også på tidens og omverdenens niveau, og i kraft af den postmoderne samtidighed kommer mange typer af fortællestof og re- ferencer til stede. Thorups litterære univers sammenskriver eventyr, personlige do- kumenter som dagbøger, breve og monologer, med scener fra Morten Korch-film, mytestof, dannelsesroman og surrealistiske stills. Verden foreligger i det litterære univers horisonten rundt som en ophobning af vidt forskellige tekster om miljøer, tider og steder. Romanværket bevæger sig ud og ind af overklassemiljø, landsby, hospital, storby, ødemark og underverden. Hos Thorup er omverdenen ikke en hie- 1 5 rarkisk struktur, men et uendeligt netværk af overraskende ind- og udgange til både trivielle og eksotiske miljøer. I Elskede ukendte passerer Rene og Karl en række ste- der og tider på deres rejse frem mod livets midte. Utopi, dystopi og sammenhæng Selv om Rene sandsynligvis for evigt forvises fra universitetets hellige haller, og Karl for bestandig trækkes ud fra sine fattige kår som børnehjemsbarn for at få en opvækst i overklassen, bliver so- ciale skel forceret – frem og tilbage igen. Mange af Thorups karakterer flytter sig fra underklasse til overklasse, fra land og til by og får smertelige erfaringer i bagagen. Omverdenen er som hos Vibeke Grønfeldt mulighedernes verden, men sjældent med lykke og held som individets udkomme. Både de kulturelle, de politiske og de sociale utopier opstår i dette opbrud og denne åbning af verden, hvor forældre- generationens tilværelsestolkninger, hvad enten de er farvet af landbokultur eller borgerlighed, smuldrer, og nye ideer og forestillinger bliver mulige. Opbrud, utopi og dystopi hænger således uløseligt sammen. Med Hans-Jørgen Nielsens formuleringer fra Nielsen og den hvide verden (1968) kan man sige, at der hos Thorup ikke findes et gyldigt perspektiv på virkeligheden, den er bare med sine mange værdipunkter horisonten rundt.27 Thorups tematik omkring utopien og den aperspektiviske sammenhæng kan gen- findes hos en række forfattere fra midten af tresserne og begyndelsen af 1970´erne, og det er faktisk først når man gør Thorup til litteraturhistorisk hovedperson, at man opdager, hvor gennemgribende det utopiske tema er i samtidens litteratur. Ud af utopierne Det er interessant at parallelisere den utopiske tematik hos Thorup med Suzanne Brøggers tilbagevendende kritiske diskussion af de vesterlandske kærlighedsutopi- er lige fra hendes debutessay s Fri os fra kærligheden (1973) til hendes skuespil Efter orgiet (1992). Suzanne Brøgger har en helt anden retorik og en anden genrebrug end Thorup, men tematisk er der ligheder. Hos Suzanne Brøgger er kritikken af bie- dermeierkulturens og kapitalismens kønsliv også en kritik af frigørelsesutopierne. I Kærlighedens veje og vildveje (1976) har et stort essay titlen “Ingenmandsland”. Betydningen af begrebet er en lidt anden. Mens ingenmandslandet hos Thorup er en transformationszone mellem utopi og dystopi, er det hos Brøgger en alternativ topos i forhold til såvel utopierne som den gældende realitet. Brøgger pointerer, at bevægelsen “udi ingenmandsland er ikke-hierarkisk, ikke-eksklusiv, ikke-fal- lisk. Den er multi-dimensional. Den siger nej til at være “den anden”, nej til at være “ovenpå”, “nedenunder” eller “udenfor” […] At bevæge sig ud i ingenmandslandet er at nægte at begrænse skaberkraften til at passe inden for rammerne af en bestemt patriarkalsk livsopfattelse eller ideologi.”28 Som hos Thorup stilles en mulig tilværelsessammenhæng og praksis op som modtræk til utopien: “Fri mig for tidens frigørelsesdogmatik/bind mig til en æble- gren/Jeg er vegetativ/Ueffektiv/Jeg gider ikke arbejde/Jeg gider kun flytte et sten- bjerg eller to/Jeg er gået i kloster for at lave grundforskning/tænke ikke-tanker/stil- le ikke-spørgsmål/og analysere ikke-data./Det er ikke mine meningsytringer I skal lægge mærke til,/men mit vidnesbyrd om at der er et andet liv.”29 I Brøggers univers bliver det fortællerens egen performative insisteren på sit ekspansive vidnesbyrd 1 6 om et andet sammenhængende liv, der strukturerer teksten og værket. Over for alle slags utopier sætter jeg’et sit eksistentielt-narrative projekt, herunder også sine for- søg med at digte sig ind i andre jeg´er og stemmeføringer og en stribe forskellige genrer: brev, interview, strøtanke, essay, novelle, monolog, digt, roman. Derfor fin- der man også i Fri os fra kærligheden et fiktivt brev fra Lou Salomé til en ung kvinde- sagskvinde med følgende pointe: “Hvis du gør noget andet, end det der forventes, så digter du. Og så er grænserne for livsytringer blevet udvidet og definitionen af det kvindelige mere vidtfavnende – forandret.”30 Senere i Brøggers forfatterskab stilles der også spørgsmålstegn ved det selvberoende og selvfremførende individ, og my- terne, der hele tiden har været en stærk undertekst hos Brøgger, træder frem som pejlemærker i en sammenhængsskabende tolkningsproces. Ud af utopierne I det dystopiske skuespil Efter orgiet (1992) om Aids-epidemien skabes der igen forbindelse mellem myte og ritual i en dramaturgi, der også inddrager tilskuerne, idet de forvandles til forestil- lingens græske kor. Forholdet mellem modernitet, myte og utopi er et centralt tema hos flere af Tho- rups jævnaldrende. Jens Smærup Sørensens forfatterskab tematiserer i lighed med Thorups og Brøggers 1960´ernes og 1970´ernes ungdoms- og oprørserfaringer. I debutromanen, At ende som eneboer, 1972, opsluges fortælleren, som Erik Svend- sen31 har vist, af sin egen tidstolkende virksomhed, og myten og forestillingen om utopia forener sig i et vanvid, der sender fortælleren helt ud af livet og nutiden og tilbage i en mandlig urscene: vejen ind i labyrinten på Knossos. Polemikken i Smærup Sørensens forfatterskab mod 1970´ernes marxistiske for- klaringsiver og sociale idealisering deles af Thorup og Brøgger, men får også en særlig version hos en lidt yngre prosaist Bent Vinn Nielsen. Hans roman Opkøb af dødsboer, 1980, former sig som en nøgtern og grum demontering af 1970´ernes so- ciale idealisering af arbejder- og underklasse. Familien Klop skildres med reference til kykloperne i Odysseen som både rå og lovløse. En vis flygtig velstand opnår fami- lien i forbindelse med sønnens talent for opkøb af dødsboer. Men tingenes mentale tilstand bliver ikke bedre af den grund, og snart er de usminkede realiteter igen helt på plads. Den eneste følelse, far Ervin nærer, gælder hans termokande: “Det var ikke nogen stor thermokande, men han havde altid været glad for den. Den var god til at holde kaffen varm og af en eller anden grund havde han altid så godt kunne lide at den var skotskternet.”32 Hvis man afprøver Thorups gamle tese om, at alle mennesker har en fantastisk historie på Vinn Nielsens karakterer, er resultatet mest af alt fantastisk grumt. Der er mindre forsonlighed og mere ironi og moralitet hos Vinn Nielsen33 end hos Thorup, men sammenhængen mellem det kulturelle opbrud og dystopien står centralt i begge forfatterskaber. De mange dystopier i Thorups romaner finder også en pendant i Henrik Bjel- kes galskabsromaner, især Saturn (1974) og Hundrede postkort fra helvede (1980), hvor skriften selv er i en lang svimlende bevægelse mellem utopi og dystopi. Bjelkes tekster kunne være forfattet af en af Thorups utopister, idet karakterernes psyke simpelthen ofte er opløst i et felt af utopiske og dystopiske punkter. Ud af utopierne Hos Vibeke Grønfeldt gør både moderne mytologi, utopi og dystopi deres ind- tog i karakterernes omverden og bevidsthed i kølvandet på nedbrydningen af den traditionelle almuekultur. I hendes storværk, Mulighedernes verden, I-II, 1989, styk- 1 7 ker den kunstneriske skrift en verden sammen af historier, situationer, monologer, dialoger og skriftlige readymades. Den kunstneriske skabelsesproces bliver her et modstykke til den dystopi, som fortællingen gang på gang fremholder. En kvinde- lig fotograf, der er født i år 1900, er en gennemgående figur, som reflekterer over sit livs oplevelser og udvikler sin dystopiske tænkning om mennesket anno 2050. Denne skabning har skinnende ham og en mave som en kemisk fabrik, “ der i løbet af et døgn nedbryder, hvad lys og luft er 1.000.000 år om at hente tilbage. Denne proces vil være det nye menneskes vigtigste funktion. En ny religion kan prædikes for fulde kirker: Den, der æder mest, skal leve videre. I bjerge af grundstoffer. Den, der æder mest, belønnes på jorden. I evighed. “ der i løbet af et døgn nedbryder, hvad lys og luft er 1.000.000 år om at hente tilbage. Denne proces vil være det nye menneskes vigtigste funktion. En ny religion kan prædikes for fulde kirker: Den, der æder mest, skal leve videre. I bjerge af grundstoffer. Den, der æder mest, belønnes på jorden. I evighed. “ De frembragte gasser vil ikke være ufarlige for mindre tilpasningsdygtige skabninger. Men som en særlig retfærdighed vil det vise sig, at de første ikke igen bliver sidst. Først kvæles de hvirvelløse dyr, leddyr, fisk, padder og krybdyr. Tilbage bliver de bevægelige pattedyr. Og når solen forsvinder, har vi midler til at søge en større sol. Og en stadig større sol. Og til at tage den blå måne med os.34 p a s s a g e \| 5 6 \| v i n t e r 2 0 0 6 Thorups kritiske tematisering af utopier og hendes søgende udforskning af en sam- menhæng kan endvidere paralleliseres med bestræbelser hos Dan Turèll, Peter Lau- gesen og Klaus Høeck. Dan Turèlls forsøg på at nærme sig, hvad han kaldte altings betydningsløse sam- menhæng,35 finder sted i den stadige improviserende sideordning af storbystem- mer, situationer, historier og genrer fra Karma Cowboy (1974) og Vangede billeder (1975). De repræsenterer ligesom Thorups Baby og Himmel og helvede forsøg på at udfolde en aperspektivisk storbyskildring, og også i Turèlls forfatterskab ligger der konstant både et forsøg med og en skarp kritik af utopisk tænkning. Karma Cowboy digter om “en ny krops smeltende tid”,36 men udpeger samtidig verdens øjeblik- kelige åbenhed: “‘Hele verden er åben’ og det ER den Endnu/ i denne nat er der ingenting der/ikke kan gøres.”37 Klaus Høecks tematisering af de politiske oprørsbevægelser i værker som Ulrike Maria Meinhof (1977), Topia eller Che Guevara (1978) og Winterreise (1979) tager fat i den marxistiske politiske utopi om social lighed. Jeg’et “forelsker sig håbløst i en ny tabt sag” og påtager sig “forsvaret af Kain”:38 de desperate unges væbnede kamp mod magthaverne. Deres nederlag og fejltagelser bliver mere og mere åben- lyse, selv om jeg’et hos Høeck søger at formulere et forsvar for deres fremtidshåb, leve sig ind i deres fortvivlelse og skildre deres ensomme endeligt. I Thorups roman Den yderste grænse (1987) tiltrækkes den unge drømmer Bien af den hærgende ty- ske byguerilla, og Jonna opdager skrækslagent, at hun måske godt kunne se sig selv i Ulrike Meinhof. Jonna tænker om Ulrike: “Hun kunne bare være blevet hjemme i sin store dyre Hamburg-villa i stedet for at gå i krig mod magthaverne. Hun havde overtrådt samfundets allerhelligste tabu: magtens urørlighed. Og det skulle selvføl- gelig straffes med døden. For at alt kunne blive som før og intet skulle forandres. Men hvad nu hvis man kom til at se sig selv (eller en del af sig selv) i hende? Den pæne, dygtige pige, der havde stirret sig blind på magten og ladet sig indfange i magtens ingenmandsland, hvor livet ikke mere var et alternativ, hvor der ikke var 1 8 andet tilbage end at kæmpe til døden. Den pæne, dygtige pige, der havde gjort sig til magtens bagside, dens skygge. p a s s a g e \| 5 6 \| v i n t e r 2 0 0 6 Og var blevet fremmed for alt menneskeligt.”39 Det er en sådan fremmedfølelse, jeg’et hos Høeck konfronterer sig med, på sin vej ned gennem Tyskland, den kuldsejlede katedral med “vredens og nattens sarkofag”40. De følgende samlinger af Høeck, Canzone. Digte fra Nørrebro (1981) og Metamor foses (1983) tager afsked med den politiske, utopiske tænkning og dens dystopiske realitet, og lader “poesiens metode”, som det lyder i Winterreise, åbne omverdenen og skabe jeg’ets nøgterne tilstedeværelse i en uforklarlig og paradoksal sammen- hæng af ånd og realitet. Den utopiske og dystopiske tematik er ligeledes til stede i Peter Laugesens digt- ning, hvor skriftens konkrete åbnende og legende forløb danner et modstykke til samfundstilstandens og den politiske tænknings stivnen i magt og selvretfærdighed. “Den ondskab/der bunder i at man er den/der har ret/ at man er den sunde rigtige sande/ det er nok bare ikke den mest almindelige/men den eneste på alle planer/ fra sovekammer skriveborg og våbensal/fra talerstol og slagmark” – lyder det ek- sempelvis i Konstrueret situation (1996). En i positiv forstand anarkistisk tilgang til omverden og medmenneske præger Laugesens digtning og er måske et fællestræk for hele generationen fra slutningen af 1960´erne og begyndelsen af 1970´erne. Thorups kunstneriske pointe og drivkraft er som omtalt ideen om, at alle men- nesker, hver og en, har en fantastisk historie. Fortællingerne ligger som Hans-Jørgen Nielsens “værdipunkter” overalt i mennesket, kulturen og sproget og kan opdages og udfoldes. En litteraturhistorisk hovedperson Noter 1 Jf. Hans Hauge: “Ti teser om en post-national dansk litteraturhistorie”, i Kritik 133 (1988). 2 Gode introduktioner til diskussionen om litteraturhistorieskrivningens problematik findes i Lise Busk Jensens artikel “Litteraturhistoriens nytte”, i Poetik 6, 1996, og i Mads Rosendahl Thomsens og Svend Erik Larsens antologi Litteraturhistoriografi (Århus, 2005). Mads Rosen- dahl Thomsens afhandling Kanoniske konstellationer. Om litteraturhistorie, kanonstudier og 1920’ernes litteratur (Odense 2003), analyserer selve kanonbegrebet og kanondannelsen ud fra 1920´ernes litteratur. 3 4 Jf. Paul de Man: “Literary History and Literary Modernity”, Blindness & Insight (London, 1983). Jeg skelner meget forsøgsvis og heuristisk mellem tre romanformer i den nyere litteratur ud fra bestemmelser af forholdet mellem individ, tid og rum: den moderne roman, der strukturerer tid og rum omkring det menneskelige subjekt og gør individet til den centrale figur i det lit- terære univers’ betydningsdannelse (et oplagt eksempel er Tage Skou-Hansens Holger Mikkel sen-serie), den eksistentielt-narrative roman, der centrerer sig omkring narrationen og flytter fokus fra individet til fortællingen, der bliver den overgribende figur i forhold til tid og rum (et oplagt eksempel er Svend Åge Madsens Syv aldres galskab) og den konstruktivistiske roman, der strukturerer sig omkring det tilfældige spil af mulige konstellationer mellem figurer, tider, rum og stykker af fortælling (et oplagt eksempel er Kirsten Thorups tidlige værker, Vibeke Grønfeldts Mulighedernes land, 1989, Peer Hultbergs Byen og verden, 1992, eller Helle Helles debut Eksem pel på liv, 1993). Den moderne roman fortæller individets historie i tid og rum, den eksisten- tielt-narrative roman fortæller om fortællingen, der fortæller individet, tiden og rummet. Den konstruktivistiske roman igangsætter tilfældets spil af muligheder i skikkelse af individ, tid, rum og fortælling, idet tilfældet samtidig peger på læseren som betydningsdanner. Det er kendeteg- nende for Kirsten Thorup og andre prosaister i det formelle gennembrud, at de har arbejdet med de tre romantyper. De tre romantyper er omtalt i artiklen, “Det formelle gennembrud”, Danske digtere i det 20. århundrede, bd. III (København, 2000), 549ff. 5 Johannes Fibiger og Gert Lütken: Litteraturens veje (København, 2003), 381. 6 Jens Anker Jørgensen m.fl. (red.): Hovedsporet. Dansk litteraturs historie (København, 2005), 630. 6 Jens Anker Jørgensen m.fl. (red.): Hovedsporet. Dansk litteraturs historie (København, 2005), 630. 8 Jon Helt Haarder kommenterer Skyum-Nielsens biografiske læsning af romanen og betegner den som en destruktiv sproghandling i artiklen, “Lugtede der hjemme hos Villy Sørensen? 10 I den afsluttende artikel i bind III, Danske digtere i det 20. århundrede (København, 2000), 9 Jørgen Leth: Det gør ikke noget (København, 2006), 19. En litteraturhistorisk hovedperson Hvis man som litteraturhistorieskriver sætter Thorup ind som en hovedperson i den nyere litteratur bliver det tydeligt, hvor fremtrædende romaneksperimenter og for- søg på at nå “den yderste grænse” i vilde og store fortællinger faktisk er i den nyere danske litteratur fra Thorup og Vibeke Grønfeldt til Henrik Bjelke, Bent Vinn Niel- sen og Jens Smærup Sørensen. Også genrekrydsene mellem lyrik, prosa, dramatik og essay istik er tydelige, når man læser Thorup i sammenhæng med Brøgger, Turèll, Høeck og Laugesen. Dansk litteratur bliver med Thorup som en hovedperson en mere livlig og sammensat størrelse med stærke og klare inspirationer i europæisk og amerikansk litteratur. Tematikken omkring opbruddet fra den gamle landbokultur, som er så stærkt i prosaen i det formelle gennembrud, får også flere dimensioner og opbruddets utopiske og dystopiske problematik bliver tydelig og både politiske, erotiske, kulturelle og religiøse utopier træder frem. Thorups danske beslægtede er utvivlsomt Herman Bang, men hun har – som hun selv pointerer – stærke forudsætninger i europæisk og amerikansk pop- og avantgar- dekunst i lighed med mange andre i generationen fra midten af 1960´erne. Thorups indlevede opgør med den utopiske tænkning gennemspilles i hendes store romanværk og en forestilling om menneskets mulighed for performativt at skabe en eksistentielt- narrativ sammenhæng bliver mere og mere fremtrædende, samtidig med at omver- denen antager karakter af at være et åbent netværk af muligheder og faldgruber, en fysisk og mental realitet, som karaktererne på godt og ondt hører hjemme i. 1 9 Måske vi netop i det nye årtusind, hvor tro og utopi igen så stærkt bindes sammen af forskellige religioner, har nye forudsætninger og et større behov for at læse eksempel- vis Thorups mesterlige Elskede ukendte om utopiens forvandling til dystopi og om en grundlæggende menneskelig længsel efter helt enkelt at møde det andet menneske. Den kritiske refleksion over den utopiske tænkning, som kunsten fra midten af 1960´erne og begyndelsen af 1970´erne har foretaget, er nutidens udfordring til hver og en. Noter Noter Om biografiske sproghandlinger og retten til det private”, Kristin Ørjasætter, Henrik Skov Nielsen og Stefan Kjerkegaa rd: Mellem autofiktion og selvfremstilling (Århus, 2006). 9 Jørgen Leth: Det gør ikke noget (København, 2006), 19. 2 0 diskuterer jeg en ny periodisering af litteraturen fra midten af 1960´erne til nutiden under overskriften “Det formelle gennembrud”. diskuterer jeg en ny periodisering af litteraturen fra midten af 1960´erne til nutiden under overskriften “Det formelle gennembrud”. Den aktuelle diskussion om selvbiografiske dobbeltkontrakter, som Poul Behrendts bog Dobbelt kontrakten. En æstetisk nydannelse (København, 2006) har rejst, viser lige ind i kernen af det for- melle gennembruds problematik om den nyere litteraturs spidsformuleringer af relationen mel- lem den læsende, den skrivende og verden. Vigtig i diskussionen af den nye forfatterfigur er også Jon Helt Haarders forskning i forfatterfigurens forandringer, blandt andet offentliggjort i artik- len, “Det forhold, vi havde til forfatteren”, Norsk Litteraturvitenskapelig Tidsskrift 1 (2005). 11 Jf. Jørn Erslev Andersens læsning af Ved havet i Anne-Marie Mai, Maria Davidsen og Jørgen Aabenhus (red.): Peter Seeberg & Hald. At åbne arkivet (Odense, 2005). 12 Kirsten Thorup: Indeni – udenfor (København, 1967), 11. 13 Jf. Maurice Merleau-Ponty: Kroppens fænomenologi (København, 1994), 92. 14 Hans-Jørgen Nielsen : “Det aperspektiviske rum”, i Nielsen og den hvide verden (København, 1968), 20. 15 Kirsten Thorup: “Vi er landet på en anden planet. Tale til Det danske Akademi”, Aktuelt, 4.12.2000. 16 Man kan bruge Jean-Marie Schaeffers begreb syntetisk generecitet om Thorups romanværker. 17 A n n e - M a r i e M a i \| I n d e n i – u d e n f o r Jf. Kirsten Thorup: Elskede ukendte (København, 1994), 47. 18 Kirsten Thorup: Den yderste grænse (København, 1987) bd. 2, 443. 19 Kirsten Thorup: Den yderste grænse (København, 1987) bd. I, 34. 20 Kirsten Thorup: Den yderste grænse (København, 1987) bd. I, 34. 20 p y g ( ø , ) , Ibid., 343. 21 Elisabeth Møller Jensen har understreget forfatterskabets dystopiske og apokalyptiske tematik i sit forfatterskabsportræt i Nordisk Kvindelitteraturhistorie, bd. IV, 28-35. 22 Thorup 1987, 459. Ibid., 459. 23 24 Thorup 1987, 459. 23 Thorup 1987, 459. 23 Ibid., 459. 24 Ibid., 462. 25 Ibid., 462. 25 Kirsten Thorup: “Vi er landet på en anden planet. Tale til Det danske Akademi”, i Aktuelt 4.12.2000. 26 Jf. Hans-Jørgen Nielsen: “Det aperspektiviske rum”, i Nielsen og den hvide verden (København, 1968), 20. Noter 27 Suzanne Brøgger: Kærlighedens veje og vildveje (København, 1975), 164-65. 28 Suzanne Brøgger: Kærlighedens veje og vildveje (København, 1975), 164-65. Ibid., 165. 28 29 Ibid., 165. 29 Ibid., 165. 29 Ibid., 105. 30 Ibid., 105. 30 Erik Svendsen: “Jens Smærup Sørensen”, i Anne-Marie Mai (red.): Danske digtere i det 20. år hundrede (København, 2000), bd. III, 188. 31 hundrede (København, 2000), bd. III, 188. Bent Vinn Nielsen: Opkøb af dødsboer (Viborg, 1980), 227. 32 Bent Vinn Nielsen: Opkøb af dødsboer (Viborg, 1980), 227. 32 Frederik Stjernfelts læsning af Bent Vinn Nielsens forfatterskab i Danske digtere i det 20. århund rede understreger netop den moralske dimension i forfatterskabet. 33 Vibeke Grønfeldt: Mulighedernes verden (København, 1989), bd. II, 300. 34 Dan Turèll: Karma Cowboy (København, 1974/1999), 191. 35 Dan Turèll: Karma Cowboy (København, 1974/1999), 191. 35 Ibid., 305. 36 Ibid., 305. 36 Ibid., 273. 37 Klaus Høeck: Winterreise (København, 1979), 187. 38 Thorup 1987, 67. 39 Thorup 1987, 67. 39 2 1 2 1 Høeck 1979, 23. 40
https://openalex.org/W2390640775	https://nottingham-repository.worktribe.com/file/792443/1/1-s2.0-S1567724916300423-main.pdf	English	null	Analysis of Mitochondrial haemoglobin in Parkinson's disease brain	Mitochondrion	2,016	cc-by	7,309	Abbreviations: Pcd5j, Purkinje Cell Degeneration mouse strain 5j; PM, post mortem; HbA, alpha haemoglobin; HbB, beta haemoglobin; BSA, bovine serum albumin; TBS-T, tris buffered saline–tween; COXIV, cytochrome c oxidase IV; HSP90, heat shock protein 90; VDAC, voltage dependent anion channel; NDUFS3, NADH dehydrogenase ubiquinone FeS; SMAC, second mitochondrial activator of caspase. ⁎ Corresponding author. E-mail address: lisa.chakrabarti@nottingham.ac.uk (L. Chakrabarti). a r t i c l e i n f o Article history: Received 1 October 2015 Received in revised form 29 April 2016 Accepted 3 May 2016 Available online 12 May 2016 Keywords: Parkinson's disease Mitochondria Haemoglobin Gender Cerebellum Hypoxia Article history: Received 1 October 2015 Received in revised form 29 April 2016 Accepted 3 May 2016 Available online 12 May 2016 Mitochondrial dysfunction is an early feature of neurodegeneration. We have shown there are mitochondrial haemoglobin changes with age and neurodegeneration. We hypothesised that altered physiological processes are associated with recruitment and localisation of haemoglobin to these organelles. To conﬁrm a dynamic localisation of haemoglobin we exposed Drosophila melanogaster to cyclical hypoxia with recovery. With a single cycle of hypoxia and recovery we found a relative accumulation of haemoglobin in the mitochondria compared with the cytosol. An additional cycle of hypoxia and recovery led to a signiﬁcant increase of mitochondrial haemoglobin (p b 0.05). We quantiﬁed ratios of human mitochondrial haemoglobin in 30 Parkinson's and matched control human post-mortem brains. Relative mitochondrial/cytosolic quantities of haemoglobin were obtained for the cortical region, substantia nigra and cerebellum. In age matched post- mortem brain mitochondrial haemoglobin ratios change, decreasing with disease duration in female cerebellum samples (n = 7). The change is less discernible in male cerebellum (n = 18). In cerebellar mitochondria, haemoglobin localisation in males with long disease duration shifts from the intermembrane space to the outer membrane of the organelle. These new data illustrate dynamic localisation of mitochondrial haemoglobin within the cell. Mitochondrial haemoglobin should be considered in the context of gender differences characterised in Parkinson's disease. It has been postulated that cerebellar circuitry may be activated to play a protective role in individuals with Parkinson's. The changing localisation of intracellular haemoglobin in response to hypoxia presents a novel path- way to delineate the role of the cerebellum in Parkinson's disease. © 2016 The Authors. Elsevier B.V. and Mitochondria Research Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). nalysis of Mitochondrial haemoglobin in Parkinson's disease brain a University of Nottingham, Faculty of Medicine, SVMS, Sutton Bonington Campus, LE12 5RD, England, UK b Division of Animal Sciences, School of Biosciences, Sutton Bonington Campus, LE12 5RD, England, UK Contents lists available at ScienceDirect Contents lists available at ScienceDirect and Mitochondria Research Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). http://dx.doi.org/10.1016/j.mito.2016.05.001 1567-7249/© 2016 The Authors. Elsevier B.V. and Mitochondria Research Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by Mitochondrion 29 (2016) 45–52 Mitochondrion 29 (2016) 45–52 Contents lists available at ScienceDirect 1. Introduction which is also found accumulated in Lewy bodies – the pathological hall- mark of this disease (Spillantini et al., 1997). Recently it has been sug- gested that mitochondrial haemoglobin in neurons is reduced by an interaction with alpha-synuclein, providing further evidence that mito- chondrial haemoglobin is important when thinking about Parkinson's disease (Yang et al., 2016). Altered haemoglobin levels affect the ex- pression of genes involved in mitochondrial function, demonstrating a link between mitochondria and haemoglobin (Chuang et al., 2012). Hypoxia, due to reduced blood perfusion, is linked to ageing (Daulatzai, 2013). This is particularly relevant to brain ageing and neu- rodegeneration, as the brain is most sensitive to reduced oxygen levels. Hypoxia affects mitochondrial structure and function as well as intracel- lular haemoglobin expression (Gleixner et al., 2012). Haemoglobin (HBA2) expression changes are now also implicated in preclinical prion disease, and Hbb has been found to interact with subunits of ATP synthase in a study of Multiple Sclerosis (Xerxa et al., 2016; Brown et al., 2016). Parkinson's disease is most frequently a sporadic occurrence with the major risk factor being advanced age. Although a collection of symp- toms deﬁne this disease, cardinal features correspond to a movement disorder including a resting tremor, rigidity and difﬁculties with gait and balance. Despite substantial efforts, the cause of idiopathic Parkinson's disease still remains unknown and treatment offers only symptomatic relief over a limited period of time. In recent years Parkinson's disease research has centred increasingly around the mito- chondrial organelle. Mitochondrial dysfunction is a feature of Parkinson's disease with many aspects of this organelle having been im- plicated in disease generation and progression (Chu, 1802). Parkinson's disease is associated with mutations of the protein alpha-synuclein Our work on the process of neurodegeneration has revealed mito- chondrial dysfunction as an early event (Chakrabarti et al., 2010). We want to know how the mitochondrial milieu is adjusted during the pe- riod of early neural decline when symptoms are just becoming F. Shephard et al. / Mitochondrion 29 (2016) 45–52 46 Table 1 Age at death varied from 58 to 87 years for Parkinson's disease brains, non- degenerative human control brains were age matched to within three years of Parkinson's disease samples, see Table 1. Early cases of Parkinson's are selected Braak 3 or 4, late cases are all Braak 5 and 6. Young onset cases are also Braak 5 or 6. Braak staging was performed ac- cording to published classiﬁcation (Alafuzoff et al., 2009). apparent. In order to do this we have utilised a classic mouse model of neurodegeneration, the pcd5j mouse, and examined its mitochondrial proteome (Chakrabarti et al., 2006). The pcd5j mouse undergoes a spon- taneous degenerative process just after weaning. In fact, within 30 days of weaning, the cerebellum of this strain is nearly devoid of Purkinje Cells. The degeneration is cell speciﬁc and autonomous and is associated with an increase in mitophagy in presymptomatic cerebellum. We showed that there are differences in the mitochondrial proteome in the pcd mouse when compared to controls. One of the proteins we iden- tiﬁed as changed is haemoglobin. We demonstrated that mitochondrial haemoglobin levels change in post-mortem Parkinson's disease brain (Shephard et al., 2013). The identiﬁcation of haemoglobin in mammalian cells, other than erythrocytes, was initially ascribed to blood contamination. However, it has been demonstrated that haemoglobin is detectable in neurons in vitro after the accepted lifespan of cultured erythrocytes has expired (Carlson et al., 2008). A growing body of evidence now demonstrates that haemoglobin is present in a range of cell types, in many of these cases the effect of intracellular haemoglobin is postulated to be protec- tive. It has been shown prior to our work that hypoxia upregulates haemoglobin in alveolar epithelial cell lines (Grek et al., 2011). α- and β-haemoglobin (HbA and HbB respectively) respond to H2O2 induced oxidative stress by increasing their levels in HEK293 and hepatic cell lines (Liu et al., 2011). Haemoglobin mRNA and protein have been de- tected in rat, mouse and human brain (Biagioli et al., 2009). Our localisation of haemoglobin to the mitochondrial compartment has now been conﬁrmed as a protective mechanism in circulating leuko- cytes (Shephard et al., 2013; Brunyanszki et al., 2015). In neurons eryth- ropoietin induction has now been shown to increase mitochondrial function with increasing intraneuronal haemoglobin, in this case a re- versal of memory impairment is recorded (Horng et al., 2015). Table 1 A study of the mitochondrial proteome in Multiple Sclerosis cortex also ﬁnds beta haemoglobin expression is altered in this neurodegenerative dis- ease (Biagioli et al., 2009). Ethical permission for the study was obtained through the brain banks from which the tissue was obtained. The entire study was also reviewed by our local ethics board. Fig. 1. A. Mitochondrial HbA migrates from the intermembrane space to the outer membrane in affected human male cerebellum. Mitochondrial samples were sub- fractioned to allow examination of HbA localisation within the organelle, a representative gel is shown for each gender and control (total n = 8). The male Parkinson's disease brain demonstrated a shift in HbA localisation from the intermembrane space to the outer membrane fraction. This was not seen in the control or female Parkinson's brain mitochondria. Levels of HbA in the IMS were quantiﬁed in control and PD samples for both male and female patients (n = 2 for each), using Image J. Please see Supplemental Fig. 7 for all gel images. Levels of HbA in the IMS were signiﬁcantly decreased in male PD compared with male control (p = 0.028 using unpaired two-tailed t-test). No signiﬁcant change in female PD compared with female control (p $ amp $gt; 0 05 unpaired two tailed t test) Mito fr mitochondrial fraction Some studies already correlate haemoglobin levels with Parkinson's disease, simultaneously commenting upon the brain iron accumulation in Parkinson's and other neurodegenerations (Abbott et al., 2012; Savica et al., 2009). Since the majority of iron in the body is haemoglobin de- rived there may be also some connection with observations that serum and brain iron levels change in Parkinson's disease (Pichler et al., 2013). Potential interactions between haemoglobin and neurode- generation are supported by the association of a functional polymor- phism in the haemoglobin binding protein haptoglobin which inﬂuences susceptibility to idiopathic Parkinson's disease (Costa- Mallen et al., 2008). Much of the ﬂuctuation in haemoglobin levels could be attributed to observed, yet unexplained anaemia in older indi- viduals (Gaskell et al., 2008). This study set out to understand localisation of haemoglobin to mi- tochondria and examine the levels of mitochondrial haemoglobin in Parkinson's disease post-mortem brains. We want to understand whether levels of mitochondrial haemoglobin change in Parkinson's disease compared with controls. Table 1 Table 1 Parkinson's disease and matched control brain samples information. Post mortem interval (PMI) is stated in hours after death. Alpha – synuclein scoring by Parkinson's Brain Bank is in- cluded where available (not applicable NA, for control brains) the alpha-synuclein data for these tissues are visualised in Supplementary Fig. 5. PD1 is early onset cases before age of 60, PD2 is early disease with onset after 60 years, PD3 long disease duration onset after 60 available for this individual and the particular brain region, red boxes indicate the sample was not in- cluded. All tissues were obtained from Human Tissue Authority approved, Nottingham Health Science Biobank (Nottingham University Hospitals NHS Trust) and Parkinson's UK Brain Bank (Imperial College London) Table 1 Parkinson's disease and matched control brain samples information. Post mortem interval (PMI) is stated in hours after death. Alpha – synuclein scoring by Parkinson's Brain Bank is in- cluded where available (not applicable NA, for control brains) the alpha-synuclein data for these tissues are visualised in Supplementary Fig. 5. PD1 is early onset cases before age of 60, PD2 is early disease with onset after 60 years, PD3 long disease duration onset after 60 available for this individual and the particular brain region, red boxes indicate the sample was not in- cluded. All tissues were obtained from Human Tissue Authority approved, Nottingham Health Science Biobank (Nottingham University Hospitals NHS Trust) and Parkinson's UK Brain Bank (Imperial College London). perial College London). F. Shephard et al. / Mitochondrion 29 (2016) 45–52 47 Research Ethics Committee for Wales ref. 08/MRE09/31+5. The tissue collection and procedures at the Nottingham University Hospitals Biobank have been ethically approved by the Greater Manchester Na- tional Research Ethics Service. This study was granted speciﬁc ethics ap- proval by both of the ethics committees serving the biobanks and also by the local ethics committee at the School of Veterinary Medicine and Science at the University of Nottingham. Tissues from human cere- bellum, cortex and substantia nigra were used for mitochondrial isola- tions and were frozen at the time of post mortem (PM). Material used for immunohistochemistry was ﬁxed in PFA at the time of PM. PM delay varied from a minimum of 2 h after death to a maximum of 6 days. Diagnoses of Parkinson's disease were conﬁrmed at PM. Table 1 We chose to examine mitochondrial haemoglobin levels in the substantia nigra - as the region of the brain where neuronal loss is most evident. We sampled cerebellar mitochon- dria for motor coordination related changes and also the cortex since it is least frequently associated with Parkinsonism. Fig. 1. A. Mitochondrial HbA migrates from the intermembrane space to the outer membrane in affected human male cerebellum. Mitochondrial samples were sub- fractioned to allow examination of HbA localisation within the organelle, a representative gel is shown for each gender and control (total n = 8). The male Parkinson's disease brain demonstrated a shift in HbA localisation from the intermembrane space to the outer membrane fraction. This was not seen in the control or female Parkinson's brain mitochondria. Levels of HbA in the IMS were quantiﬁed in control and PD samples for both male and female patients (n = 2 for each), using Image J. Please see Supplemental Fig. 7 for all gel images. Levels of HbA in the IMS were signiﬁcantly decreased in male PD compared with male control (p = 0.028 using unpaired two-tailed t-test). No signiﬁcant change in female PD compared with female control (p $_amp_$gt; 0.05, unpaired two-tailed t-test). Mito fr – mitochondrial fraction, OM – outer membrane, IMS – inter membrane space, IM – inner membrane, M - matrix. B. Cycles of hypoxia result in increased hb in Drosophila mitochondrial fractions. Mito- chondrial/cytoplasmic Hb levels determined using Western blotting, normalised to beta- actin. Hypoxia conditions: 2.5% O2 30 min 25 °C followed by normoxia 30 min 25 °C (mid- dle bar) 2.5% O2 30 min 25 °C followed by normoxia 30 min 25 °C × 2 (right hand bar). 40– 100 ﬂies per condition. n = 3, * p $_amp_$lt; 0.05 (1 tailed t-test). 2.4.1. Immunohistochemistry Immunohistochemistry was performed as previously described (Shephard et al., 2013). Slides were visualised using a Pannoramic P250 scanner (3D Histech) with Lumencor Spectra Light Engine illu- mination source. Images were captured using a PCO Edge 5MP sCMOS monochrome camera and Carl Zeiss Plan ApoChromat 20x/ 0.8NA lens. The ﬁlter conﬁguration used is shown in Supplementary Table 4. 3. Results 3.1. Mitochondrial HbA migrates from the intermembrane space to the out- er membrane in affected male cerebellum 3.1. Mitochondrial HbA migrates from the intermembrane space to the out- er membrane in affected male cerebellum Sub-mitochondrial fractions were prepared from cerebella and in- terrogated for HbA content (Fig. 1A). Fractions were veriﬁed using ap- propriate antibodies SMAC and NDUFS3 for intermembrane space and inner membrane respectively. Control (male 80 years) and affected (male, 82 years, 18 years of disease) cerebella mitochondria were pre- pared and subfractionated. A female (85 years old, 18 years of disease) sub-fractionated sample is included for comparison. HbA content of the fractions was determined by western blotting. We found HbA to be present in the inter membrane space of the control sample which corroborates earlier ﬁndings (Shephard et al., 2013). In the affected sample there is little evidence of HbA in the inter membrane space. 2.1. Human tissues Human brain sections and frozen brain samples were obtained from, Human Tissue Authority approved, Nottingham Health Science Biobank (Nottingham University Hospitals NHS Trust) and Parkinson's UK Brain Bank (Imperial College London). The tissue banks granted us use of the tissue as end users. The Parkinson's tissue bank has approval from the F. Shephard et al. / Mitochondrion 29 (2016) 45–52 48 Representative gel images for mitochondrial/cytoplasmic Hb ratios - normalised to beta actin, were determined by Western blotting. COXIV antibody was utilised for quality cont ochondrial versus cytoplasmic fractions. To summarise data, samples were grouped into age ranges (1 $_amp_$lt; 70, 2 70–79, 3 ≥80) and separated by sex and diagnosis ty rol, late PD: late stage disease diagnosed over the age of 60, early PD: early stage disease diagnosed over the age of 60, young PD: early onset disease diagnosed under the age alues were visualised as boxplots. Boxplots show the median (line), interquartile range (box) and whiskers extend to 1.5× the Inter quartile range. Extreme values beyond t ers are shown as circles. Ctx – Cortex, Cer – Cerebellum, SN – Substantia nigra. C M - cytoplasmic and mitochondrial fractions extracted from the same sample. Mito/cyto – ra ochondrial Hb compared with cytoplasmic Hb. F-female, M – male. Full densitometry dataset is provided in Supplementary Table 2. Fig. 2. Representative gel images for mitochondrial/cytoplasmic Hb ratios - normalised to beta actin, were determined by Western blotting. COXIV antibody was utilised for quality control of mitochondrial versus cytoplasmic fractions. To summarise data, samples were grouped into age ranges (1 $_amp_$lt; 70, 2 70–79, 3 ≥80) and separated by sex and diagnosis type (control, late PD: late stage disease diagnosed over the age of 60, early PD: early stage disease diagnosed over the age of 60, young PD: early onset disease diagnosed under the age of 60). Values were visualised as boxplots. Boxplots show the median (line), interquartile range (box) and whiskers extend to 1.5× the Inter quartile range. Extreme values beyond the whiskers are shown as circles. Ctx – Cortex, Cer – Cerebellum, SN – Substantia nigra. C M - cytoplasmic and mitochondrial fractions extracted from the same sample. Mito/cyto – ratio of mitochondrial Hb compared with cytoplasmic Hb. F-female, M – male. Full densitometry dataset is provided in Supplementary Table 2. 2.3. Immunoblotting Western blotting was conducted as previously described (Shephard et al., 2013). Primary antibodies used were: Hba sc-21005 (Santa Cruz) 1:1000; and ab102758 (Abcam) 1:500 – for ﬂy hypoxia; Hbb sc-22718 (Santa Cruz) 1:1000; COXIV ab16056 (Abcam) 1:1000; beta-actin ab8227 (Abcam) 1:4000; HSP-90 ab13495 (Abcam) 1:500; VDAC/ Porin ab15895 (Abcam) 1:2000; NDUFS3 ab110246 (Abcam) 1:1000; SMAC/Diablo ab8115 (Abcam) 1:1000; dilution in 3% (w/v) BSA in TBS-T. Band densities were measured using Image J and samples were normalised to beta-actin. Using the normalised values the ratio of mito- chondrial/cytoplasmic HbA and HbB were calculated. Data were analysed using the R statistical package http://www.r-project.org/ see supplemental data for script used to generate Fig. 3. 2.2. Mitochondrial isolation to the following hypoxic conditions: 2.5% O2 30 min 25 °C followed by normoxia 30 min 25 °C or 2.5% O2 30 min 25 °C followed by normoxia 30 min 25 °C × 2. Mitochondria were isolated as described above and subjected to immunoblotting. Mitochondria were prepared as previously described (Shephard et al., 2013). The quality of the crude fractions was conﬁrmed using standard Western blotting techniques with nuclear, mitochondrial and cytoplasmic markers (Histone H3, ab 1791 (Abcam); COX IV ab16056 (Abcam); and HSP-90 ab13495 (Abcam) respectively). Sub-fractions were conﬁrmed using outer membrane, inner membrane and inter- membrane space markers. 2.4. Hypoxic treatment Drosophila melanogaster were maintained using standard tech- niques. Mixed populations of approx. 100 wild type ﬂies were subjected F. Shephard et al. / Mitochondrion 29 (2016) 45–52 49 Fig. 3. Scatterplots of mitochondrial/cytoplasmic Hb ratio versus disease duration in years. Scatterplots of the ratio of mitochondrial/cytoplasmic heamoglobin A and B in three tissues. Patient samples are split by sex and a linear model used to ﬁt a trendline. The shaded areas show the 95% conﬁdence limits of the ﬁtted line. Calculations and plotting was conducted using ggplot in R (see supplemental R script which was used for generating ﬁgures). Orange circles (female), blue circles (male). Yr – years. Ctx – Cortex, Cer – Cerebellum, SN – Substantia nigra. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) 49 F. Shephard et al. / Mitochondrion 29 (2016) 45 52 F. Shephard et al. / Mitochondrion 29 (2016) 45 52 Fig. 3. Scatterplots of mitochondrial/cytoplasmic Hb ratio versus disease duration in years. Scatterplots of the ratio of mitochondrial/cytoplasmic heamoglobin A and B in three tissues. Patient samples are split by sex and a linear model used to ﬁt a trendline. The shaded areas show the 95% conﬁdence limits of the ﬁtted line. Calculations and plotting was conducted using ggplot in R (see supplemental R script which was used for generating ﬁgures). Orange circles (female), blue circles (male). Yr – years. Ctx – Cortex, Cer – Cerebellum, SN – Substantia nigra. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 4. The cellular distribution of HbB appears changed in female cerebellum with disease duration. HbB and COXIV antibodies were utilised to visualise the physical location of mitochondria and HbB in the cerebellum of female individuals with different disease durations. It appears that with longer disease duration the overall quantity of COXIV is reduced and the location of HbB shifts in the longest disease course from in and around Purkinje cells observed in controls, to the granule cell layer in sections from 18 years disease duration. DAPI is used to stain nuclei. Filter sets used are detailed in the methodology. Fig. 4. The cellular distribution of HbB appears changed in female cerebellum with disease duration. 4. Discussion Our observations on the dynamic location of Hb led us to ask what could be the drivers of this proteins mobility within the cell and its or- ganelles (Shephard et al., 2013). An obvious place to start is the manip- ulation of oxygen saturation and or delivery as has been done previously in vitro (Grek et al., 2011). We devised an experimental regime where we subject Drosophila to hypoxia. In order to emulate longer term ﬂuc- tuations in oxygen availability we exposed ﬂies to single or double hyp- oxic events, each followed by an equal period of recovery. Examination of mitochondrial fractions isolated from the ﬂies revealed a signiﬁcant increased ratio of mitochondrial Hb in response to multiple cycles of hypoxia and recovery. We suggest that this is indicative of a possible protective mechanism whereby Hb is sequestered to the mitochondrial organelle in conditions of hypoxia, perhaps in order to maintain an es- sential oxygen supply to the organelle. In long-lived brains this could occur in response to age related vascular changes when suboptimal quantities of oxygenated blood could be delivered to this most impor- tant organ (Qiu and Fratiglioni, 2015). It is possible that this is present at some level in multiple sclerosis where it is shown that HbB is found at higher levels in cortical neurons (Broadwater et al., 1812). However over a lifetime, unregulated sequestering of Hb in the organelle could lead to toxic levels within the intermembrane space. At the tipping point, overloaded organelles might be targeted for removal by mitophagy (Chakrabarti et al., 2009). In order to examine the localisation of Hb in human brains we carefully examined mitochondri- al haemoglobin levels in control and age matched Parkinson's disease mitochondria. 3.4. Mitochondrial haemoglobin ratios exhibit a dynamic range with dis- ease duration We re-examined our data set of mitochondrial/cytoplasmic ratios of alpha and beta haemoglobin, this time against disease duration. Speciﬁc immunoblotting of HbA and HbB were veriﬁed by LC-MSMS of trypsin digested proteins from immunopositive and negative gel bands (data provided in Supplementary Fig. 2). Scatter plots were generated from the whole set of immunoblotting data and show highly variable rela- tionships as one might expect from post-mortem unrelated human brains with a sporadic disease (Fig. 3). However, two regions of the brain describe a trend towards a change when mitochondrial haemoglobin is measured. 2.4. Hypoxic treatment HbB and COXIV antibodies were utilised to visualise the physical location of mitochondria and HbB in the cerebellum of female individuals with different disease durations. It appears that with longer disease duration the overall quantity of COXIV is reduced and the location of HbB shifts in the longest disease course from in and around Purkinje cells observed in controls, to the granule cell layer in sections from 18 years disease duration. DAPI is used to stain nuclei. Filter sets used are detailed in the methodology. F. Shephard et al. / Mitochondrion 29 (2016) 45–52 50 disease duration is not an effect of tissue changes after death has oc- curred. Brain banks usually have fewer female Parkinson's disease brains in their collections. We have analysed the full dataset we obtain- ed but in order to be sure that differences in the numbers of females and males analysed did not skew our dataset we disease stage and age matched each female Parkinson's brain mitochondrial fraction (n = 7) with a male brain (n = 7) mitochondrial fraction and re-ran the data to show the gender difference is upheld with equal numbers of samples from each gender (Supplementary Fig. 6). Multiple linear regression analysis (software Genstat) was performed to verify that PMI is not a confounding factor in our analyses with disease duration – Supplemen- tary Table 3. The HbA in the affected sample appears in the outer membrane fraction. Though the amount of HbA was not absolutely quantiﬁed, relative ratios suggest that there is a substantial quantity of this protein in or associat- ed with the outer membrane of the affected sample mitochondria. 3.2. Hb levels increase in Drosophila mitochondrial fractions in response to cyclical hypoxia In order to demonstrate an in vivo response of haemoglobin regula- tion in mitochondria we exposed fruit ﬂies to cycles of hypoxia. Flies were in the group normoxic if they were not exposed to any hypoxic events. We tested two groups where the ﬂies were exposed to two or three cycles of hypoxia (30 min at 2.5% oxygen), each followed by a recovery period (30 min) in normoxia. Mitochondria were prepared from whole ﬂies in each of the three groups and Hb content ascertained in both cytoplasmic and mitochondrial fractions (Fig. 1B, Supplementary 1). 2.4. Hypoxic treatment The ratios of Hb content were plotted to reveal a signiﬁcant increase (p b 0.05) in mitochondrial samples undergoing three rounds of hypoxia. Samples undergoing two rounds of hypoxia ﬁtted with a trend towards increased mitochon- drial Hb with hypoxia. 3.5. The cellular distribution of HbB changes in female cerebellum with dis- ease duration Using antibodies to HbB and COXIV (for mitochondria) we localised mitochondrial haemoglobin in the female cerebellum (Fig. 4). In the fe- male control cerebellum large Purkinje cells are labelled with both anti- bodies demonstrating the presence of mitochondrial HbB in the cell body. The cerebellum examined from a female patient 9 years into the disease suggests an overall decrease of mitochondrial HbB and mito- chondria too. The patient shown with disease duration of 18 years ap- pears to show COXIV staining in a different pattern again through the cerebellum, moving away from the Purkinje cell layer. HbB staining is increasingly distant from the Purkinje cell layer region of the cerebel- lum. Purkinje cell staining is relatively reduced. The brightness of the COXIV stain appears decreased in both affected brains. All the images presented were stained and imaged in a single batch to ensure valid qualitative comparison between images. The changes appear to be re- lated to HbB protein localisation and this is something that needs to be looked into further. 3.3. Mitochondrial versus cytoplasmic HbA and HbB ratios in early onset, early stage and late stage Parkinson's Data for calculating alpha and beta globin ratios were collected by western blot. Fractionated cell extracts were produced and run to give cytoplasmic versus mitochondrial ratios (Fig. 2). Ratios were calculated with reference to beta actin levels in the same lane. COXIV antibody in- dicated enrichment of mitochondrial fractions. Cortex, cerebellum and substantia nigra regions of the brain were examined for each sample. Using the arbitrary grouping of early Parkinson's (within 10 years of onset), late Parkinson's (beyond 10 years of disease) and young Parkinson's (onset before 60 years) we were unable to detect any major differences in mitochondrial/cytoplasmic ratios. 4. Discussion In the cortex, the gender speciﬁc scatter plots can be summarised by lines demonstrating an increase in mito- chondrial HbA as the disease duration increases, this is most marked in the female brain tissues examined (red line). In the cerebellum mito- chondrial HbB shows little dynamic change in mitochondrial HbB in male samples (blue line). The cerebellum was subsequently interrogat- ed more carefully by sub-fractionation of the mitochondrial sample (Fig. 1A). In female samples mitochondrial HbB trends towards a de- crease with disease duration going from a ratio of up to 1.5 at 10 years duration and then decreasing to a ratio ~0.3 by 30 years of disease course. Interestingly mitochondrial HbB ratio in substantia nigra indi- cates mostly low levels of this protein. This may reﬂect the proportion of neuron loss in this area of the brain and suggest that the losses in this area occur early in the disease process. We show that the levels of mitochondrial Hb trend towards a de- crease in the cerebellum of females with Parkinson's. In males we ﬁnd that the Hb content of the mitochondria does not appear decreased overall, sub-mitochondrial fractionation reveals that the Hb content though maintained, has moved into a different mitochondrial In order to account for any protein changes with regard to post mortem interval (PMI) we plotted HbB levels and also the mitochondri- al marker COXIV against the delay after death in hours (Supplementary Figs. 3 and 4). These conﬁrm that the levels of HbB we see against F. Shephard et al. / Mitochondrion 29 (2016) 45–52 51 Ethics Burmester, T., Storf, J., Hasenjäger, A., Klawitter, S., Hankeln, T., 2006. The hemoglobin genes of Drosophila. FEBS J. 273, 468–480. http://dx.doi.org/10.1111/j.1742-4658. 2005.05073.x. Human brain sections and frozen brain samples were obtained from, Human Tissue Authority approved, Nottingham Health Science Biobank (Nottingham University Hospitals NHS Trust) and Parkinson's UK Brain Bank (Imperial College London). The tissue banks granted us use of the tissue as end users. The Parkinson's tissue bank has approval from the Research Ethics Committee for Wales ref. 08/MRE09/31+5. The tissue collection and procedures at the Nottingham University Hospitals Biobank have been ethically approved by the Greater Manchester Na- tional Research Ethics Service. This study was granted speciﬁc ethical approval by both of the ethics committees serving the biobanks and also by the School of Veterinary Medicine and Science Local Ethics Com- mittee at the University of Nottingham. Caranci, G., Piscopo, P., Rivabene, R., Traﬁcante, A., Riozzi, B., Castellano, A.E., et al., 2013. Gender differences in Parkinson's disease: focus on plasma α-synuclein. J. Neural Transm. 120, 1209–1215. http://dx.doi.org/10.1007/s00702-013-0972-6. Carlson, E.S., Magid, R., Petryk, A., Georgieff, M.K., 2008. Iron deﬁciency alters expression of genes implicated in Alzheimer disease pathogenesis. Brain Res. 1237, 75–83. http://dx.doi.org/10.1016/j.brainres.2008.07.109. Caslake, R., Taylor, K., Scott, N., Gordon, J., Harris, C., Wilde, K., et al., 2013. Age-, gender-, and socioeconomic status-speciﬁc incidence of Parkinson's disease and parkinsonism in northeast Scotland: the PINE study. Parkinsonism Relat. Disord. 19, 515–521. http://dx.doi.org/10.1016/j.parkreldis.2013.01.014. Cereda, E., Cilia, R., Klersy, C., Canesi, M., Zecchinelli, A.L., Mariani, C.B., et al., 2014. Swallowing disturbances in Parkinson's disease: a multivariate analysis of contribut- ing factors. Parkinsonism Relat. Disord. 20, 1382–1387. http://dx.doi.org/10.1016/j. parkreldis.2014.09.031. Chakrabarti, L., Neal, J.T., Miles, M., Martinez, R.A., Smith, A.C., Sopher, B.L., et al., 2006. The Purkinje cell degeneration 5J mutation is a single amino acid insertion that destabi- lizes Nna1 protein. Mamm. Genome 17, 103–110. http://dx.doi.org/10.1007/ s00335-005-0096-x. Funding Our sam- ple set for this study is small, in particular there is some difﬁculty in obtaining large sample sets of female brains since the disease is less fre- quently encountered by women (Caslake et al., 2013). We would be in- terested to see whether other groups with similar or larger size sample sets can replicate these ﬁndings. Gender differences in Parkinson's dis- ease are well documented, with females having a lower risk and a trem- or dominant phenotype as well as reduced motor symptoms (Caslake et al., 2013). The cause of this is currently unknown but could be impor- tant in terms of generating neuroprotective therapies exploiting gender speciﬁc mechanisms. Our ﬁnding that mitochondrial Hb may be modu- lated differently according to gender could be a result of gender speciﬁc systemic availability of oxygen. Females in their reproductive years are particularly prone to anaemia (Pasricha et al., 2014). A recent article now connects haptoglobin a haemoglobin binding protein with low levels of serum iron, also with a greater effect when stratifying by gen- der (Costa-Mallen et al., 2015). There is gathering evidence that iron levels and anaemia are likely to be important players in the area of neu- rodegeneration, it is interesting to note that it is still unknown where the iron accumulation, seen for example in Parkinson's, originates Hametner et al., 2013; Mochizuki and Yasuda, 2012. This study was supported by a Michael J Fox Foundation ‘Rapid Re- sponse Innovation Award’. OGH is supported by the Biotechnology and Biological Sciences Research Council (GB) Doctoral Training Pro- gramme at the University of Nottingham and supplementary funding came from the University of Nottingham. Author contribution statement FS, OGH, SL and LC conducted the research and analysed the data. RE analysed the data. LC and FS designed the study and obtained ethical ap- proval and samples. LC and FS wrote the paper. Acknowledgements High quality brain tissues were provided for this study from the Parkinson's UK Brain Bank (Imperial College London) and Nottingham Health Science Biobank (Nottingham University Hospitals NHS Trust). We would like to acknowledge the efforts of these providers, in partic- ular Djordje Gveric at the Parkinson's UK Brain Bank, and also we would like to express our gratitude to the donors and their families that make this resource an actuality. Our thanks to the people at Laser 2000 for their time and the use of their slide scanning facilities. We would also like to thank RJ Stöger for discussions during the preparation of this paper. This study is important in identifying molecular changes in the cer- ebellum of PD post mortem brain. Recently the cerebellum has piqued the interest of PD researchers and has been postulated as a potential source of compensatory signalling (Lewis et al., 2013; Wu and Hallett, 2013; Rodríguez-Cueto et al., 2014). Change in glucose metabolism has been found to be an early event in the PD cerebellum and there have been a number of imaging studies that implicate the importance of this part of the brain in PD (Dunn et al., 2014). Our discovery of gen- der related molecular changes in PD mitochondria is an essential step in understanding the observed differences in symptomology between males and females with PD (Caslake et al., 2013; Colombo et al., 2015; Caranci et al., 2013; Solla et al., 2012; Cereda et al., 2014). As we are able to deﬁne the gender effect, common pathways can be better de- ﬁned and compensatory loops, such as those proposed involving the cerebellum, can be targeted for therapy (Wu and Hallett, 2013). References Abbott, R.D., Ross, G.W., Tanner, C.M., Andersen, J.K., Masaki, K.H., Rodriguez, B.L., et al., 2012. Late-life hemoglobin and the incidence of Parkinson's disease. Neurobiol. Aging 33, 914–920. http://dx.doi.org/10.1016/j.neurobiolaging.2010.06.023. Alafuzoff, I., Ince, P.G., Arzberger, T., Al-Sarraj, S., Bell, J., Bodi, I., et al., 2009. Staging/typing of Lewy body related alpha-synuclein pathology: a study of the BrainNet Europe Con- sortium. Acta Neuropathol. 117, 635–652. http://dx.doi.org/10.1007/s00401-009- 0523-2. Biagioli, M., Pinto, M., Cesselli, D., Zaninello, M., Lazarevic, D., Roncaglia, P., et al., 2009. Un- expected expression of alpha- and beta-globin in mesencephalic dopaminergic neu- rons and glial cells. Proc. Natl. Acad. Sci. U. S. 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In fact the localisation in the outer membrane fraction does not allow us to specify whether Hb is merely associated with the external surface of the organelle or contained within the membrane or- ganelle. Importantly, in males Hb is no longer detected in the inter membrane space where it is found in controls and in affected female cerebellum, this takes it away from the site of Complex 1 activity where it is shown to have an effect (Brunyanszki et al., 2015). Our sam- ple set for this study is small, in particular there is some difﬁculty in obtaining large sample sets of female brains since the disease is less fre- quently encountered by women (Caslake et al., 2013). We would be in- terested to see whether other groups with similar or larger size sample sets can replicate these ﬁndings. Gender differences in Parkinson's dis- ease are well documented, with females having a lower risk and a trem- or dominant phenotype as well as reduced motor symptoms (Caslake et al., 2013). The cause of this is currently unknown but could be impor- tant in terms of generating neuroprotective therapies exploiting gender speciﬁc mechanisms. Our ﬁnding that mitochondrial Hb may be modu- lated differently according to gender could be a result of gender speciﬁc systemic availability of oxygen. Females in their reproductive years are particularly prone to anaemia (Pasricha et al., 2014). A recent article now connects haptoglobin a haemoglobin binding protein with low levels of serum iron, also with a greater effect when stratifying by gen- der (Costa-Mallen et al., 2015). There is gathering evidence that iron levels and anaemia are likely to be important players in the area of neu- rodegeneration, it is interesting to note that it is still unknown where the iron accumulation, seen for example in Parkinson's, originates Hametner et al., 2013; Mochizuki and Yasuda, 2012. compartment. 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Testes-speciﬁc he- moglobins in Drosophila evolved by a combination of sub- and neofunctionalization after gene duplication. BMC Evol. Biol. 12, 34. http://dx.doi.org/10.1186/1471-2148- 12-34. Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q., Jakes, R., Goedert, M., 1997. Alpha-synuclein in Lewy bodies. Nature 388, 839–840. http://dx.doi.org/10.1038/ 42166. Grek, C.L., Newton, D.A., Spyropoulos, D.D., Baatz, J.E., 2011. Hypoxia up-regulates expres- sion of hemoglobin in alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 44, 439–447. http://dx.doi.org/10.1165/rcmb.2009-0307OC. Wu, T., Hallett, M., 2013. The cerebellum in Parkinson's disease. Brain 136, 696–709. http://dx.doi.org/10.1093/brain/aws360. Declarations of interest Chakrabarti, L., Eng, J., Ivanov, N., Garden, G.A., La Spada, A.R., 2009. Autophagy activation and enhanced mitophagy characterize the Purkinje cells of pcd mice prior to neuro- nal death. Mol. 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https://openalex.org/W4381284640	https://www.ijfmr.com/papers/2023/3/3081.pdf	English	null	Predictive Maintenance for Nasa’s Turbofan Engine	International Journal For Multidisciplinary Research	2,023	cc-by-sa	3,489	Abstract NASA's turbofan engine is a vital equipment used in its aircraft fleet. This engine is designed to provide the required thrust for various missions, from scientific research to astronaut training. However, this engine requires regular maintenance to ensure its optimal performance and safe operation. In this paper, we will find the remaining useful life of the turbofan engine by applying data science techniques and machine learning algorithms for predicting more accurate maintenance requirements. We will examine the performance metrics of different machine learning models and tune the parameters of the best model using random search. We will be deployed as an application using Streamlit. The final result of the web application is that it provides the results of the predictions done by the model as a csv file along with the model loss and accuracy. Keywords: Predictive maintenance, Remaining useful life, Machine Learning, Streamlit, Web Application Predictive Maintenance for Nasa’s Turbofan Engine Kamalkumaar V P1, Veda Keerthi A2, Navaneetha Kannan A Yogesh T4, Dr. B. Kirubagari5 1,2,3,4UG Student, Department of CSE, Annamalai University, Chidambaram, India 5Associate Professor, Department of CSE, Annamalai University, Chidambaram, India 2. Literature review Predictive Maintenance (PdM) has been widely studied and applied in various industries. Many studies have shown that PdM can significantly reduce maintenance costs, increase equipment uptime, and improve safety. In the aviation industry, PdM is particularly important for ensuring the safe and reliable operation of aircraft engines. PdM techniques in aviation typically involve collecting data from various sensors and systems on the aircraft, analyzing the data using statistical and machine learning algorithms, and using the results to predict when maintenance should be performed. Despite the benefits of PdM, there are also some challenges and limitations to its implementation. One of the main challenges is the complexity of the data collected from aircraft systems, which can make it difficult to identify patterns and predict failures accurately, so for the feature extraction part of the model we have considered using the reference [2], and since we are predicting the Remaining useful life of an engine, the formula has coined from the reference [3], and moreover, there are also research papers that have made remaining useful life for engines with operational data [4], and considering all these research papers and studies, there are also studies for predictive maintenance on lithium-ion batteries using machine learning techniques [5], which is different from the turbofan maintenance, and with keeping all these studies in mind, we have made a research paper for the predictive maintenance of the NASA’s turbofan engine, in the aviation industry. Moreover, there is an additional research paper on Deep learning based RUL prediction using an optimized decision tree [6], which has been referred to calculate the RUL formula, and has also used machine learning techniques to predict the RUL of an engine. Additionally, PdM requires significant data processing and analysis capabilities, which can be challenging for smaller organizations. There is also a need for continuous monitoring of the data to ensure that the predictions remain accurate over time. Finally, there are issues related to data privacy and security, which must be carefully addressed to ensure that sensitive data is not compromised. Machine learning-based approaches have been widely studied and applied in PdM. These approaches involve training machine learning models on historical data to predict when maintenance should be performed. Many studies have shown that machine learning-based approaches can outperform traditional statistical approaches in terms of accuracy and reliability. International Journal for Multidisciplinary Research (IJ The Remaining useful life metric has been calculated with a difference of the whole engine life cycle from the cycle that the engine has completed, with the help of this feature we are going to fit the dataset with different machine learning models. The purpose of this research paper is to present a Streamlit app developed for the Predictive Maintenance of NASA turbofan engines. The app uses the best machine learning algorithm to analyze real-time data from the engines and predict when maintenance should be performed. The scope of the research paper includes a literature review of existing PdM techniques, a description of the methodology used to develop the app, a presentation of the app's results, and a discussion of the implications of the app for Predictive Maintenance in the aviation industry. 1. Introduction Predictive Maintenance (PdM) is a proactive maintenance approach that uses data analysis and machine learning algorithms to predict when maintenance should be performed on a machine or system. By analyzing real-time data from sensors and other sources, PdM can help identify potential failures before they occur, minimize downtime, and reduce maintenance costs. The data that we have considered for predictive maintenance is online and it is available on kaggle [1]. We are going to predict the Remaining useful life of NASA’s turbofan engine using various machine learning models since PdM has become increasingly important in the aviation industry, where the safety and reliability of aircraft are of paramount importance. NASA turbofan engines are a type of high-bypass turbofan engine used in aircraft, including commercial airliners, military aircraft, and space vehicles. These engines are designed to provide high thrust and fuel efficiency while minimizing noise and emissions. They are critical components of modern aviation and require high maintenance to ensure their safe and reliable operation. The dataset contains simulated aircraft engine run-to-failure events, operational settings, and 21 sensor measurements provided by Microsoft. It is assumed that the engine progressing degradation pattern is reflected in its sensor measurements, which are provided in a text format. Volume 5, Issue 3, May-June 2023 IJFMR23033081 1 1 3. Methodology Use either SI or CGS as primary units. (SI units are preferred.) English units may be used as secondary units (in parentheses). An exception would be the use of English units as identifiers in trade, such as “3.5 inch disk drive”. The dataset has been acquired from the online resource [1], and it refers to the NASA turbofan engine degradation simulation data, which contains sensor data from multiple turbofan engines, including information on temperature, pressure, and rotational speed, as well as data on the health and performance of the engines over time. The dataset includes both nominal and degraded engine conditions, allowing for the development of machine-learning models capable of predicting when maintenance should be performed, the methodology of how this problem has been approached has shown in figure 1. Figure 1: Architecture diagram Figure 1: Architecture diagram The dataset has gone through several preprocessing techniques, starting from treating null values, followed by treating duplicate values since duplicate values in a dataset don’t do any contribution to the learning of the model, the duplicate values are dropped from the dataset, then feature engineering has been performed in the data to extract meaningful features from the raw sensor data, this involves calculating statistical features such as mean, median, standard deviation, and skewness for each sensor signal along with finding how the sensor have been distributed using histogram, finding the outliers present in each sensor using a box plot, and finally a time series plot of each sensor value on how it performs over the sensor value. Using a heatmap, the correlation of the features has been plotted to show which of the features is more correlated, and once the resulting features are found they have been normalized to ensure that all the features had a similar scale and range using standard scaler function. Followed by the preprocessing of data, then the data have been split into training and testing sets so that they can be used to fit the model. Then several machine learning algorithms were evaluated for their ability to predict the remaining useful life of the turbofan engines. These include regression analysis models such as linear regression, lasso regression, ridge regression, decision trees, random forests, support vector machines, gradient boost regressor, and deep learning algorithms such as artificial neural networks, recurrent neural networks, and lstms. 2. Literature review Some of the most commonly used machine learning algorithms for PdM include decision trees, random forests, neural networks, and support vector machines. Deep learning techniques, such as long short term memory and recurrent neural networks, have also been applied in PdM with promising results. However, machine learning- based approaches also have their own set of challenges, including the need for large amounts of high- quality data and the risk of overfitting the model to the data. Volume 5, Issue 3, May-June 2023 2 IJFMR23033081 2 International Journal for Multidisciplinary Research (IJFMR) directly predict the data. This model will be used in the web application, to predict the data from the user, so that the user can get the RUL predictions from the model. directly predict the data. This model will be used in the web application, to predict the data from the user, so that the user can get the RUL predictions from the model. The web application for predictive maintenance for NASA turbofan engines was developed using the Python framework Streamlit. The app takes as input from the user of the real-time sensor values from the engine and uses the model which has been imported as a pickle file to predict the remaining useful life of an engine. The app includes a user-friendly interface for inputting data and displaying the results of the analysis, including the predicted remaining useful life of the engine. The app also allows users to visualize the sensor data over time and view diagnostic plots and other relevant information. The app was designed to be scalable and customizable, allowing for easy integration with other systems and data sources. 3. Methodology The models were evaluated using a range of performance metrics, including mean squared error, root mean squared error and r2 score, to determine the most effective algorithm for the task. IJFMR23033081 Volume 5, Issue 3, May-June 2023 3 Then the final best algorithm will be tuned with the best parameters using the Random Search CV function, this step is crucial to increase the accuracy of the model for the specific problem statement, once it is done, then the model is saved as a pickle file so that the pre-trained model can be used to 3 International Journal for Multidisciplinary Research (IJFMR) as RNN and LSTM performed well by having a slight higher MSE value, overall both the algorithms performed well with less MSE values but have overfitted the training data, so that we can’t able to select the deep learning models as the best model. Finally, the results of our analysis suggest that machine learning based approaches can be effective for predictive maintenance of NASA turbofan engines. However, it is important to carefully evaluate the performance of each model and consider the limitations and potential biases of the dataset and modeling techniques used, keeping that in mind, we have selected Random forest model has the best model and to increase the accuracy of the model hyperparameter tuning has been done over the model. In conclusion, the parameters of the base random forest model have been tuned with some of its parameters such as the number of trees in the forest, the maximum depth of each trees in the forest, the minimum number of samples required to split an internal node, the minimum number of samples required to be at a leaf node, and the parameter specifies the maximum number of features to consider when splitting a node, all these parameters values are specified in a search space, and Randomized Search CV fits all the values randomly into the model with the training data and gives the parameters that have best fitted the model, by this the base model have an improved accuracy of 2%, which is shown in the figure 3. Figure 3: Base model vs Tuned model 4. Results Use equation editor feature of your word processing software to create equation if equation contains division, or multiple lines. In this section, we present the performance of several machine learning algorithms evaluated for predicting the remaining useful life of the NASA turbofan engine. Since being a regression problem, the models were evaluated using several performance metrics, including mean squared error (MSE), root mean squared error (RMSE), R-squared (R2), and the accuracy score of the model on how well it has performed on the training data, all these metrics have been stored in a data frame which is shown in figure 2, to get a clear insight on which model has made the accurate predictions of the remaining useful life of an engine without underfitting or overfitting. Figure 2: Results of various models Figure 2: Results of various models Figure 2: Results of various models Figure 2: Results of various models Based on the results, the linear regression model has not performed very well on the training data and has made a lesser accuracy score with a higher MSE value, the same goes for the lasso and ridge regression models. The decision tree model achieved the lowest MSE value, indicating that it provided the best predictions overall. However, it is important to note that the decision tree model achieved a perfect accuracy score of 100% which may suggest overfitting to the training data, so to overcome this overfitting problem we have considered using the Random forest model, and it has produced a great result with least MSE value and with the highest accuracy without overfitting the data. IJFMR23033081 Volume 5, Issue 3, May-June 2023 4 The Support vector regressor and gradient boosting models achieved similar results to the linear regression models, while the ANN have achieved this result with the help of a simple model but increasing the layers of the model would help in increased accuracy, the other deep learning models such Volume 5, Issue 3, May-June 2023 4 4 rnational Journal for Multidisciplinary Research (IJFMR) However, it is important to note that the accuracy of the predictions made by the machine learning models depends on the quality and completeness of the input data. Therefore, it is important for maintenance teams to ensure that they are collecting and inputting high-quality data into the app. Additionally, further testing and validation of the app would be necessary before it can be fully integrated into the maintenance workflow of NASA turbofan engines. While the results of this study are promising, there are several limitations and potential directions for future research. First, the dataset used in this study was relatively small and may not be representative of all NASA turbofan engines. Therefore, it would be beneficial to evaluate the performance of machine learning models on larger and more diverse datasets. Second, the models developed in this study were trained and evaluated on historical data and may not be able to predict previously unseen failure modes. Therefore, it would be useful to test the models on real-time data to evaluate their performance in a real-world setting. Finally, while the app developed in this study is user- friendly and accessible, additional features could be added to enhance its functionality and usability, such as the ability to visualize engine data. Figure 4: Predictive Maintenance application 6. Conclusion In this study, we have developed a Streamlit application for predictive maintenance of NASA turbofan engines using machine learning algorithms, our results showed that the Random forest Figure 4: Predictive Maintenance application Figure 4: Predictive Maintenance application Figure 4: Predictive Maintenance application 5. Model Deployment Once the best model have been tuned with the best parameters, then the model have been dumped into a pickle file, so that the pre-trained model can be used to predict the new data, and so it will be used in the web application to predict the user’s data. The Streamlit application developed in this study provides a user-friendly interface for predictive maintenance of NASA turbofan engine, one of the key features of the app is the visualization of accuracy vs predicted values in the form of a chart, this chart allows maintenance teams to quickly access the accuracy of the predictions made by the machine learning models and make informed decisions about maintenance schedules and downtime. In addition to the accuracy vs predicted chart, the app also allows users to download the predictions as a csv file, this feature enables maintenance teams to easily integrate the predictions into their existing maintenance management systems, further streamlining the maintenance workflow. The input from the user has been got through a drop down bar, where the user can drop the data in the specified column, and he can drop upto 200 mb file, and the specified file should be dropped in the specified field, so that the model can able to predict the value, once the files have been dropped and if the user presses the predict button, the backend application works and it returns the chart of actual vs predicted along with the predictions in a csv file, which the user can able to download and along with it the application display the loss and the accuracy of the model, and the final model is deployed in the streamlit website for further use of the application, and a view of the application is shown in the figure 4. Volume 5, Issue 3, May-June 2023 IJFMR23033081 IJFMR23033081 5 International Journal for Multidisciplinary Research (IJFMR) regressor have out performed all the other models such as Linear Regression, Lasso Regression, Ridge Regression, Decision tree, Gradient boosting, ANN, RNN, and LSTM. The application we developed provides a user-friendly interface for maintenance teams to input engine data and generate accurate predictions of engine failures, the accuracy vs predicted chart and the ability to download predictions as a csv file provide valuable tools for maintenance teams to make informed decisions about maintenance schedules and remaining useful life. In future research, it would be valuable to explore the use of other machine learning algorithms or combinations of algorithms to improve the accuracy of predictions. Additionally, incorporating other factors such as weather data or pilot behavior could provide a more comprehensive understanding of engine failures. Finally, research could also focus on the integration of Predictive Maintenance systems with other aircraft maintenance management systems to further streamline the maintenance workflow. 6. Conclusion In this study, we have developed a Streamlit application for predictive maintenance of NASA turbofan engines using machine learning algorithms, our results showed that the Random forest In this study, we have developed a Streamlit application for predictive maintenance of NASA turbofan engines using machine learning algorithms, our results showed that the Random forest In this study, we have developed a Streamlit application for predictive maintenance of NASA turbofan engines using machine learning algorithms, our results showed that the Random forest Volume 5, Issue 3, May-June 2023 IJFMR23033081 6 7. References 1. https://www.kaggle.com/c/predictive-maintenance/data 2. Wang, S., Li, X., & Zhao, L. (2021). "A hybrid feature extraction and machine learning approach fo remaining useful life prediction of wind turbine bearings." Renewable Energy, 166, 1056-1066. 3. Lee, J., & Choi, J. (2018). "A machine learning approach to remaining useful life prediction of in dustrial machines with limited data." Sensors, 18(8), 2479. 4. He, Z., Zhu, P., & Xu, J. (2019). "Deep learning-based remaining useful life prediction for rotating machinery using only operational data." Mechanical Systems and Signal Processing, 130, 155-170. 5. Khan, F. A., & Kim, D. H. (2019). "Predicting remaining useful life of lithium-ion batteries using machine learning techniques: A review." Renewable and Sustainable Energy Reviews, 112, 649-657 6. Liu, Y., Li, L., & Wang, H. (2019). "Deep learning-based remaining useful life prediction using an optimized decision tree." IEEE Access, 7, 101685-101697. 2. Wang, S., Li, X., & Zhao, L. (2021). "A hybrid feature extraction and machine learning approach for remaining useful life prediction of wind turbine bearings." Renewable Energy, 166, 1056-1066. 3. Lee, J., & Choi, J. (2018). "A machine learning approach to remaining useful life prediction of in- dustrial machines with limited data." Sensors, 18(8), 2479. 7. References 4. He, Z., Zhu, P., & Xu, J. (2019). Deep learning-based remaining useful life prediction for rotating machinery using only operational data." Mechanical Systems and Signal Processing, 130, 155-170. 5. Khan, F. A., & Kim, D. H. (2019). "Predicting remaining useful life of lithium-ion batteries using machine learning techniques: A review." Renewable and Sustainable Energy Reviews, 112, 649-657. 6. Liu, Y., Li, L., & Wang, H. (2019). "Deep learning-based remaining useful life prediction using an optimized decision tree." IEEE Access, 7, 101685-101697. 5. Khan, F. A., & Kim, D. H. (2019). Predicting remaining useful life of lithium ion batteries using machine learning techniques: A review." Renewable and Sustainable Energy Reviews, 112, 649-657. 6. Liu, Y., Li, L., & Wang, H. (2019). "Deep learning-based remaining useful life prediction using an optimized decision tree." IEEE Access, 7, 101685-101697. 6. Liu, Y., Li, L., & Wang, H. (2019). "Deep learning-based remaining useful life prediction using an optimized decision tree." IEEE Access, 7, 101685-101697. 6. Liu, Y., Li, L., & Wang, H. (2019). "Deep learning-based remaining useful life prediction using an optimized decision tree." IEEE Access, 7, 101685-101697. Volume 5, Issue 3, May-June 2023 IJFMR23033081 7
https://openalex.org/W2791025612	https://serval.unil.ch/resource/serval:BIB_4EF44BEFF689.P001/REF.pdf	English	null	Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis	Nature communications	2,018	cc-by	17,228	\| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications ARTICLE \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications Results Concerning the visual ﬁeld, tests on healthy subjects under pixelated vision indicated that an array of 25 × 25 pixels and 30 degrees of visual angle (about 8.5 mm in diameter) could provide adequate mobility skills7,8. However, the size of the prosthesis is typically limited by the maximal allowed sclerotomy, which is about 6–7 mm long; available prostheses are therefore in the range of 1 to 5 mm. Argus II™, the largest implanted electrode array in humans, is a 6 × 10 array with a 575 µm electrode pitch4 and a theoretical ﬁeld of view of 10 × 18 degrees. Increasing the size of the array is associated with two main challenges: it requires a large scleral incision and it may not conform to the eye cur- vature. In a ﬂat prosthesis placed over the retina, central and peripheral electrodes may not be at the same distance from the retina. A large distance will inevitably increase the stimulation threshold and the cross-talk between adjacent electrodes9. Pre- liminary attempts in designing wide-ﬁeld retinal prosthesis have been proposed9,10. However, these approaches are based on materials (i.e., polyimide) with high elastic modulus (GPa), very thin substrates (e.g., 10 µm), and complex shapes (e.g., star) that could create challenges in manipulation, implantation, and ﬁxation. Concerning visual acuity, previous researches estimated that, to be useful in daily life, a retinal prosthesis should have 500 pixels distributed in the central area of approximately 5 mm in dia- meter11,12. More recently, a trial on healthy subjects showed that the number of pixels required to recognize common objects is on the order of 3000–500013. Despite microfabrication techniques allow such electrode density, a limitation remains due to the routing of the connection tracks in the active area and the size of the ﬂat cable connection to the implantable electronics/stimu- lator. To overcome these issues, in photovoltaic stimulation14, the light projected into the pupil is wirelessly converted into electrical stimuli delivered to the retina. After the ﬁrst demonstration of vision restoration in blind rats with a silicon photovoltaic sub- retinal prosthesis15, a second major step was achieved with the exploitation of conjugated polymers and organic semiconductors (i.e., poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), PEDOT:PSS; regioregular poly(3-hexylthiophene-2,5-diyl), P3HT; [6,6]-phenyl-C61-butyric acid methyl ester, PCBM) to build an organic photovoltaic subretinal interface16–18. In the latter, despite the capability of improving visual acuity in dys- trophic rats after 1 month of implantation19, several issues remain unsolved. Design and validation of a foldable and photovoltaic wide-ﬁeld epiretinal prosthesis Laura Ferlauto1, Marta Jole Ildelfonsa Airaghi Leccardi 1, Naïg Aurelia Ludmilla Chenais1, Samuel Charles Antoine Gilliéron1, Paola Vagni1, Michele Bevilacqua1, Thomas J. Wolfensberger2, Kevin Sivula 3 & Diego Ghezzi 1 Retinal prostheses have been developed to ﬁght blindness in people affected by outer retinal layer dystrophies. To date, few hundred patients have received a retinal implant. Inspired by intraocular lenses, we have designed a foldable and photovoltaic wide-ﬁeld epiretinal pros- thesis (named POLYRETINA) capable of stimulating wireless retinal ganglion cells. Here we show that within a visual angle of 46.3 degrees, POLYRETINA embeds 2215 stimulating pixels, of which 967 are in the central area of 5 mm, it is foldable to allow implantation through a small scleral incision, and it has a hemispherical shape to match the curvature of the eye. We demonstrate that it is not cytotoxic and respects optical and thermal safety standards; accelerated ageing shows a lifetime of at least 2 years. POLYRETINA represents signiﬁcant progress towards the improvement of both visual acuity and visual ﬁeld with the same device, a current challenging issue in the ﬁeld. 1 Medtronic Chair in Neuroengineering, Center for Neuroprosthetics, Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. 2 Department of Ophthalmology, University of Lausanne, Hôpital Ophtalmique Jules-Gonin, Fondation Asile des Aveugles, Lausanne, Switzerland. 3 Laboratory for Molecular Engineering of Optoelectronic Nanomaterials, Institute of Chemical Sciences and Engineering, School of Basic Science, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. Laura Ferlauto, Marta J. I. Airaghi Leccardi and Naïg A. L. Chenais contributed equally to this work. Correspondence and requests for materials should be addressed to D.G. (email: diego.ghezzi@epﬂ.ch) 1 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:992 NATURE COMMUNICATIONS\| (2018) 9:992 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 ARTICLE B B lindness affects more than 30 million people worldwide1, and it is deﬁned as visual acuity of less than 20/400 or a corresponding visual ﬁeld loss to less than 10 degrees, in the better eye with the best possible correction2. In North America and most of European countries, legal blindness is deﬁned as visual acuity of 20/200 or visual ﬁeld no greater that 20 degrees. In the last decade, various visual prostheses have been developed to ﬁght blindness in case of retinal dystrophies, such as Retinitis pigmentosa3 and more recently age-related macular degeneration (Clinical Trial NCT02227498). Results Design and fabrication. POLYRETINA is a novel foldable and photovoltaic wide-ﬁeld epiretinal prosthesis based on poly (dimethylsiloxane) (PDMS) as substrate material, because of its transparency, elasticity, low Young’s modulus, and high strain to failure21,22. Moreover, PDMS is available as medical grade elas- tomer already in use in medical device applications. The device consists in a PDMS–photovoltaic interface (Fig. 1a, c), embedding 2215 stimulating pixels (80 and 130 µm in diameter) distributed on an active area of 12.7 mm (Supplementary Fig. 1a). Each pixel is composed by a PEDOT:PSS bottom anode, a P3HT:PCBM (referred also as Blend) semiconductor layer, and a top cathode in titanium (Ti). Another PDMS layer encapsulates the prosthesis, avoiding the delamination and degradation of the organic materials and extending its lifetime (Supplementary Fig. 1b). Openings of 67 and 120 µm in diameter have been made in the encapsulation layer to expose the cathodes (Fig. 1f). Titanium is a mechanically and electrochemically stable metal, it is widely used in implantable devices, it has an appropriate work function for the photovoltaic mechanism, and it is a capacitive charge- injection material (also due to the thin layer of titanium oxide formed at the surface). The latter is desirable with mono-phasic pulses, as in this photovoltaic approach, because no chemical species are created or consumed during a stimulation pulse23, thus avoiding undesired tissue reactions. Under this condition, the electrode/electrolyte interface can be modeled as pure elec- trical capacitor without electron transfer from the metal to the solution24. To verify this hypothesis, we measured the pH with a microelectrode positioned above the titanium electrode of the PDMS–photovoltaic interface (Supplementary Fig. 2) upon 1 h of pulsed illumination (20 Hz, 10 ms, 3.4 mW mm−2; N = 3 devi- ces). The irradiance has been set to a value above the maximum allowed for prosthetic application (see Optical and thermal safety). During illumination, a negligible pH shift of about 0.002 pH units has been detected, which could be explained by a recording artifact due to the local temperature increase induced by the prosthesis (see Optical and thermal safety). Local heating could reduce the resistivity of the solution and decrease the vol- tage difference between the pH microelectrode and the local reference. Design and validation of a foldable and photovoltaic wide-ﬁeld epiretinal prosthesis Several multi-center clinical trials showed the feasibility of restoring a coarse form of vision with retinal implants, such as single letters discrimination and simple objects recognition4,5. However, several challenges remain open, such as the improvement of visual acuity and the enlargement of the visual ﬁeld above the thresholds of blindness6. An agreed upon strategy to improve visual acuity is to increase the electrode density, while a large visual ﬁeld could be attained by enlarging the retinal coverage with a larger prosthesis. ﬁeld, unless implanting multiple devices20. Some concerns remain about the risks associated with the implantation of multiple devices in the subretinal space (e.g., retinal detachment, move- ments of the devices, and device overlaps). Thus, increasing both visual acuity and visual ﬁeld size with a single retinal prosthesis remains one of the main unsolved challenges in the ﬁeld11. \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 a b Dome-shaped PDMS support PDMS–photovoltaic interface g f a Anchoring wing Tack hole b Dome-shaped PDMS support PDMS–photovoltaic interface c d e Fig. 1 Foldable and photovoltaic wide-ﬁeld retinal prosthesis. a 3D model of the fabricated PDMS-interface and of the dome-shaped PDMS support. b 3D model of the retinal prosthesis after boding the PDMS-interface to the PDMS support. c Fabricated PDMS–photovoltaic interface with pixels arranged in three areas of different sizes and densities: central area (red), diameter of 5 mm, 967 electrodes in hexagonal arrangement, electrode diameter 80 µm and pitch 150 µm, density 49.25 px mm−2; ﬁrst ring (green), diameter of 8 mm, 559 electrodes in hexagonal arrangement, electrode diameter 130 µm and pitch 250 µm, density 17.43 px mm−2; second ring (blue), diameter 12.7 mm, 719 electrodes, electrode diameter 130 µm, density 9.34 px mm−2. Circles show an enlarged view of the pixels distribution. Scale bar is 2.5 mm. d Picture of POLYRETINA. Four anchoring wings with holes are present for attaching the prosthesis with retinal tacks. e POLYRETINA folded before injection. f Scanning electron microscope image (40° tilted view) of a photovoltaic pixel. Scale bar is 10 µm. g 3D model after epiretinal placement Anchoring wing Tack hole d b c d c a Dome-shaped PDMS support f e f e g g Fig. 1 Foldable and photovoltaic wide-ﬁeld retinal prosthesis. a 3D model of the fabricated PDMS-interface and of the dome-shaped PDMS support. b 3D model of the retinal prosthesis after boding the PDMS-interface to the PDMS support. c Fabricated PDMS–photovoltaic interface with pixels arranged in three areas of different sizes and densities: central area (red), diameter of 5 mm, 967 electrodes in hexagonal arrangement, electrode diameter 80 µm and pitch 150 µm, density 49.25 px mm−2; ﬁrst ring (green), diameter of 8 mm, 559 electrodes in hexagonal arrangement, electrode diameter 130 µm and pitch 250 µm, density 17.43 px mm−2; second ring (blue), diameter 12.7 mm, 719 electrodes, electrode diameter 130 µm, density 9.34 px mm−2. Circles show an enlarged view of the pixels distribution. Scale bar is 2.5 mm. d Picture of POLYRETINA. Four anchoring wings with holes are present for attaching the prosthesis with retinal tacks. e POLYRETINA folded before injection. f Scanning electron microscope image (40° tilted view) of a photovoltaic pixel. Scale bar is 10 µm. g 3D model after epiretinal placement a b Anchoring wing c Fig. Results Conjugated polymers are well tolerated when exposed to the subretinal space18, but they start to delaminate a few months after placement leading to an unavoidable degradation of the organic materials. Moreover, in the cases of both silicon and organic photovoltaic subretinal prostheses, the limited size of the devices (1–2 mm) will not allow the recovery of a large visual The hemispherical shape of POLYRETINA (Fig. 1b, d) is obtained by bonding the PDMS–photovoltaic interface on a dome-shaped PDMS support (Fig. 1a) with a radius of curvature of 12 mm, corresponding to the standard human eye. The bonding induces a radial elongation in the PDMS–photovoltaic interface of about 3% (in diameter), which has been considered to determine the covered retinal surface (Supplementary Fig. 1c). Four anchoring wings, with holes for tacks, have been included for the ﬁxation of the prosthesis (Fig. 1d). The folding of POLYRETINA, its insertion, and covering of the retinal surface have been evaluated in simulated surgeries with plastic models of the human eye (Fig. 2a). The prosthesis can be folded prior implantation (Figs. 1e, 2a, top- left), inserted through an aperture of 6.5 mm (Fig. 2a, top- right), released within the posterior chamber (Figs. 1g, 2b, bottom-right and bottom-left), and attached in epiretinal conﬁguration (Fig. 2b). The same surgical approach has been also validated in enucleated pig eyes (Fig. 2c). NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 ARTICLE \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 2 Simulated surgical implantation. a Picture sequence of the implantation in a human eye plastic model. The white line in top-right panel shows the incision of 6.5 mm. b Picture of POLYRETINA placed in epiretinal conﬁguration. c Picture sequence of the implantation in a pig eye a Anchoring wing b b c Fig. 2 Simulated surgical implantation. a Picture sequence of the implantation in a human eye plastic model. The white line in top-right panel shows the incision of 6.5 mm. b Picture of POLYRETINA placed in epiretinal conﬁguration. c Picture sequence of the implantation in a pig eye Fig. 2 Simulated surgical implantation. a Picture sequence of the implantation in a human eye plastic model. The whi incision of 6.5 mm. b Picture of POLYRETINA placed in epiretinal conﬁguration. c Picture sequence of the implantatio layer of P3HT:PCBM, and aluminum (Al) top cathodes. We initially used aluminum since it is one of the most common cathode material in organic photovoltaics. KPFM measures (Fig. 3c) across several devices showed that the variation of the surface potential upon illumination (white LED, light from the top, 0.4 mW mm−2) is about 15 folds higher (Fig. 3d) with alu- minum cathodes with respect to P3HT:PCBM only. When Optimization of the photovoltaic pixel. First, using Kelvin probe force microscopy (KPFM), we evaluated the changes in the sur- face potential generated at the cathode upon illumination for different conditions of fabrication (Fig. 3a, b). To assess the role of the bottom anode, we fabricated photovoltaic interfaces onto glass substrates including a bottom anode made of indium tin oxide (ITO), an injection layer of PEDOT:PSS, a semiconductor NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 a c d e Glass ITO PEDOT:PSS P3HT:PCBM Al / Ti 25 nm 1.10 1.00 0.95 0.90 0.85 0.80 0.75 0.70 Surface potential (V) 50 ms 50 mV 1.05 Voltage change (V) 0 0.01 0.02 0.15 0.20 0.25 ** + Al – Al ITO/PEDOT:PSS/Blend/Al ITO/PEDOT:PSS/Blend PEDOT:PSS/Blend/Al PEDOT:PSS/Blend Blend/Al Blend f 500 nm P3HT:PCBM Al Ti Al Voltage change (V) 0.075 0.100 0.125 0.200 0.225 0.250 0.150 0.175 100 150 Electrode diameter (µm) * * +PDMS/Ti +PDMS/Al b 100 nm 10 nm 4 nm ig. 3 Optimization of the photovoltaic pixel. a Picture of the KPFM measures. b Sketch of the fabricated device. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 Glass substrates have been coated with a hin ﬁlm of ITO (200 nm), a thin ﬁlm of PEDOT:PSS (50 nm), a thin ﬁlm of P3HT:PCBM (100 nm), and last aluminium (100 nm) or titanium (150 nm). c Representative KPFM map on a Glass/PEDOT:PSS/Blend/Al device obtained by repeating a line scan of 100 nm (vertical direction). The horizontal bar ndicates period of dark (black) and light (white). The bottom panel shows the average potential ﬂuctuation during time; each point is the average potential n a single line scan. d Surface potential variations (voltage in light—voltage in dark) for 6 different architectures. Each bar is the mean (±s.e.m.) of at least N = 3 devices, in which at least n = 3 electrodes/points has been measured and averaged. ITO/PEDOT:PSS/Blend/Al: 0.2106 ± 0.0092 V, N = 5, n = 3; PEDOT:PSS/Blend/Al: 0.2259 ± 0.0085 V, N = 5, n = 3; Blend/Al: 0.1334 ± 0.0090 V, N = 3, n = 3; ITO/PEDOT:PSS/Blend: 0.0128 ± 0.0032 V, N = 3, n = 3; PEDOT:PSS/Blend: 0.0091 ± 0.0025 V, N = 3, n = 4; Blend: 0.0052 ± 0.0007 V, N = 3, n = 4. One-way ANOVA, p < 0.0001, F = 177.9. e Surface potential variations with/without a bottom PDMS layer and with Al or Ti top contacts of 100 and 150 µm in diameter. Each point is the mean (±s.e.m.) of at east N = 3 devices, in which at least n = 3 electrodes has been measured and averaged. PEDOT:PSS/Blend/Al-100 µm: 0.1984 ± 0.0043 V, N = 3, n = 3; PEDOT:PSS/Blend/Al-150 µm: 0.2232 ± 0.0082 V, N = 3, n = 3; PDMS/PEDOT:PSS/Blend/Al-100 µm: 0.1927 ± 0.0115 V, N = 5, n = 3; PDMS/PEDOT: PSS/Blend/Al-150 µm: 0.2163 ± 0.0150 V, N = 5, n = 3; PDMS/PEDOT:PSS/Blend/Ti-100 µm: 0.1055 ± 0.0063 V, N = 3, n = 6; PDMS/PEDOT:PSS/ Blend/Ti-150 µm: 0.1342 ± 0.0068 V, N = 3, n = 3. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 f Representative AFM images of PEDOT:PSS/Blend, PEDOT:PSS/Blend/Al, and PEDOT:PSS/Blend/Ti urfaces d Glass ITO PEDOT:PSS P3HT:PCBM Al / Ti 0 0 Voltage change (V) 0 0.01 0.02 0.15 0.20 0.25 ** + Al – Al ITO/PEDOT:PSS/Blend/Al ITO/PEDOT:PSS/Blend PEDOT:PSS/Blend/Al PEDOT:PSS/Blend Blend/Al Blend Glass ITO PEDOT:PSS P3HT:PCBM Al / Ti b a b a d Voltage change (V) 0 0.01 0.02 0.15 0.20 0.25 + Al – Al d c 25 nm 1.10 1.00 0.95 0.90 0.85 0.80 0.75 0.70 Surface potential (V) 50 ms 50 mV 1.05 d c e Al Voltage change (V) 0.075 0.100 0.125 0.200 0.225 0.250 0.150 0.175 100 150 Electrode diameter (µm) * * +PDMS/Ti +PDMS/Al f f e e 500 nm P3HT:PCBM Al Ti 100 nm 10 nm m Fig. 3 Optimization of the photovoltaic pixel. a Picture of the KPFM measures. b Sketch of the fabricated device. Glass substrates have been coated with a thin ﬁlm of ITO (200 nm), a thin ﬁlm of PEDOT:PSS (50 nm), a thin ﬁlm of P3HT:PCBM (100 nm), and last aluminium (100 nm) or titanium (150 nm). c Representative KPFM map on a Glass/PEDOT:PSS/Blend/Al device obtained by repeating a line scan of 100 nm (vertical direction). The horizontal bar indicates period of dark (black) and light (white). The bottom panel shows the average potential ﬂuctuation during time; each point is the average potential in a single line scan. d Surface potential variations (voltage in light—voltage in dark) for 6 different architectures. Each bar is the mean (±s.e.m.) of at least N = 3 devices, in which at least n = 3 electrodes/points has been measured and averaged. ITO/PEDOT:PSS/Blend/Al: 0.2106 ± 0.0092 V, N = 5, n = 3; PEDOT:PSS/Blend/Al: 0.2259 ± 0.0085 V, N = 5, n = 3; Blend/Al: 0.1334 ± 0.0090 V, N = 3, n = 3; ITO/PEDOT:PSS/Blend: 0.0128 ± 0.0032 V, N = 3, n = 3; PEDOT:PSS/Blend: 0.0091 ± 0.0025 V, N = 3, n = 4; Blend: 0.0052 ± 0.0007 V, N = 3, n = 4. One-way ANOVA, p < 0.0001, F = 177.9. e Surface potential variations with/without a bottom PDMS layer and with Al or Ti top contacts of 100 and 150 µm in diameter. Each point is the mean (±s.e.m.) of at least N = 3 devices, in which at least n = 3 electrodes has been measured and averaged. \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 In pa g, the PC density and PV on every device (N = 3) has been measured for all electrodes (n = 6) and data have been averaged \| / PC density (µA cm–2) a Time (ms) 0 100 200 300 400 50 0 –50 –100 –150 –200 –250 Photovoltage (mV) Time (ms) 0 100 200 300 400 20 0 –20 –40 –60 –80 –100 –120 –140 b c PC density (µA cm–2) Time (ms) 0 100 200 300 400 50 0 –50 –100 –150 –200 –250 b Photovoltage (mV) Time (ms) 0 100 200 300 400 20 0 –20 –40 –60 –80 –100 –120 –140 c b a Photovoltage (mV) d 180 120 100 80 60 0 140 40 20 160 0 800 1000 200 400 600 Irradiance (µW mm–2) PC density (µA cm–2) d Photovoltage (mV) 225 175 125 50 0 200 150 100 75 25 180 120 100 80 60 0 140 40 20 160 0 800 1000 200 400 600 Irradiance (µW mm–2) 0 800 1000 200 400 600 PC density (µA cm–2) Irradiance (µW mm–2) e Photovoltage (mV) 225 175 125 50 0 200 150 100 75 25 0 800 1000 200 400 600 Irradiance (µW mm–2) e d e f 10 50 100 200 Pulse duration (ms) 160 120 80 40 0 180 140 100 60 20 PC density (µA cm–2) Photovoltage (mV) 225 125 75 50 0 10 50 100 200 Pulse duration (ms) 200 175 150 100 25 g f g Fig. 4 Characterization of the photo-current and photo-voltage. a Drawing of the experimental setup for the measure of PC and PV; the light pulse comes from the bottom. b, c Examples of PC density (b) and PV (c) measures obtained from 1 electrode (diameter 100 µm) at maximal light intensity (565 nm, 943.98 µW mm−2) and for increasing pulse durations (10, 50, 100, and 200 ms). Horizontal bars represent the light pulses. d,e, Mean (±s.e.m) PC density (d) and PV(e) measured upon illumination with 10 ms pulses at increasing light intensities. f, g, Mean (±s.e.m) PC density (f) and PV (g) measured for increasing light intensities (12.75, 111.11, 225.00, 430.56, 616.67, 785.65, and 943.98 µW mm−2) and pulse durations (10, 50, 100, and 200 ms). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 PEDOT:PSS/Blend/Al-100 µm: 0.1984 ± 0.0043 V, N = 3, n = 3; PEDOT:PSS/Blend/Al-150 µm: 0.2232 ± 0.0082 V, N = 3, n = 3; PDMS/PEDOT:PSS/Blend/Al-100 µm: 0.1927 ± 0.0115 V, N = 5, n = 3; PDMS/PEDOT: PSS/Blend/Al-150 µm: 0.2163 ± 0.0150 V, N = 5, n = 3; PDMS/PEDOT:PSS/Blend/Ti-100 µm: 0.1055 ± 0.0063 V, N = 3, n = 6; PDMS/PEDOT:PSS/ Blend/Ti-150 µm: 0.1342 ± 0.0068 V, N = 3, n = 3. f Representative AFM images of PEDOT:PSS/Blend, PEDOT:PSS/Blend/Al, and PEDOT:PSS/Blend/Ti surfaces aluminum is present (Fig. 3d, left), the absence of any anode (ITO or ITO/PEDOT:PSS) signiﬁcantly reduces the surface potential variation upon illumination (ITO/PEDOT:PSS/Blend/Al vs. Blend/Al, p < 0.0001; PEDOT:PSS/Blend/Al vs. Blend/Al, p < 0.0001; one-way ANOVA, Tukey’s multiple comparison test). No signiﬁcant difference has been found with or without the ITO anode if the PEDOT:PSS injection layer is present (ITO/PEDOT: PSS/Blend/Al vs. PEDOT:PSS/Blend/Al, p = 0.6219; one-way ANOVA, Tukey’s multiple comparison test). In the absence of aluminum cathodes (Fig. 3d, right), the architectures with dif- ferent bottom anodes do not induce any signiﬁcant difference (ITO/PEDOT:PSS/Blend vs. PEDOT:PSS/Blend, p = 0.9997; ITO/ PEDOT:PSS/Blend vs. Blend, p = 0.9890; PEDOT:PSS/Blend vs. Blend, p = 0.9995; one-way ANOVA, Tukey’s multiple compar- ison test). The maximization of the surface potential variation has been obtained with aluminum cathodes and both ITO/PEDOT: PSS or only PEDOT:PSS anodes. Therefore, to simplify the fab- rication process, we implemented PEDOT:PSS alone as bottom layer. We also veriﬁed that the surface potential variation was not altered (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 3e) when the interface was built over a PDMS sub- strate instead of bare glass with aluminium cathode diameters of both 100 and 150 µm (●PEDOT:PSS/Blend/Al and ○PDMS/ PEDOT:PSS/Blend/Al); no statistical differences have been found among the groups (two-way ANOVA, Tukey’s multiple NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 PC density (µA cm–2) a Time (ms) 0 100 200 300 400 50 0 –50 –100 –150 –200 –250 Photovoltage (mV) Time (ms) 0 100 200 300 400 20 0 –20 –40 –60 –80 –100 –120 –140 b f d Photovoltage (mV) 225 175 125 50 0 200 150 100 75 25 180 120 100 80 60 0 140 40 20 160 0 800 1000 200 400 600 Irradiance (µW mm–2) 0 800 1000 200 400 600 Photovoltage (mV) 225 125 75 50 0 10 50 100 200 Pulse duration (ms) 10 50 100 200 Pulse duration (ms) 200 175 150 100 25 160 120 80 40 0 180 140 100 60 20 PC density (µA cm–2) PC density (µA cm–2) Irradiance (µW mm–2) g e c 4 Characterization of the photo-current and photo-voltage. a Drawing of the experimental setup for the measure of PC and PV; the light pulse co m the bottom. b, c Examples of PC density (b) and PV (c) measures obtained from 1 electrode (diameter 100 µm) at maximal light intensity (565 .98 µW mm−2) and for increasing pulse durations (10, 50, 100, and 200 ms). Horizontal bars represent the light pulses. d,e, Mean (±s.e.m) PC den and PV(e) measured upon illumination with 10 ms pulses at increasing light intensities. f, g, Mean (±s.e.m) PC density (f) and PV (g) measured easing light intensities (12.75, 111.11, 225.00, 430.56, 616.67, 785.65, and 943.98 µW mm−2) and pulse durations (10, 50, 100, and 200 ms). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 In panels d to g, the PC density and PV on every device (N = 3) has been measured for all electrodes (n = 6) and data have been averaged comparison test, interaction p = 0.9633; factor 1, diameter, p = 0.0887; factor 2, substrate, p = 0.6385). When titanium replaces aluminium (Δ PDMS/PEDOT:PSS/Blend/Ti), the surface potential is slightly reduced (for 100 µm: one-way ANOVA, F = 25.43, p < 0.001; PDMS/PEDOT:PSS/Blend/Ti vs. both PEDOT:PSS/Blend/Al and PDMS/PEDOT:PSS/Blend/Al, p < 0.001, Tukey’s multiple comparison test; for 150 µm: one-way ANOVA, F = 9.266, p < 0.01; PDMS/PEDOT:PSS/Blend/Ti vs. both PEDOT:PSS/Blend/Al and PDMS/PEDOT:PSS/Blend/Al, p < 0.05, Tukey’s multiple comparison test). comparison test, interaction p = 0.9633; factor 1, diameter, p = 0.0887; factor 2, substrate, p = 0.6385). When titanium replaces aluminium (Δ PDMS/PEDOT:PSS/Blend/Ti), the surface potential is slightly reduced (for 100 µm: one-way ANOVA, F = 25.43, p < 0.001; PDMS/PEDOT:PSS/Blend/Ti vs. both PEDOT:PSS/Blend/Al and PDMS/PEDOT:PSS/Blend/Al, p < 0.001, Tukey’s multiple comparison test; for 150 µm: one-way ANOVA, F = 9.266, p < 0.01; PDMS/PEDOT:PSS/Blend/Ti vs. both PEDOT:PSS/Blend/Al and PDMS/PEDOT:PSS/Blend/Al, p < 0.05, Tukey’s multiple comparison test). current (PC) and the photo-voltage (PV) generated in the presence of electrolyte solution upon illumination. We fabricated chips embedding six electrodes, each of them connected to a contact pad for measuring the signal with respect to an Ag/AgCl reference electrode immersed in solution (Fig. 4a). Both PC and PV have been measured with illumination (565 nm) at increasing light intensities (12.75, 111.11, 225.00, 430.56, 616.67, 785.65, and 943.98 µW mm−2) and pulse duration (10, 50, 100, and 200 ms). The PC (Fig. 4b) generated by pulsed illumination (943.98 µW mm−2) has a typical capacitive proﬁle, peaking in about 10 ms and then decreasing with an exponential decay, while the PV (Fig. 4c) reaches a steady-state value and remains constant. This is in agreement with the capacitive nature of the electrode/ electrolyte interface. Moreover, the PV generated (about 180 mV) is largely below the redox potential of titanium (or titanium oxide), thus ensuring that no irreversible reactions occur at the KPFM measurements have been performed in air in non- contact mode; therefore, the measured variations in the surface potential may be slightly different with respect to the electric potential generated by the double layer capacitive charging occurring at an electrode–electrolyte interface, as in the case of an implanted retinal prosthesis. \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:992 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 ectrodes from N = 1 device RTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03 a 25 0 –200 Photovoltage (mV) –25 –50 –75 –100 –125 –150 –175 Time (ms) 0 50 100 150 b Normalized photocurrent 1.1 0.9 0.8 0.7 0.6 0.5 1.0 Pulse number (n) 0 1000 800 600 400 200 Stimulation rate 1 Hz b Photovoltage (mV) c 20 0 –20 –60 –100 –140 –180 Time (ms) Time (ms) 0 600 800 400 200 1000 Stimulation rate 10 Hz ( ) Stimulation rate 20 Hz Photovoltage (mV) d 20 0 –20 –60 –100 –140 –180 Time (ms) 0 600 800 400 200 1000 d c Normalized PC 1.1 0.9 0.8 1.0 Pulse number (n) 20 10 8 6 4 2 16 14 12 18 f Normalized PC 1.1 0.9 0.8 1.0 Pulse number (n) 10 5 4 3 2 1 8 7 6 9 e f C f e Pulse number (n) Pulse number (n) Pulse number (n) Normalized PC 1.1 0.9 0.8 1.0 g 0.7 Pulse number (n) 220k 200k 180k 160k 0 280k 260k 240k 300k 80k 60k 40k 20k 140k 120k 100k Stimulation rate 20 Hz g Fig. 5 High-frequency train stimulation. a Mean PV trace obtained at maximal light intensity (565 nm, 10 ms, 943.98 µW mm−2). The trace is the mean of N = 6 devices; in which n = 6 electrodes have been measured and averaged. The horizontal bars represent the light pulse. The dotted lines highlight the discharging rate of the electrode. b Evolution of the PC density peaks during 1000 stimuli delivered at 1 Hz (10 ms, 943.98 µW mm−2). Each point is the mean (±s.e.m.) of N = 3 devices, in which n = 6 electrodes have been measured and averaged. c Representative PV recording upon 10 pulses at 10 Hz (565 nm, 10 ms, 943.98 µW/mm2). d Representative PV recording upon 20 pulses at 20 Hz (565 nm, 10 ms, 943.98 µW mm−2). e Evolution of the PC density peaks normalized to the ﬁrst pulse. Each point is the mean ± s.e.m. of N = 10 devices, in which n = 6 electrodes have been measured and averaged. f Evolution of the PC density peaks normalized to the ﬁrst pulse. Each point is the mean ± s.e.m. of N = 8 devices, in which n = 6 electrodes have been measured and averaged. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 a 25 0 –200 Photovoltage (mV) b Normalized photocurrent 1.1 0.9 0.8 0.7 0.6 0.5 1.0 Pulse number (n) 0 1000 800 600 400 200 Stimulation rate 1 Hz Stimulation rate 20 Hz –25 –50 –75 –100 –125 –150 –175 Photovoltage (mV) c 20 0 –20 –60 –100 –140 –180 Time (ms) 0 50 100 150 Time (ms) 0 600 800 400 200 1000 Stimulation rate 10 Hz Photovoltage (mV) d 20 0 –20 –60 –100 –140 –180 Time (ms) 0 600 800 400 200 1000 Normalized PC 1.1 0.9 0.8 1.0 Pulse number (n) 20 10 8 6 4 2 16 14 12 18 Normalized PC 1.1 0.9 0.8 1.0 Pulse number (n) 10 5 4 3 2 1 8 7 6 9 Normalized PC 1.1 0.9 0.8 1.0 g 0.7 Pulse number (n) 220k 200k 180k 160k 0 280k 260k 240k 300k 80k 60k 40k 20k 140k 120k 100k Stimulation rate 20 Hz f e 5 High-frequency train stimulation. a Mean PV trace obtained at maximal light intensity (565 nm, 10 ms, 943.98 µW mm−2). The trace is the m 6 devices; in which n = 6 electrodes have been measured and averaged. The horizontal bars represent the light pulse. The dotted lines highlig harging rate of the electrode. b Evolution of the PC density peaks during 1000 stimuli delivered at 1 Hz (10 ms, 943.98 µW mm−2). Each point n (±s.e.m.) of N = 3 devices, in which n = 6 electrodes have been measured and averaged. c Representative PV recording upon 10 pulses at 10 Hz 10 ms, 943.98 µW/mm2). d Representative PV recording upon 20 pulses at 20 Hz (565 nm, 10 ms, 943.98 µW mm−2). e Evolution of the PC d s normalized to the ﬁrst pulse. Each point is the mean ± s.e.m. of N = 10 devices, in which n = 6 electrodes have been measured and averag ution of the PC density peaks normalized to the ﬁrst pulse. Each point is the mean ± s.e.m. of N = 8 devices, in which n = 6 electrodes have sured and averaged. g PC generated with 320,000 stimuli delivered at 20 Hz (565 nm, 10 ms, 943.98 µW mm−2). Each point is the mean ± s.d. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 Therefore, we measured the photo- NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 5 ARTICLE \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications Stimulation rate 20 Hz NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 Given the possibility to stimulate at 20 Hz, we tested Ti- based photovoltaic electrodes over a long operation period (Fig. 5g). Upon 320,000 stimuli (20 Hz, 10 ms, 943.98 µW mm −2), the stable steady state response (average of the last 1000 pulses/ﬁrst response) is only slightly affected (88.6%). the irradiance tested (Supplementary Fig. 3b and Supplementary Fig. 4b). As already demonstrated by others29, we also veriﬁed in a second subset of cells (n = 6, N = 5; 209.4 ± 37.14 days) that the prosthetic activation of both ML and LL spikes is abolished by using synaptic blockers (Supplementary Fig. 5). This conﬁrms the hypothesis that ML and LL are induced by the activation of the internal retinal circuit. Spatial selectivity. We then addressed the spatial selectivity by using an experimental/computation hybrid approach. First, using a glass microelectrode (Fig. 7a, b) we measured the radial voltage spreading in three directions (D1, D2, and D3) upon illumination of a single pixel (Fig. 7c). For each illuminated pixel (n = 4 pix- els), the normalized voltage spreading in the three principal directions have been averaged. The mean (±s.e.m.) voltage dis- tribution across all the pixel tested has been plotted and inter- polated with a Gaussian function (Fig. 7d). Experimental data match with the normalized voltage proﬁle obtained by a ﬁnite element analysis (FEA) model (Fig. 7d, dotted blue line). The full width at half maximum (FWHM) of the simulated curve (Fig. 7d, dotted gray line) has been taken as the effective activation area, which is about 100 µm. FEA simulations have been used to characterize the normalized voltage proﬁle induced by illumina- tion of increasing diameters (Fig. 7e). Increasing the spot size from 1 pixel to 7 and 19 pixels increases the potential. Last, we simulate the effect of different patterns of activation (Fig. 7f). In all cases, a spatially selective potential proﬁle corresponding to light pattern is shown. Validation ex vivo with explanted retinas from blind mice. Next, we tested the ex vivo efﬁcacy of the PDMS–photovoltaic interface in stimulating RGCs. For this purpose, we used the retinal degeneration 10 mouse model26, that is recognized as an excellent model for Retinitis pigmentosa27. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 Extracellular record- ings of prosthetic-evoked spiking activity of RGCs have been collected from retinas explanted from old mice to avoid as much as possible the natural responses from surviving photoreceptors (n = 39 cells, N = 15 mice; mean ± s.d. age 140.87 ± 20.35 days). Retinas have been layered on the central 5-mm area of the PDMS–photovoltaic interface mimicking the epiretinal conﬁg- uration (Fig. 6a). According to the PC density measures, we tested only 10-ms pulses (peak of the PC response) with a broad range of irradiance (from 47.35 to 29.07 mW mm−2). Light pulses induced a prosthetic-evoked spiking activity in the recorded RGC (Supplementary Fig. 3a and Fig. 6b). Spikes have been detected with a threshold algorithm (red lines in Fig. 6b and Supple- mentary Fig. 3a), converted into a raster plot (Supplementary Fig. 3a, middle), and presented as peri-stimulus time histogram (PSTH; Supplementary Fig. 3a, bottom). As previously repor- ted28, we observed three types of responses, classiﬁed as short, medium, and long latency (SL, ML, and LL). The presence of SL spikes (elicited in the 10-ms window after the light onset, 1 bin) indicates a direct electrical stimulation of RGCs; while the pre- sence of ML and LL spikes indicates a network-mediated acti- vation. We have found that SL spikes can be evoked starting from the ﬁrst irradiance tested (47.35 µW mm−2), then the ﬁring rate slowly increases and it remains stable above 1.08 mW mm−2 till the highest irradiance tested (Fig. 6c and Supplementary Fig. 3c). However, the mean ( ± s.e.m.) latency (Fig. 6d) at this ﬁrst irra- diance is relatively long (6.05 ± 0.23 ms); it decreases with the increase of the irradiance, and it stabilizes at 4.12 ± 0.07 ms for irradiance higher than 1.08 mW mm−2 (Fig. 6d and Supple- mentary Fig. 3d). In this range (higher than 1.08), the mean ( ± s. e.m.) jitter of the ﬁrst SL spike is 0.39 ± 0.05 ms. This suggests that the SL response is saturated for irradiance higher than 1.08 mW mm−2, as predicted by the measure of the PC densities. For irradiance lower than 1.08 mW/mm2 the mean latency appears shorter but the jitter is more variable, indicating a more instable response (Fig. 6d). The ﬁring rate of ML (Fig. 6e and Supple- mentary Fig. 3e) and LL (Fig. 6f and Supplementary Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 g PC generated with 320,000 stimuli delivered at 20 Hz (565 nm, 10 ms, 943.98 µW mm−2). Each point is the mean ± s.d. of n = 2 electrodes from N = 1 device solution (stored in dark). The mean (±s.e.m.) ratio before/after has been measured in 94.44 ± 12.28, 95.11 ± 13.07, 93.36 ± 13.26, 94.99 ± 12.48%, respectively, for 10, 50, 100, and 200-ms pulses; no signiﬁcant differences have been found (10 ms: p = 0.4423; 50 ms: p = 0.5798; 100 ms: p = 0.5798; 200 ms: p = 0.5526; N = 3 devices, n = 6 electrodes per device; Wilcoxon matched-pairs signed rank test). interface. The PC density increases with irradiance, with a mean (±s.e.m.) peak value of 135.51 ± 26.74 µA cm−2 (10 ms) for 943.98 µW mm−2 (Fig. 4d, f). According to the literature in the ﬁeld22, these current values should be able to induce epiretinal stimulation of retinal ganglion cells (RGCs). The slope of the PC density proﬁle is decreasing while increasing irradiance, and a saturation of the response could be expected for irradiance higher than 1–2 mW mm−2. We also measured the PC density (10 ms, 943.98 µW mm−2) after 48 h of immersion in physiological Ti-based photovoltaic electrodes show a full discharge (97.7%) after 100 ms (Fig. 5a) when illuminated with 10-ms pulses NATURE COMMUNICATIONS\| (2018) 9:992 6 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 (943.98 µW mm−2); while they are discharged of 65.4 and of 89.9% after 25 ms and 50 ms respectively. This suggests that POLYRETINA could operate in the 1–20 Hz range without the need of an external shunting resistor25. To characterize the stimulation efﬁciency over repetitive stimuli, we measured the PC over 1000 stimuli (Fig. 5b) delivered at 1 Hz (10 ms, 943.98 µW mm−2). The mean (±s.e.m.) steady state response (average of the last 20 pulses/ﬁrst response) is almost unchanged (96.99 ± 1.51%). At a higher stimulation frequency, such as 10 Hz, the electrodes are entirely discharged between pulses (Fig. 5c), therefore the PC density is not largely affected by repetitive stimulations; in a train of 10 pulses at 10 Hz, the mean (±s.e.m.) ratio last/ﬁrst responses is 92.20 ± 1.52 % (Fig. 5e). Also, in a train of 20 pulses at 20 Hz, the mean (±s.e.m.) ratio last/ﬁrst responses is 90.21 ± 4.96 % (Fig. 5f). \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 10 ms b 25 ms 20 µV * a PDMS Ti PEDOT:PSS P3HT:PCBM e 80 60 40 20 0 0.0 1.0 2.0 3.0 c Firing rate (Hz)/probability (%) 60 50 40 30 0 Irradiance (mW mm–2) 0 1.0 1.5 SL spikes 3.0 0.5 20 10 2.0 2.5 0.5 1.5 2.5 d Time (ms) 8 6 4 2 0 0.0 1.0 2.0 3.0 Latency Jitter 0.5 1.5 2.5 ML spikes 70 50 30 10 f 60 40 20 0 0.0 1.0 2.0 3.0 0.5 1.5 2.5 LL spikes 50 30 10 Rate Probability SL spikes 1.08 mW mm–2 Firing rate (Hz) Firing rate (Hz) Bath Irradiance (mW mm–2) Irradiance (mW mm–2) Irradiance (mW mm–2) . 6 Evaluation ex vivo with retinal explants. a Sketch of the recording set-up together with a picture of a retinal explant over the PDMS–photovolt erface with the metal electrode used for recordings. Scale bar is 100 µm. b Representative single-sweep recording from a retinal ganglion cell over MS–photovoltaic interface upon 10-ms illumination at 1081.7 µW mm−2. The red dotted line is the threshold set for spike detection. The green ba resents the light pulse. The blue insert shows a magniﬁcation of the period around the light pulse. The asterisk indicates the over-threshold spike ected, while the gray arrows are the on-set and off-set stimulation artifacts. c Mean (±s.e.m.) ﬁring rate (circles) and ﬁring probability (squares) of kes, computed across all the recorded cells (n = 39, 10 sweeps each) on the PDMS–photovoltaic interface. For each cell, the probability has been deﬁn the percentage of sweeps with at least a SL spike over the 10 consecutive trials. d Mean (±s.e.m.) latency (circles) and jitter (squares) of the ﬁrst sp curring in the 10 ms window after the light onset, computed across all the recorded cells (n = 39, 10 sweeps each) on the PDMS–photovoltaic interfa each cell, the mean latency and jitter has been computed over the ten consecutive trials. e, f Mean (±s.e.m.) ﬁring rate of medium (e) and long ency spikes, computed across all the recorded cells (n = 39, ten sweeps each) on the PDMS–photovoltaic interface. In panels c–f values have bee tted up to 3 mW mm−2, while the full proﬁles are shown in Supplementary Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 3c–f \| / 10 ms b 25 ms 20 µV * a PDMS Ti PEDOT:PSS P3HT:PCBM 1.08 mW mm–2 Bath 10 ms b 25 ms 20 µV * 1.08 mW mm–2 Ti P3HT:PCBM a PDMS PEDOT:PSS Bath b a c Firing rate (Hz)/probability (%) 60 50 40 30 0 Irradiance (mW mm–2) 0 1.0 1.5 SL spikes 3.0 0.5 20 10 2.0 2.5 Rate Probability d Time (ms) 8 6 4 2 0 0.0 1.0 2.0 3.0 Latency Jitter 0.5 1.5 2.5 SL spikes Irradiance (mW mm–2) d c f 60 40 20 0 0.0 1.0 2.0 3.0 0.5 1.5 2.5 LL spikes 50 30 10 Firing rate (Hz) Irradiance (mW mm ) Irradiance (mW mm–2) e 80 60 40 20 0 0.0 1.0 2.0 3.0 ( ) 0.5 1.5 2.5 ML spikes 70 50 30 10 Firing rate (Hz) Irradiance (mW mm–2) f e Fig. 6 Evaluation ex vivo with retinal explants. a Sketch of the recording set-up together with a picture of a retinal explant over the PDMS–photovoltaic interface with the metal electrode used for recordings. Scale bar is 100 µm. b Representative single-sweep recording from a retinal ganglion cell over PDMS–photovoltaic interface upon 10-ms illumination at 1081.7 µW mm−2. The red dotted line is the threshold set for spike detection. The green bar represents the light pulse. The blue insert shows a magniﬁcation of the period around the light pulse. The asterisk indicates the over-threshold spike detected, while the gray arrows are the on-set and off-set stimulation artifacts. c Mean (±s.e.m.) ﬁring rate (circles) and ﬁring probability (squares) of SL spikes, computed across all the recorded cells (n = 39, 10 sweeps each) on the PDMS–photovoltaic interface. For each cell, the probability has been deﬁned as the percentage of sweeps with at least a SL spike over the 10 consecutive trials. d Mean (±s.e.m.) latency (circles) and jitter (squares) of the ﬁrst spike occurring in the 10 ms window after the light onset, computed across all the recorded cells (n = 39, 10 sweeps each) on the PDMS–photovoltaic interface. For each cell, the mean latency and jitter has been computed over the ten consecutive trials. e, f Mean (±s.e.m.) ﬁring rate of medium (e) and long (f) latency spikes, computed across all the recorded cells (n = 39, ten sweeps each) on the PDMS–photovoltaic interface. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 3f) spikes growth more progressively, but they also become stable after 1.08 mW mm−2. As a control, when retinas have been layered on bare PDMS substrates (n = 34, N = 13; 143.08 ± 32.09 days), no light-evoked responses have been detected for all Cytotoxicity and long-term functioning. To validate the long- term functioning of POLYRETINA, we tested the mechanical impact of the hemispherical shape. For this purpose, the PDMS–photovoltaic interface has been bonded on the dome- shaped PDMS support. The bonding procedure induces tensile stresses in the PDMS–photovoltaic interface leading to the for- mation of cracks in the polymers and the titanium cathodes (Fig. 8a, top row). To avoid cracks in the titanium cathodes, SU-8 rigid platforms30 have been integrated below each cathode in the substrate of the interface (Supplementary Fig. 1b). With this precaution, the pixel above the SU-8 rigid platforms is protected from cracks (Fig. 8a, bottom row); images correspond to the green area in Fig. 1c. Cracks are still formed within the blend ﬁlm in the area between SU-8 rigid platforms, however this is less critical since that area is encapsulated in PDMS to prevent delamination and the carriers photo-generated outside of the area deﬁned by the cathode do not signiﬁcantly contribute to the photo-potential/current generated at the electrode/electrolyte interface. Then, we measured the changes in the surface potential by using KPFM (Fig. 8b). Due to the hemispherical shape, only the electrodes at the top of the prosthesis (80 µm in diameter / 67 µm openings) can be reached by the AFM tip. The surface potential change induced by illumination (white LED, light from the top, 0.4 mW mm−2) is not statistically different (Mann–Whitney test, p = 0.8182) with respect to the planar PDMS-interface (Fig. 8c). To simulate the lifetime of POLYRETINA once implanted, we performed a functional accelerating ageing test by immersion in physiological saline solution hold at 87 °C (Fig. 8d). The changes of the surface potential upon illumination have been measured with KPFM before starting the ageing and at several time points during the protocol (Fig. 8e). No statistically signiﬁcant changes in the mean (±s.d.) surface potential have been detected till 24 months of accelerated ageing (one-way ANOVA, F = 0.1252, p = 0.9731). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 Last, according to ISO 10993-5: Biological Evaluation of Medical Devices, in vitro cytotoxicity has been evaluated via an 7 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 7 NATURE COMMUNICATIONS\| (2018) 9:992 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 a Light stimulus Bath Electrode 1 2 3 4 5 b d e D1 D2 D3 1 0 0.5 –150 –110 –75 –40 0 40 75 110 150 0 0.5 1 Data Fitting Simulation Eccentricity (µm) Normalized voltage 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 f –800 –600 –400 –200 0 200 400 600 800 Eccentricity (µm) 0 1 Normalized voltage 0.5 1.5 Eccentricity (µm) –800 –600 –400 –200 0 200 400 600 800 0 1 Normalized voltage 0.5 1.5 c g. 7 Spatial conﬁnement of the prosthetic stimulation. a Sketch of the experimental setting. The green circle corresponds to the area illuminated around e central pixel. Gray circles represent the illuminated pixel and the six surrounding ones. The voltage has been measured in nine positions (red dots) for ch direction (D1, D2, and D3), all cantered in the center of the illuminated pixel. b Picture during recordings. The light spot is visible (brighter area). The ale bar is 100 µm. c Voltage spreading colour map generated by interpolating the experimental measures with a triangulation-based linear interpolation. each point ten consecutive recordings have been averaged and the voltage peaks have been normalized with respect to the value obtained in the central xel (position 1 in a). The green circle is the illuminated area, while the gray circles represent the pixels. d Mean (±s.e.m.) normalized PV peaks from n = 4 xels. For each pixel, the data from the three directions have been averaged. The red line shows a Gaussian ﬁtting, while the blue dotted line represents the rmalized voltage proﬁle obtained by FEA simulations. The gray dotted lines show the FWHM value for the simulated proﬁle. e FEA simulations for three am sizes, normalized to the potential corresponding to the illumination of the single central pixel. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 f FEA simulations for various patterns of activation rmalized to the potential corresponding to the illumination of the single central pixel d –150 –110 –75 –40 0 40 75 110 150 0 0.5 1 Data Fitting Simulation Eccentricity (µm) Normalized voltage 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.9 b a Light stimulus Bath Electrode b d b a 1 0 0.5 c Light stimulus 1 2 3 4 5 D1 D2 D3 c Eccentricity (µm) 0 f Eccentricity (µm) –800 –600 –400 –200 0 200 400 600 800 0 1 Normalized voltage 0.5 1.5 e –800 –600 –400 –200 0 200 400 600 800 Eccentricity (µm) 0 1 Normalized voltage 0.5 1.5 f e Normalized voltage Normalized voltage Eccentricity (µm) Fig. 7 Spatial conﬁnement of the prosthetic stimulation. a Sketch of the experimental setting. The green circle corresponds to the area illuminated around the central pixel. Gray circles represent the illuminated pixel and the six surrounding ones. The voltage has been measured in nine positions (red dots) for each direction (D1, D2, and D3), all cantered in the center of the illuminated pixel. b Picture during recordings. The light spot is visible (brighter area). The scale bar is 100 µm. c Voltage spreading colour map generated by interpolating the experimental measures with a triangulation-based linear interpolation. At each point ten consecutive recordings have been averaged and the voltage peaks have been normalized with respect to the value obtained in the central pixel (position 1 in a). The green circle is the illuminated area, while the gray circles represent the pixels. d Mean (±s.e.m.) normalized PV peaks from n = 4 pixels. For each pixel, the data from the three directions have been averaged. The red line shows a Gaussian ﬁtting, while the blue dotted line represents the normalized voltage proﬁle obtained by FEA simulations. The gray dotted lines show the FWHM value for the simulated proﬁle. e FEA simulations for three beam sizes, normalized to the potential corresponding to the illumination of the single central pixel. f FEA simulations for various patterns of activation normalized to the potential corresponding to the illumination of the single central pixel than the saturation value measured with retinal explants (1.08 mW mm−2). corresponds to the extreme case of projecting a constant full white frame, which is not realistic in daily operation when images will be presented as black and white. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 In panels c–f values have been plotted up to 3 mW mm−2, while the full proﬁles are shown in Supplementary Fig. 3c–f implant should not exceed 2 °C above the normal surrounding body temperature of 37 °C31. We measured in air the increase in temperature on the POLYRETINA surface (Fig. 9a, b) due to continuous operation for 2 h under full-ﬁeld pulsed illumination (20 Hz, 10 ms, 1.22 mW mm−2). The irradiance has been set to the maximal allowed by the LED. The mean ( ± s.d., N = 4 prostheses) thermal increase at steady state is 1.24 ± 0.29 °C, which is below the standard limit of 2 °C. We veriﬁed also that the temperature increases on the electrodes and on the polymer surface are not different (Fig. 9c, d). Anyhow, this experiment extraction test on the murine ﬁbroblastic L929 cells. Cell viability has been estimated via an XTT cell viability assay. Results on the prosthesis showed a 100% viability, while positive control has 0.3% viability and negative control has 100% viability (averages of three repetitions; see Certiﬁcate in Supplementary Information). Thermal and optical safety. According to the thermal safety standard for active implantable medical devices (ISO 14708-1 / EN 45502-1), the maximum temperature on the surface of the NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 8 8 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 Under this condition, the average light dose is lower and therefore the related increase in temperature will be lower. In addition, the eye vitreous has a thermal conductivity about 30 times higher than air, therefore heat sinking is more efﬁcient. In case of POLYRETINA, the incident light is ﬁrst absorbed by the P3HT:PCBM layer. The mean (±s.d., N = 4 prostheses) transmittance of POLYRETINA has been experimentally mea- sured as 49.07 ± 5.25% (Supplementary Fig. 6). Therefore, only part of the incident light reaches the retina and the retinal pigmented epithelium (RPE), thus reducing the effect of retinal heating due to light absorption in the RPE. However, the light absorbed by P3HT:PCBM generates heat, that should be taken into account when evaluating the MPE. We performed FEA simulations to estimate the temperature increase in the retina upon illumination of POLYRETINA. First, we veriﬁed the temperature increase at the RPE–retina interface using the MPE obtained without POLYRETINA (328 and 1.64 mW mm−2), respectively, for continuous and pulsed (10 ms pulses at 20 Hz) illumination. After 150 s of continuous illumination (560 nm, Regarding optical safety, photovoltaic prostheses are limited by retinal damage upon light exposure32 (ANSI Z136.1 / ISO 60825 / ISO 15004). According to the standards, the maximum permissible exposure (MPE) during chronic illumination of the full POLYRETINA (equivalent to a full white frame) is controlled by the photothermal damage and equal to 328.75 µW mm−2 (see Methods). However, photovoltaic prostheses operate with pulsed illumination. With pulses of 10 ms and duty cycle of 5, 10, or 20% (respectively for 5, 10, or 20 Hz), the MPE is increased to 6.58, 3.29, or 1.64 mW mm−2, respectively. These values are higher NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 a b c Voltage change (V) Planar 3D+SU-8 0.15 0.13 0.11 0.09 0.07 0.05 Ageing time (months) – SU-8 200 µm 200 µm 200 µm 200 µm + SU-8 d e Voltage change (V) 0.20 0.16 0.12 0.08 0.04 0.00 0 6 12 24 18 Fig. 8 Lifetime of the retinal prosthesis. a Pictures of the titanium cathodes before (left column) and after (right column) bonding on the dome-shaped PDMS support. The top row is without SU-8 rigid platforms, while the bottom row is with SU-8 rigid platforms. b Picture of a KPFM measure on bonded prostheses integrating SU-8 rigid platforms. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 c Comparison of KPFM measures on bonded prostheses integrating SU-8 rigid platforms (99.35 ± 25.26 mV, mean ± s.d., n = 15; electrode diameter 80 µm) with respect to measures on PDMS-interface bonded to a planar glass substrate (105.50 ± 17.79 mV, mean ± s.d., n = 36; electrode diameter 100 µm). d Sketch of the accelerated ageing tests. KPFM measures have been performed at the beginning of the experiment, then prostheses have been immersed in saline solution at 87 °C and 100% humidity for 135 h, after that KPFM has been repeated, and on for four cycles. e Quantiﬁcation (mean ± s.d., N = 4 prostheses, n = 4 electrodes per prosthesis) of the surface potential changes (voltage in light—voltage in dark) during accelerated ageing tests over a simulated period of 24 months (months: 0, 110.5 ± 33.53 mV; 6, 108.5 ± 33.37 mV; 12, 109.8 ± 44.59 mV; 18, 103.8 ± 25.73 mV; 24, 111.1 ± 35.48 mV) a – SU-8 200 µm 200 µm 200 µm 200 µm + SU-8 b d d b a c Voltage change (V) Planar 3D+SU-8 0.15 0.13 0.11 0.09 0.07 0.05 Ageing time (months) e Voltage change (V) 0.20 0.16 0.12 0.08 0.04 0.00 0 6 12 24 18 e c Fig. 8 Lifetime of the retinal prosthesis. a Pictures of the titanium cathodes before (left column) and after (right column) bonding on the dome-shaped PDMS support. The top row is without SU-8 rigid platforms, while the bottom row is with SU-8 rigid platforms. b Picture of a KPFM measure on bonded prostheses integrating SU-8 rigid platforms. c Comparison of KPFM measures on bonded prostheses integrating SU-8 rigid platforms (99.35 ± 25.26 mV, mean ± s.d., n = 15; electrode diameter 80 µm) with respect to measures on PDMS-interface bonded to a planar glass substrate (105.50 ± 17.79 mV, mean ± s.d., n = 36; electrode diameter 100 µm). d Sketch of the accelerated ageing tests. KPFM measures have been performed at the beginning of the experiment, then prostheses have been immersed in saline solution at 87 °C and 100% humidity for 135 h, after that KPFM has been repeated, and on for four cycles. Discussion 328 µW mm−2), the temperature increase is stable at 0.42 °C (Supplementary Fig. 7a, b). Pulsed illumination (10 ms pulses at 20 Hz, 1.64 mW mm−2) generates temperature spikes of about 0.04 °C, oscillating around the proﬁle corresponding to the continuous illumination (Supplementary Fig. 7c, d). This demonstrate that the scaling factor of 5 to estimate the MPE during pulsed stimulation (20% duty cycle) is correct. Continuous illumination has been used in the following simulations to reduce the computational cost. With POLYRETINA the temperature increase after 150 s of continuous illumination (560 nm, 328 µW mm−2) is slightly reduced to 0.37 °C (Fig. 10a, b). In this case, the critical interface is the one between the retina and the prosthesis (Supplementary Fig. 8a, b) giving a slightly higher temperature increase with respect to the RPE-retina interface (0.37 vs. 0.35 ° C). POLYRETINA has been simulated in direct contact with the retina because this represents the worst-case scenario. A thin space of vitreous (100 µm) between the retina and POLYRETINA reduces the temperature increase by 0.009 °C, which is negligible. Thermal damage of the retina requires a local rise in temperature higher than 10 °C33; the 50% of probability of retinal damage (ED50) has been previously deﬁned for a temperature rise of 12.5 °C31. In our model, we estimated the ED50 with (red) and without (black) POLYRETINA (Fig. 10c). As expected the ED50 for continuous illumination is slightly higher when POLYRE- TINA is present (10.6 vs. 9.4 mW mm−2), which correspond to 53 mW mm−2 for pulsed illumination. A comparison with and without POLYRETINA showed that over the broad range of irradiances the temperature increase in the retina is reduced by 11% with POLYRETINA. Therefore, the MPE could be slightly increased to 1.84 mW mm−2 and POLYRETINA can safely operates at 1 mW mm−2. One of the most important open questions in the ﬁeld of retinal prostheses concerns how to increase both visual acuity and visual ﬁeld size together. From the engineering point of view this implies to increase the density of the stimulating electrodes and enlarge the size of the prosthesis. POLYRETINA is a novel foldable and photovoltaic wide-ﬁeld epiretinal prosthesis with a remarkable increase in its size (46.3 degrees) and in the number of stimu- lating pixels (2215) compared to other epiretinal prostheses4,34. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 e Quantiﬁcation (mean ± s.d., N = 4 prostheses, n = 4 electrodes per prosthesis) of the surface potential changes (voltage in light—voltage in dark) during accelerated ageing tests over a simulated period of 24 months (months: 0, 110.5 ± 33.53 mV; 6, 108.5 ± 33.37 mV; 12, 109.8 ± 44.59 mV; 18, 103.8 ± 25.73 mV; 24, 111.1 ± 35.48 mV) \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 d Mean (±s.d.) changes in surface temperature in the average surface, the electrode area or the polymer area are not signiﬁcantly different (1.24 ± 0.29, 1.23 ± 0.20, 1.31 ± 0.21, respectively; one-way ANOVA, F = 0.0569, p = 0.9451) Fig. 9 Temperature variation during operation. a The top surface of POLYRETINA has been imaged with a thermal camera while pulsed illumination has been provided from the bottom, as in the epiretinal conﬁguration. The camera has been focused on the top electrodes and a ROI has been selected to measure the changes in surface temperature (cyan circle). Electrodes show higher value of baseline temperature because the metallic surface reﬂects part of the IR light used for the measurement. b Mean (±s.d.) changes in surface temperature measured in N = 4 prostheses. Data have been plotted has difference with respect to the baseline temperature measured for 5 min before pulsed illumination. The green bar represents the period of 2 h when light pulses have been applied (10 ms pulses, 20 Hz repetition rate, 1.22 mW mm−2). The dotted red line represents the maximal allowed temperature increase. c Mean (±s.e.m.) changes in surface temperature measured on the electrodes (left, N = 4 prostheses) or on the polymer area (right, N = 4 prostheses). For each prosthesis, n = 3 electrodes/areas have been sampled and averaged. d Mean (±s.d.) changes in surface temperature in the average surface, the electrode area or the polymer area are not signiﬁcantly different (1.24 ± 0.29, 1.23 ± 0.20, 1.31 ± 0.21, respectively; one-way ANOVA, F = 0.0569, p = 0.9451) a Time (s) b 0 25 75 100 150 0 0.7 Temperature increase (°C) 0 0.4 0.1 0.2 0.3 125 50 Irradiance (mW mm–2) c 0.1 1 100 1000 Damage probability (%) 0 100 20 40 60 80 10 CW, 328 µW mm–2, 1 Hz Log Gaussian fit ED50 + POLYRETINA – POLYRETINA Fig. 10 FEA simulation of thermal effects with POLYRETINA. a Temperature increase in the modeled eye with POLYRETINA after 150 s of continuous illumination (CW, 560 nm, 328 µW mm−2). The insert shows a larger view of the modeled retina and POLYRETINA. b Time course of the temperature increase in the modeled retina during 150 s of continuous illumination (CW, 560 nm, 328 µW mm−2). The simulation frequency has been set to 1 Hz. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 a 3.0 2.5 2.0 1.5 1.0 0.5 Temperature change (°C) b Time (s) 0 1000 2000 3000 4000 5000 0 –0.5 –1.0 29.3 26.1 c 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0 3.0 2.5 2.0 1.5 1.0 0.5 Temperature change (°C) Time (s) 0 1000 2000 3000 4000 5000 0 –0.5 –1.0 3.0 2.5 2.0 1.5 1.0 0.5 Time (s) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0 –0.5 –1.0 d 9000 8000 7000 6000 9000 8000 7000 6000 Area Electrode Polymer Fig. 9 Temperature variation during operation. a The top surface of POLYRETINA has been imaged with a thermal camera while pulsed illumination has been provided from the bottom, as in the epiretinal conﬁguration. The camera has been focused on the top electrodes and a ROI has been selected to measure the changes in surface temperature (cyan circle). Electrodes show higher value of baseline temperature because the metallic surface reﬂects part of the IR light used for the measurement. b Mean (±s.d.) changes in surface temperature measured in N = 4 prostheses. Data have been plotted has difference with respect to the baseline temperature measured for 5 min before pulsed illumination. The green bar represents the period of 2 h when light pulses have been applied (10 ms pulses, 20 Hz repetition rate, 1.22 mW mm−2). The dotted red line represents the maximal allowed temperature increase. c Mean (±s.e.m.) changes in surface temperature measured on the electrodes (left, N = 4 prostheses) or on the polymer area (right, N = 4 prostheses). For each prosthesis, n = 3 electrodes/areas have been sampled and averaged. Discussion Concerning visual ﬁeld, POLYRETINA has the potential to cover a retinal surface corresponding to a visual angle of 46.3 degrees, which is larger than the threshold for both legal blind- ness (20 degrees) and adequate mobility skills (30 degrees). Concerning spatial resolution, the presence of a continuous semiconductor layer does not represent a limitation. In organic photovoltaics, the low carrier mobility and lifetime limit the carrier–transport length to tens of nm for holes and few hun- dreds of nm for electrons35. It has been shown by another group that the PC detected at the cathode is reduced to about 10% of the maximum if the illumination spot (size 1 µm) is moved laterally by about 12 µm from the electrode edge36. This large decay length, beyond the simple diffusion processes, has been explained by a steady state nonlocal electric ﬁeld inducing a lateral ﬂow of the separated carriers. For this reason, an internal cross-talk between electrodes due to charge carriers generated under one electrode traveling laterally towards an adjacent electrode can be excluded (at least down to an edge-to- edge distance of about 20 µm). By measuring the voltage spread in solution together with FEA simulations we showed that the area of activation (about 100 µm) of 1 pixel is comparable to the pixel size. NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 10 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 ARTICLE \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 d Mean (±s.d.) changes in surface temperature in the average surface, the electrode area or the polymer area are not signiﬁcantly different (1.24 ± 0.29, 1.23 ± 0.20, 1.31 ± 0.21, respectively; one-way ANOVA, F = 0.0569, p = 0.9451) a 3.0 2.5 2.0 1.5 1.0 0.5 Temperature change (°C) b Time (s) 0 1000 2000 3000 4000 5000 0 –0.5 –1.0 29.3 26.1 9000 8000 7000 6000 b a Time (s) c 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0 3.0 2.5 2.0 1.5 1.0 0.5 Temperature change (°C) Time (s) 0 1000 2000 3000 4000 5000 0 –0.5 –1.0 3.0 2.5 2.0 1.5 1.0 0.5 Time (s) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0 –0.5 –1.0 d 9000 8000 7000 6000 Area Electrode Polymer ( ) 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0 3.0 2.5 2.0 1.5 1.0 0.5 Time (s) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0 –0.5 –1.0 d Area Electrode Polymer c 3.0 2.5 2.0 1.5 1.0 0.5 Temperature change (°C) Time (s) 0 1000 2000 3000 4000 5000 0 –0.5 –1.0 9000 8000 7000 6000 d c Time (s) Time (s) Fig. 9 Temperature variation during operation. a The top surface of POLYRETINA has been imaged with a thermal camera while pulsed illumination has been provided from the bottom, as in the epiretinal conﬁguration. The camera has been focused on the top electrodes and a ROI has been selected to measure the changes in surface temperature (cyan circle). Electrodes show higher value of baseline temperature because the metallic surface reﬂects part of the IR light used for the measurement. b Mean (±s.d.) changes in surface temperature measured in N = 4 prostheses. Data have been plotted has difference with respect to the baseline temperature measured for 5 min before pulsed illumination. The green bar represents the period of 2 h when light pulses have been applied (10 ms pulses, 20 Hz repetition rate, 1.22 mW mm−2). The dotted red line represents the maximal allowed temperature increase. c Mean (±s.e.m.) changes in surface temperature measured on the electrodes (left, N = 4 prostheses) or on the polymer area (right, N = 4 prostheses). For each prosthesis, n = 3 electrodes/areas have been sampled and averaged. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 A further NATURE COMMUNICATIONS\| (2018) 9:992 11 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 POLYRETINA will help in deﬁning the appropriate stimulation parameters to obtain a more focal stimulation. improvement consists in reducing the size of the electrode (i.e., 60 µm) with a pitch of 80 µm, thus approaching a theoretical visual acuity of 20/300, similar to the silicon photovoltaic sub- retinal prosthesis15. However, these values come from theoretical computation, and therefore must be validated with proper in vivo experiments in animals and later in humans. Moreover, the reduction of the pixel size will reduce the PC generated by the interface, therefore the efﬁciency in stimulating RGCs should be validated again. Taking advantage of accelerated ageing experiments, we demonstrated that POLYRETINA preserves its optoelectronic functions unaltered for at least 2 years. More experiments and additional time points will be added to investigate the entire lifetime of the prosthesis. Last, POLYRETINA fulﬁls the requirements for in vitro cytotoxicity according to ISO 10993-5 and for thermal safety (ISO 14708-1/EN 45502-1). y POLYRETINA is foldable to allow implantation through a small scleral incision and it self-opens once released into the eye. Although it could operate in both epiretinal and subretinal con- ditions, it has been designed as an epiretinal prosthesis, since the implantation of a large retinal prosthesis in the subretinal space may result in an excessive damage to the remaining retinal tissue. Moreover, an epiretinal placement may allow an easier replace- ment in case of malfunction (e.g., due to ageing or detachment). The hemispherical shape has been obtained by bonding the PDMS-photovoltaic interface on a dome-shaped PDMS support with a radius of curvature of 12 mm. However, the ﬂexibility in the fabrication process of the dome-shaped PDMS support (PDMS molding) allows the fabrication of prostheses designed to ﬁt the real eye curvature/shape of a patient44. This opens up the possibility to an optimized retinal prosthesis according to per- sonal needs. Last, the shape of the prosthesis and the insertion strategy have been inspired by the widely use intra ocular lenses. With further investigations, a similar ‘injection’ approach could also be envisaged for POLYRETINA, simplifying even more the surgical approach. A future improvement for human use may include the removal of electrodes in correspondence of the optic nerve head and the creation of small holes within the substrate to allow metabolic exchange between the vitreous and the retina. Methods P th i Prosthesis micro-fabrication. PDMS-photovoltaic interfaces were fabricated on silicon wafers. A thin sacriﬁcial layer of poly(4-styrenesulfonic acid) solution (561223, Sigma-Aldrich) was spin-coated on the wafers (1000 rpm, 40 s) and baked (120 °C, 15 min). Degassed PDMS pre-polymer (10:1 ratio base-to-curing agent, Sylgard 184, Dow-Corning) was then spin-coated (1000 rpm, 60 s) and cured in oven (80 °C, 2 h). After surface treatment with oxygen plasma (30 W, 30 s), a 6-µm thick SU-8 (GM1060, Gersteltec) layer was spin-coated (3800 rpm, 45 s), soft- baked (130 °C, 300 s), exposed (140 mJ cm−2, 365 nm), post-baked (90 °C, 1800 s; 60 °C, 2700 s), developed in propylene glycol monomethyl ether acetate (48443, Sigma-Aldrich) for 2 min, rinsed in isopropyl alcohol, and dried with nitrogen. After surface treatment with oxygen plasma (30 W, 30 s), a second layer of degassed PDMS pre-polymer (10:1) was spin-coated (3700 rpm, 60 s) and cured in oven (80 °C, 2 h). PEDOT:PSS (HTL Solar, Ossila) was ﬁltered (1 μm PTFE ﬁlters) and then spin-coated (3000 rpm, 60 s) onto the O2-plasma treated (30 W, 30 s) PDMS surface. Subsequent annealing (120 °C, 30 min) was performed. The pre- paration of the organic semiconductor blend was performed in a glovebox under argon atmosphere. Twenty milligrams of P3HT (698997, Sigma Aldrich) and 20 mg of PCBM (M111, Ossila) were dissolved in 1 ml of anhydrous chlorobenzene each and let stirring overnight at 70 °C. The solutions were then ﬁltered (0.45 μm PTFE ﬁlters) and blended [1:1 v:v]. The P3HT:PCBM blend was then spin-coated at 1000 rpm for 60 s. Titanium cathodes were deposited by DC sputtering through a shadow mask aligned with the SU-8 pattern. After surface treatment with oxygen plasma (30 W, 15 s), the encapsulation layer of degassed PDMS pre-polymer (5:1 ratio) was spin-coated (4000 rpm, 60 s) and cured in oven (80 °C, 2 h). To expose the cathodes, photolithography and PDMS dry etching were performed. The wafers were then placed in deionized water to allow dissolution of the sacriﬁcial layer and the release of the PDMS-photovoltaic interfaces. The ﬂoating membranes were ﬁnally collected and dried in air. The dome-shaped PDMS supports were fabricated using a milled PMMA mold, ﬁlled with PDMS pre-polymer (10:1), which was then degassed and cured in oven (80 °C, 2 h). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 The solid line is the log Gaussian ﬁt (R2 = 0.9934). c Probability of retinal damage as a function of irradiance with (red) and without (black) POLYRETINA. ED50 corresponds to a temperature increase of 12.5 °C. The irradiance has been expressed for pulsed illumination (20% of duty cycle). The solid lines are the Sigmoidal ﬁts (R2 = 0.9971 for black and 0.9977 for red) Time (s) b 0 25 75 100 150 Temperature increase (°C) 0 0.4 0.1 0.2 0.3 125 50 CW, 328 µW mm–2, 1 Hz Log Gaussian fit a 0 0.7 a Time (s) b 0 25 75 100 150 0 0.7 Temperature increase (°C) 0 0.4 0.1 0.2 0.3 125 50 c Damage probability (%) CW, 328 µW mm–2, 1 Hz Log Gaussian fit Irradiance (mW mm–2) c 0.1 1 100 1000 Damage probability (%) 0 100 20 40 60 80 10 ED50 + POLYRETINA – POLYRETINA b c a Fig. 10 FEA simulation of thermal effects with POLYRETINA. a Temperature increase in the modeled eye with POLYRETINA after 150 s of continuous illumination (CW, 560 nm, 328 µW mm−2). The insert shows a larger view of the modeled retina and POLYRETINA. b Time course of the temperature increase in the modeled retina during 150 s of continuous illumination (CW, 560 nm, 328 µW mm−2). The simulation frequency has been set to 1 Hz. The solid line is the log Gaussian ﬁt (R2 = 0.9934). c Probability of retinal damage as a function of irradiance with (red) and without (black) POLYRETINA. ED50 corresponds to a temperature increase of 12.5 °C. The irradiance has been expressed for pulsed illumination (20% of duty cycle). The solid lines are the Sigmoidal ﬁts (R2 = 0.9971 for black and 0.9977 for red) Concerning visual acuity, with a pitch of 150 µm the theoretical visual acuity restored by POLYRETINA is in the order of 20/6006; which is better than the current epiretinal prostheses (e.g., Argus II) but still below the threshold of legal blindness. However, the technology of POLYRETINA is highly scalable. Based on mechanical simulations (not shown), the pitch can be reduced down to a value of 110 µm, keeping the same electrode size (80 µm), thus approaching the theoretical value of 20/400. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 On the functional point of view, the next step is the electro- physiological validation in vivo with large animal models, such as swine models. g To be used as retinal prosthesis, POLYRETINA must operate with a stimulation rate higher that 1 Hz. The subretinal prosthesis Alpha IMS operates in a frequency range of 1 to 20 Hz (variable from patient to patient) with a pulse duration of 1–4 ms37. Available pulse rates in the Argus II are in the range of 3–60 Hz38; however, also in this case the effect of pulse rate have been reported to be very variable among subjects39. This suggests that, even if overall the variation in the pulse rate does not have a signiﬁcant effect, an optimal pulse rate can be deﬁned for each subject. Moreover, the recent identiﬁcation of an optimal pulse duration of 25 ms per phase40 may limit the operating range of Argus II to a theoretical limit of 20 Hz. For the silicon photo- voltaic subretinal prosthesis, the stimulation frequency is mainly limited by the discharge rate of the electrode, therefore a shunt resistor has been included to allow faster stimulations (20–40 Hz) 25 up to ﬂicker fusion15. POLYRETINA shows a fast discharge of the Ti-based photovoltaic electrodes (probably due to the high shunting capacity of the P3HT:PCBM layer), and we demon- strated its functioning up to 20 Hz of stimulation rate without an additional shunting resistor. This is within the operation range of other epiretinal (e.g., Argus II) and subretinal (e.g., Alpha IMS) prostheses. The activation of RGCs has be obtained already at 47.35 µW mm−2 with a response saturation above 1.08 mW/mm2. How- ever, recording ex vivo with retinal explants may not be repre- sentative of the complexity of retinal stimulation in vivo in humans, where the electrode-to-cell distance could be larger and increase during years of implantation41, thus increasing the perceptual threshold. The hemispherical design is a solution to reduce the electrode-cell distance over the area of the prosthesis. Moreover, the capability of activating RGCs at low irradiance is promising in perspective of an in vivo application. In a future development, titanium/titanium nitride electrodes can be fabri- cated in order to improve the stimulation efﬁciency (because of their higher charge injection capacity). \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications ARTICLE Each accelerated ageing session between KPFM measures lasted for 135 h, corresponding to 6 months. Before KPFM, samples were removed from the sealed falcon tube, rinsed with deionized water, and dried under nitrogen ﬂow. Spatial selectivity measures and modeling. Measures of the voltage spread have been performed in Ames’ medium (A1420, Sigma-Aldrich) at 32 °C with a glass micropipette (tip diameter about 15 μm). Data were ampliﬁed (Model 3000, A-M System), ﬁltered (DC-1000 Hz), and digitalized at 30 kHz (Micro1401–3, CED Ltd.). Illumination was carried out on a Nikon Ti-E inverted microscope (Nikon Instruments) by the Spectra X system (Emission ﬁlter 560/32, Lumencor). The microscope was equipped with a dichroic ﬁlter (FF875-Di01–25 × 36, Semrock) and a 10 × objective. A pin-hole was used to limit the spot diameter to about 150–170 μm. After alignment of the illumination spot on a target pixel of the central area of POLYRETINA, ten pulses of 10 ms were delivered at 1 Hz with an irradiance of 29.07 mW mm−2. The resulting voltage has been measured at nine positions in three directions around the illuminated pixel. Data analysis was conducted in Matlab (Mathworks). Voltage peaks above noise level (mean noise threshold 6.2 μV) have been detected and their amplitude normalized respect to the central pixel value. Simulations were performed in COMSOL Multiphysics 5.2, with a stationary electric currents study. The titanium cathodes were set at 0.1 V, while PEDOT:PSS was put at 0 V. The ground was situated at the bath top and lateral walls that were placed 2 mm and 1 mm away from the central pixel, respectively (cylindrical geometry). Line plots shown in the results were taken at a distance of 20 µm from the titanium surface. For each material, the conductivity (S m−1) and relative permittivity is listed: titanium (2.6 × 106/1), P3HT:PCBM (0.1/ 3.4), PEDOT:PSS (30/3), Saline (1/80), PDMS (2 × 10−14/2.75). Measure of PV and PC. In this experiment, the photovoltaic interface has been fabricated directly on glass (without PDMS) to avoid breaking the connecting lines from the electrode to the pad when contacted. The titanium electrodes have been fabricated with a diameter of 100 µm; however, when evaluating the PC density generated by the interface, also the area of the connecting line exposed to light has been considered. A plastic reservoir was attached to the chip using PDMS as adhesive. ARTICLE Chips were placed on a holder, and each pad was sequentially contacted. Silver paste was used to improve the electrical contact. An Ag/AgCl pellet immersed in physiological saline solution (NaCl 0.9 %) was used as reference electrode. Light pulses were delivered by a 565-nm Green LED (Thorlabs, M565L3- C5) focused at the sample level. PV was measured using a voltage ampliﬁer (DL- Instruments, 1201; gain 20, band DC-3000 Hz) and PC using a current ampliﬁer (DL-Instruments, 1212; gain 10−6 A/V). Data sampling (16 kHz) and instrument synchronization were obtained via a DAQ board (PCIe-6321, National Instru- ments) and a custom-made software. Data analysis was performed in Matlab (Mathworks). Due to a limitation in the acquisition system, long pulse trains (Fig. 5e) have been delivered in packages of 20 pulses at 20 Hz (total of 1 s), while each package was separated by 1 s needed by the system to save data before starting the next package. Optical safety. Retinal damage upon light exposure can occur because of three main factors: photo-thermal damage, photo-chemical damage, and thermo- acoustic damage31. The ﬁrst one is related to retinal heating upon light absorption by the melanin in the RPE. The second one occurs at short wavelengths (less than 600 nm) and for exposures longer that 1 s. The latter occurs for short pulses (less than 1 ns) and is associated with nonlinear photo-mechanical effects. POLY- RETINA functions with 10 ms green light pulses; therefore, this limit could be controlled by the photo-thermal or photo-chemical damage. According to the ANSI Z136.1 Standard32, the MPE allowed for ophthalmic applications can be calculated (in W) according to equation (1) for photo-thermal damage (MPET) and equation (2) for photo-chemical damage (MPEC). Those equations are valid for λ = 560 nm and α = 808.12 mrad (Supplementary Fig. 1c). Electrophysiology. Experiments were conducted under the animal authorizations VD3055 and GE3717. Retinas were explanted in normal light conditions from mice sacriﬁced by injection of Sodium Pentobarbital (150 mg kg−1). After eye enuclea- tion, retinas were dissected in carboxygenated (95% O2 and 5% CO2) Ames’ medium (A1420, Sigma-Aldrich) and transferred to the microscope stage for recordings. In the experiment with synaptic blockers, Ames’ medium was sup- plemented with DL-AP4 (250 μM l−1, No. 0101, Tocris Bioscience), DL-AP5 (50 μM l−1, No. 0105, Tocris Bioscience), DNQX (10 μM l−1, No. 0189, Tocris Bioscience), Carbenoxolone (100 μM l−1, No. 3096, Tocris Bioscience). ARTICLE PEDOT:PSS (HTL Solar, Ossila) was ﬁltered (1 μm PTFE ﬁlters) then spin-coated at 3000 rpm for 60 s on each chip. Subsequent annealing at 120 °C for 30 min was performed. The preparation of the organic semiconductor blend was performed as described before. The P3HT:PCBM blend was then spin-coated at 1000 rpm for 60 s on each chip. Aluminum cathodes were deposited by thermal evaporation using a shadow mask; titanium cathodes were deposited by DC sputtering using a shadow mask. When present, degassed PDMS pre-polymer (10:1) was deposited on the glass substrate by spin-coating (1000 rpm, 60 s) and then cured in oven (80 °C, 2 h). Kelvin probe force microscopy. KPFM characterization was performed in ambient conditions with an Asylum Research Cypher S microscope using PtIr coated tips (AC240TM, Asylum Research, Oxford Instrument) in surface potential imaging mode. To measure the surface potential variation, KPFM images were collected by repetitively scanning a single line of 100 nm under dark and illumi- nation conditions. The white LED of the microscope positioned above the tip and sample was acting as light source and it was manually turned 100 % off and 100 % on to reach the desired conditions. KPFM images were analyzed using Gwyddion 2.36 software. For each image, the average surface potential variation value was obtained by subtracting the surface potential under illumination to the one under dark (voltage in light–voltage in dark). pH measurements. Experiments have been performed in phosphate-buffered saline at room temperature. Illumination was carried out on a Nikon Ti-E inverted microscope (Nikon Instruments) by the Spectra X system (Emission ﬁlter 560/32, Lumencor). The microscope was equipped with a dichroic ﬁlter (FF875-Di01–25 × 36, Semrock) and a 10× (diameter of the illumination spot 2.2 mm; CFI Plan Apochromat Lambda) objective. Light pulses of 10 ms where delivered at 20 Hz for 1 h with an irradiance of 3.4 mW mm−2. pH was measured with a microelectrode (tip diameter of 200 µm) with internal reference (pH-200C, Unisense). Data were sampled at 1 H. Accelerated ageing tests. Accelerated ageing was performed in a dry oven set to 87 °C. Samples were immersed in physiological saline solution (0.9 % NaCl, pH 7.4) within a sealed 50-ml falcon tube. Under this condition, the acceleration factor was 3245,46. KPFM measures were obtained before starting the ageing protocol and at several time points during ageing. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 µW mm−2), 2% + ND4 (47.38 µW mm−2), 3% + ND4 (107.91 µW mm−2), 2% (189.50 µW mm−2), 3% (421.12 µW mm−2), 3% (815.92 µW mm−2), 5% (1081.75 mW mm−2), 10% (2.81 mW mm−2), 20% (5.89 mW mm−2), 40% (11.98 mW mm −2), 60% (17.92 mW mm−2), 80% (23.56 mW mm−2), and 100% (29.08 mW mm −2). Spike detection and sorting were performed by threshold detection using the Matlab-based algorithm Wave_clus47 and further data processed in Matlab (Mathworks). The threshold for spike detection has been deﬁned as 3.7 times the standard deviation of the background noise. The minimum refractory period between spikes of the same class was set to 1.4 ms. To ensure the rejection of artifacts, an exclusion period of ± 1 ms around light onset and offset was applied. However, the spikes in the ﬁrst 10 ms after the light onset (SL) have been manually veriﬁed. PSTHs for each condition of illumination were computed discretizing and averaging spike raster obtained over ten stimulations repetitions into bins of 10 ms. Spikes were sorted from individual PSTHs and classiﬁed according to their timing after light onset (cyan bars in Supplementary Fig. 3a) in SL (<10 ms), ML (from 40 to 120 ms), and LL (from 150 to 350 ms)28. Firing rates in the three groups were measured as follow. For SL spikes the ﬁrst bin (10 ms) after the pulse was used. For ML spikes 3 bins (30 ms) in the deﬁned time range, centered in the highest bin, were used. For LL spikes 5 bins (50 ms) in the deﬁned time range, centered in the highest bin, were used. stretching of the ﬁxed membrane. The excessive PDMS used to clamp the array was removed by laser cutting. stretching of the ﬁxed membrane. The excessive PDMS used to clamp the array was removed by laser cutting. stretching of the ﬁxed membrane. The excessive PDMS used to clamp the array was removed by laser cutting. Chips micro-fabrication. Chips for KPFM and PC/PV measurements were fab- ricated on 20 × 24 mm2 glass substrates (2947–75 × 50, Corning Incorporated). Before micro-fabrication, glass chips were cleaned by ultra-sonication in deionized water, acetone and isopropyl alcohol for 15 min each and then dried with nitrogen. ITO (200 nm) was deposited on glass chips by RF sputtering. Methods P th i The supports were released from the molding parts and perforated with a hole-puncher (330 µm in diameter) at the locations dedicated to the insertion of retinal tacks. The released PDMS- photovoltaic interfaces were clamped between two O-rings and, together with the PDMS supports, were exposed to oxygen plasma (30 W, 30 s). The activated PDMS surfaces were put in contact and allowed to uniformly bond thanks to radial g g j y The presence of SL spikes is an evidence in support of a direct activation of RGCs. On the contrary, ML and LL spikes are due to the activation of the internal retinal circuit. In literature, SL spikes are reported to be very close (i.e., 0.5–4 ms) to the stimulus28, which is typically a sharp squared pulse. The photo voltage/cur- rent generated by POLYRETINA have a less shaper transition from 0 to the peak (in about 10 ms). This may explain why the average latency is 4.12 ± 0.07 ms and we considered as SL spikes those with a latency in the 0–10 ms window. It is known that brief (hundreds of µs) cathodic epiretinal stimulation preferentially excite RGCs, while pulses longer than 1 ms excite both RGCs and bipolar cells42,43. It has been recently demonstrated that the use of pulses shorter than 8 ms results in the activation of axons of passage that causes streak responses, while longer pulses results in a more focal activation40. Using calcium imaging techniques, authors explained this result via a shift from direct to indirect activation of RGCs. We showed by electrophysiological record- ings and pharmacological experiments that the cathodic stimu- lation provided by POLYRETINA is also indirectly activating RGCs. This represents a promising result for the in vivo trans- lation of POLYRETINA in order to obtain a focal activation. Further experiments aiming at dissecting the circuit activated by NATURE COMMUNICATIONS\| (2018) 9:992 12 References Med. 21, 476–482 (2015). 16. Ghezzi, D. et al. A hybrid bioorganic interface for neuronal photoactivation. Nat. Commun. 2, 166 (2011). 17. Ghezzi, D. et al. A polymer optoelectronic interface restores light sensitivity in blind rat retinas. Nat. Photonics 7, 400–406 (2013). 18. Antognazza, M. et al. Characterization of a Polymer‐based, fully organic prosthesis for implantation into the subretinal space of the rat. Adv. Healthc. Mater. 5, 2271–2282 (2016). 19. Maya-Vetencourt, J. et al. A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness. Nat. Mater. 16, 681–689 (2017). 20. Lee, D. Y., Lorach, H., Huie, P. & Palanker, D. Implantation of modular photovoltaic subretinal prosthesis. Ophthalmic Surg. Lasers Imaging Retin. 47, 171–174 (2016). Surgical implantation. Plastic eye models (Eyelabinnovations, Austria) and enucleated pig eyes were used. First three 23-gauge transconjunctival valved canulas (DORC, Zuidland, The Netherlands) were inserted into the eye at 4 mm from the limbus at the following positions: nasal superior, temporal superior and temporal inferior. A balanced salt solution infusion was hooked up to the eye to maintain a constant intraocular pressure through one of the cannulas. A 6.5-mm long incision was then performed using a 15° scalpel. The implant was folded using special forceps and then inserted through the incision into the posterior cavity. Once inside the eye the forceps grip was released and the implant could unfold. Using a light pipe and an intraocular 23-gauge forceps inserted through the other two cannulas the implant was then manipulated and ﬁxed in epi- retinal conﬁgurations. 21. Minev, I. et al. Electronic dura mater for long-term multimodal neural interfaces. Science 347, 159–163 (2015). 22. Bareket, L. et al. Semiconductor nanorod-carbon nanotube biomimetic ﬁlms for wire-free photostimulation of blind retinas. Nano. Lett. 14, 6685–6692 (2014). 23. Cogan, S. Neural stimulation and recording electrodes. Annu. Rev. Biomed. Eng. 10, 275–309 (2008). g 24. Merrill, D. R., Bikson, M. & Jefferys, J. G. Electrical stimulation of excitable tissue: design of efﬁcacious and safe protocols. J. Neurosci. Methods 141, 171–198 (2005). 25. Boinagrov, D. et al. Photovoltaic pixels for neural stimulation: circuit models and performance. IEEE Trans. Biomed. Circuits Syst. 10, 85–97 (2016). Statistical analysis and graphical representation. Statistical analysis and gra- phical representation were performed with Prism (GraphPad Software Inc.). The normality test (D’Agostino & Pearson omnibus normality test) was performed in each dataset to justify the use of a parametric or non-parametric test. References 1. Bourne, R. R. et al. Causes of vision loss worldwide. 1990–2010: a Syst. Anal. Lancet Glob. Health 1, e339–e349 (2013). Thermal modeling. Simulations were performed in COMSOL Multiphysics 5.2 with the Bioheat module for the heat transfer equation and the General PDE module for the Beer–Lambert light propagation. Illumination has been modeled as a uniform beam with a diameter of 13 mm. The eye is a 2D axi-symmetric model composed of several spheres representing each domain (Supplementary Fig. 9). A total of 8 domains (Cornea, Aqueous Humor, Lens, Vitreous Humor, Retina, RPE, Choroid and Sclera) are deﬁned in the model, with the parameters listed in Sup- plementary Table 1. POLYRETINA was modeled as a single composite material, with volume averaged properties of PDMS, Pedot:PSS, P3HT:PCBM and Titanium (Supplementary Table 2). It was simpliﬁed into 5 domains with homogeneous properties: the center, the ﬁrst ring, the second ring, the domains where no tita- nium is present, and PDMS only (Supplementary Fig. 9). A volume average has been performed on each of this domain, to obtain the parameters for the aggre- gated material. To account for the non-uniform distribution of titanium, the fraction area of titanium was considered. To validate the parameters of the aggregated model, a simulation has been performed with POLYRETINA in air exposed to continuous illumination (560 nm, 244 µW mm−2) corresponding to pulsed illumination of 1.22 mW mm−2. The heat losses at the prosthesis interface- air were radiative (emissivity = 0.9) and convective (heat transfer coefﬁcient = 38.5 W m−2 K−1). In agreement with our experimental results, the average transmit- tance was measured to be 51.67% (49.07% in Supplementary Fig. 6) and the steady- state temperature increase was 1.25 °C (1.24 °C in Fig. 9). 2. World Health Organization. The 11th Revision of the International Classiﬁcation of Diseases (ICD-11) http://www.who.int/classiﬁcations/icd/en/ (2018). 3. Ghezzi, D. Retinal prostheses: progress toward the next generation implants. Front. Neurosci. 9, 290 (2015). 4. Luo, Y. & da Cruz, L. The Argus® II Retinal Prosthesis System. Prog. Retin. Eye Res. 50, 89–107 (2016). 5. Stingl, K. et al. Subretinal visual Implant Alpha IMS—clinical trial interim report. Vision Res. 111, 149–160 (2015). 6. Palanker, D., Vankov, A., Huie, P. & Baccus, S. Design of a high-resolution optoelectronic retinal prosthesis. J. Neural Eng. 2, S105 (2005). 7. Cha, K., Horch, K. & Normann, R. Mobility performance with a pixelized vision system. Vision. Res. References 32, 1367–1372 (1992). y 8. Dagnelie, G. et al. Real and virtual mobility performance in simulated prosthetic vision. J. Neural Eng. 4, S92 (2007). 9. Ameri, H. et al. Toward a wide-ﬁeld retinal prosthesis. J. Neural Eng. 6, 035002 (2009). 10. Waschkowski, F. et al. Development of very large electrode arrays for epiretinal stimulation (VLARS). Biomed. Eng. Online 13, 11 (2014). 11. The Lasker/IRRF Initiative for Innovation in Vision Science Restoring vision to the blind: the new age of implanted visual prostheses. Transl. Vis. Sci. Technol. 3, 3–3 (2014). 12. Fornos, A., Sommerhalder, J., Rappaz, B., Safran, A. & Pelizzone, M. Simulation of artiﬁcial vision, III: do the spatial or temporal characteristics of stimulus pixelization really matter? Invest. Ophthalmol. Vis. Sci. 46, 3906–3912 (2005). In vitro cytotoxycity test. The study validation was performed by an accredited company (Medistri SA). The test was conducted according to the requirement of ISO 10993-5: Biological Evaluation of Medical Devices, in vitro cytotoxicity test; ISO 10993-12: Test article preparation and reference materials; USP 35-NF30 (87): Biological Reactivity test, invitro; Medistri internal procedure WI 47 and WI 56. Prostheses were sterilized with EtO prior the test. The test on extraction was performed with two retinal prostheses for a total surface area of 3.54 cm2, with a ratio of the product to extraction vehicle of 3 cm2 ml−1. Extraction vehicle was Eagle’s Minimum Essential Medium supplemented with fetal bovine serum, penicillin–streptomycin, amphotericin B, and L-glutamine. The extraction was performed for 24 h at 37 °C. The extract was added on triplicate cultures wells containing a sub-conﬂuent L929 cell monolayer (1:1 dilution). The test samples and the control wells were incubated at 37 °C in 5 % CO2 for 24 h. Following incubation, the cell cultures were examined for quantitative cytotoxic evaluation. 50 µl per well of XTT reagent was added to the cells then incubated at 37 °C in 5 % CO2 for further 3–5 h. An aliquot of 100 µl was then transferred from each well into the corresponding wells of a new plate and the optical density was measured at 450 nm. 13. Jung, J.-H. H., Aloni, D., Yitzhaky, Y. & Peli, E. Active confocal imaging for visual prostheses. Vision. Res. 111, 182–196 (2015). 14. Mathieson, K. et al. Photovoltaic retinal prosthesis with high pixel density. Nat. Photonics 6, 391–397 (2012). 15. Lorach, H. et al. Photovoltaic restoration of sight with high visual acuity. Nat. Received: 11 July 2017 Accepted: 8 February 2018 Received: 11 July 2017 Accepted: 8 February 2018 Received: 11 July 2017 Accepted: 8 February 2018 Thermal measurements. Measures have been performed with a thermal camera (FLIR A325sc Infrared Camera, FLIR Systems, Inc.) focused on the top surface of the POLYRETINA prosthesis. Images have been acquired at 1 frame per second. Light pulses (10 ms, 20 Hz, 1.22 mW mm−2) were delivered by a 565-nm Green LED (Thorlabs, M565L3-C5) focused at the sample level. \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications ARTICLE Retinas were placed mimicking an epiretinal conﬁguration, therefore with RGCs facing the substrate (bare PDMS or prosthesis). On the prosthesis, retinas were layered in the central part of the array with electrodes of 80 µm in diameter and 150 µm pitch. Recordings were performed in dim light at 32 °C with a sharp metal electrode (PTM23BO5KT, World Precision Instruments), ampliﬁed (Model 3000, A-M System), ﬁltered (300–3000 Hz), and digitalized at 30 kHz (Micro1401–3, CED Ltd.). Illumination was carried out on a Nikon Ti-E inverted microscope (Nikon Instruments) by the Spectra X system (Emission ﬁlter 560/32, Lumencor). The microscope was equipped with a dichroic ﬁlter (FF875-Di01–25 × 36, Semrock) and a 10× (diameter of the illumination spot 2.2 mm; CFI Plan Apochromat Lambda) objective. The stimulation protocol consisted in a repetition of ten pulses at 1 Hz for each irradiance; irradiance was increased sequentially: LED at 0% (0 MPET ¼ 6:93 ´ 105CECT 1 P with CE ¼ 6:67 ´ 103α2 forα>100mrad CT ¼ 1 for 400<λ<700 P ¼ 5:44 for 400<λ<600 andt 0:7s 8 > < > : ð1Þ ð1Þ MPEC ¼ 5:56 ´ 1010CBα2 with CB ¼ 100:02 λ450 ð Þ ð2Þ ð2Þ MPET results in 47.41 mW, which corresponds to 328.75 µW mm−2 for an exposed area of 144.22 mm2. MPEC results in 57.55 mW, which corresponds to 399.02 µW mm−2. MPET results in 47.41 mW, which corresponds to 328.75 µW mm−2 for an exposed area of 144.22 mm2. MPEC results in 57.55 mW, which corresponds to 399.02 µW mm−2. \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications 13 13 NATURE COMMUNICATIONS\| (2018) 9:992 NATURE COMMUNICATIONS\| (2018) 9:992 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications Author contributions 34. Ahnood, A. et al. Diamond devices for high acuity prosthetic vision. Adv. Biosyst. 1, 1600003 (2017). L.F. fabricated the devices and performed/analyzed KPFM, PV/PC, temperature, and accelerated ageing tests. M.J.I.A.L. designed, fabricated, and characterized the devices and the retinal prostheses; she performed/analyzed PV/PC measures and electrical simula- 35. Tumbleston, Ko, Samulski, EdwardT. & Lopez, Rene Analyzing local exciton generation proﬁles as a means to extract transport lengths in organic solar cells. Phys. Rev. B 82, 205325 (2010). tions. N.A.L.C. performed/analyzed pH, voltage spreading, and electrophysiological experiments. M.B. performed/analyzed PV and PC measures. S.C.A.G. performed ther- mal simulations. T.J.W. performed the simulated surgeries. P.V. performed the simulated surgeries. K.S. participated in the fabrication and characterization of the prostheses. D.G. designed and led the entire study, validate the data analysis, and wrote the manuscript. All the authors read and accepted the manuscript. y 36. Gupta, D., Bag, M. & Narayan. Area dependent efﬁciency of organic solar cells. Appl. Phys. Lett. 93, 384 (2008). pp y 37. Zrenner, E. et al. Subretinal electronic chips allow blind patients to read letters and combine them to words. Proc. R. Soc. Lond. B Biol. Sci. 278, 1489–1497 (2011). 37. Zrenner, E. et al. Subretinal electronic chips allow blind patients to read letters and combine them to words. Proc. R. Soc. Lond. B Biol. Sci. 278, 1489–1497 (2011). 38. Dorn, J. et al. The detection of motion by blind subjects with the epiretinal 60- electrode (argus ii) retinal prosthesis JAMA Ophthalmol 131 183 189 38. Dorn, J. et al. The detection of motion by blind subjects with the epiretinal 60- electrode (argus ii) retinal prosthesis. JAMA Ophthalmol. 131, 183–189 (2013). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 42. Jensen, R. & Rizzo, J. Thresholds for activation of rabbit retinal ganglion cells with a subretinal electrode. Exp. Eye. Res. 83, 367–373 (2006). 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. 43. Behrend, M. R., Ahuja, A. K., Humayun, M. S., Chow, R. H. & Weiland, J. D. Resolution of the epiretinal prosthesis is not limited by electrode size. Ieee. Trans. Neural Syst. Rehabil. Eng. 19, 436–442 (2011). 44. Kuo, A. et al. Posterior eye shape measurement with retinal OCT compared to MRI Posterior eye shape measurement with retinal OCT. Invest. Ophthalmol. Vis. Sci. 57, 196–203 (2016). 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/. 45. Lei, X. et al. SiC protective coating for photovoltaic retinal prosthesis. J. Neural Eng. 13, 046016 (2016). 46. Hukins, D. W. L., Mahomed & Kukureka, S. N. Accelerated aging for testing polymeric biomaterials and medical devices. Med. Eng. Phys. 30, 1270–1274 (2008). 47. Quiroga, Q., Nadasdy, Z. & Ben-Shaul, Y. Unsupervised spike detection and sorting with wavelets and superparamagnetic clustering. Neural Comput. 16, 1661–1687 (2006). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03386-7 her advices. This work has been supported by École polytechnique fédérale de Lausanne, Medtronic, European Commission (EU project 701632), Fondation Pierre Mercier pour la science, and Velux Stiftung (Project 1102). 32. Delori, F., Webb, R., Sliney, D. & Institute, A. Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices. J. Opt. Soc. Am. 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Temporal properties of visual perception on electrical stimulation of the retina. Invest. Ophthalmol. Vis. Sci. 53, 2720–2731 (2012). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-03386-7. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-03386-7. References In each ﬁgure p-values were represented as: p < 0.05, p < 0.01, p < 0.001, and **p < 0.0001. Data are reported as mean ± s.e.m. or mean ± s.d., n is used to identify the number of electrodes or cells used; N is used to identify the number of devices or animals. 26. Chang et al. Two mouse retinal degenerations caused by missense mutations in the β-subunit of rod cGMP phosphodiesterase gene. Vision. Res. 47, 624–633 (2007). 27. Chang et al. Retinal degeneration mutants in the mouse. Vision Res. 42, 517–525 (2002). 28. Boinagrov, D., Pangratz-Fuehrer, S., Goetz, G. & Palanker, D. Selectivity of direct and network-mediated stimulation of the retinal ganglion cells with epi- , sub- and intraretinal electrodes. J. Neural Eng. 11, 026008 (2014). 29. Stett, A., Barth, W., Weiss, S., Haemmerle, H. & Zrenner, E. Electrical multisite stimulation of the isolated chicken retina. Vision Res. 40, 1785–1795 (2000). stimulation of the isolated chicken retina. Vision Res. 40, 1785–1795 (2000). 30. Romeo, A., Liu, Q., Suo, Z. & Lacour, S. Elastomeric substrates with embedded stiff platforms for stretchable electronics. Appl. Phys. Lett. 102, 131904 (2013). ( ) 30. Romeo, A., Liu, Q., Suo, Z. & Lacour, S. Elastomeric substrates with embedded stiff platforms for stretchable electronics. Appl. Phys. Lett. 102, 131904 (2013). Data availability. The authors declare that all other relevant data supporting the ﬁndings of the study are available in this article and in its Supplementary Infor- mation ﬁle. Access to our raw data can be obtained from the corresponding author upon reasonable request. p pp y 31. Lorach, H. et al. Retinal safety of near infrared radiation in photovoltaic restoration of sight. Biomed. Opt. Express 7, 13–21 (2016). NATURE COMMUNICATIONS\| (2018) 9:992 14 Acknowledgements We would like to acknowledge the EPFL center of micronanotechnology for the support. Prof. Matthias Lütolf for having reviewed our manuscript and Prof. Stéphanie Lacour for NATURE COMMUNICATIONS\| (2018) 9:992 15 \| DOI: 10.1038/s41467-018-03386-7\| www.nature.com/naturecommunications
https://openalex.org/W2972121182	https://jatiswara.unram.ac.id/index.php/js/article/download/23/21	Indonesian	null	Menata Keterwakilan Perempuan Di Dpr Ri Berdasarkan Prinsip Keadilan, Ham, dan Demokrasi	Jatiswara	2,017	cc-by	5,905	1 1 • Hasil Penelitian Disertasi, Program Doktor Ilmu Hukum, Malang Tahun 2012 Dosen Fakultas Hukum Universitas Mataram, Alamat Korespondensi :a. cahyowati@gmail.com * Doesen Fakultas Hukum Universitas Mataram, Alamat Korespondensi : rodliyah fh@yahoo.co.id Abstrak Persoalan yang diangkatdalamtulisaniniberkaitandenganPengaturan keterwakilan perempuan di DPR RI, berdasarkan prinsip keadilan, HAM, dan demokrasi. DenganmenganalisisUndang-Undang PartaiPolitikdanUndang-Undang Pemilu. Metode penelitian, penelitian ini merupakan penelitian hukum normatif, dengan pendekatan perundang-undangan (statute approach1), pendekatan konsep (conceptual approach),pendekatan filsafat (philosophical approach), danpendekatan perbandingan (comparative approach). Simpulanpengaturan keterwakilan perempuan di DPR RI, berdasarkan prinsip keadilan, HAM,dan demokrasi, yaitu dengan merumuskan kembali tindakan khusus sementara/affirmative action dalam Undang-Undang Partai Politik, dan Undang-Undang Pemilu, wajib memperhatikan keterwakilan perempuan minimal 40%, dengan pertimbangan semakin banyak perempuan yang direkrut oleh parpol maka peluang perempuan menjadianggota DPR RI lebih besar. Dalam penempatan daftar calon menggunakan zipper system tidak bolong-menyusun caleg secara silang menyilang antara laki-laki dan perempuan, secara bergantian,dan adanya sanksi administratif bagi parpol yang tidak memenuhi kuota sekurang-kurangnya 40% perempuan dalam daftar bakal calon legislatif, sehingga parpol tidak berhak mengikuti pemilu. Kata kunci: KeterwakilanPerempuan di DPR RI Hasil Penelitian Disertasi, Program Doktor Ilmu Hukum, Malang Tahun 2012 Dosen Fakultas Hukum Universitas Mataram, Alamat Korespondensi :a. cahyowati@gmail.com * Doesen Fakultas Hukum Universitas Mataram, Alamat Korespondensi : rodliyah fh@yahoo.co.id 2 Jimly Asshidiqie, Demokrasi dan Nomokrasi Prasyarat Menuju Indonesia Baru, dalam Hukum Tata Negara dan Pilar-pilar Demokrasi Serpihan Pemikiran Hukum Media dan HAM, (Jakarta: Konstitusi Press, 2005), hlm.242. JUSTICE, HR, AND DEMOCRACY PRINCIPLES The issues raised in this writing due to the regulating on the women representation in DPR RI (the Parliament of Republic Indonesia) based on the justice, HR, and democracy principles. By analyzing the Act of Political Party, and the Act of General Election basedon the justice, HR, and democracy principles, Research methods, this studyis anormativelegal research, with theapproachof legislation, approachesthe concept, approach tophilosophy, andthe comparative approach.May be concluded that the regulating of based on the justice, HR, and democracy principles, may be concluded that the regulating the women representation in DPR RI based on the justice, HR, and democracy principles, namely by reformulating temporary special action/affirmative actionin the Act of Political Party, and the Act of General Election, must care to minimal 40% of the 2 women representation, by considering the more women recruited by parpol (political parties) the more opportunity of women to the members of DPR RI. The placement of the list of candidates uses zipper system so that no vacuum in ordering the list of the candidates by crossing between women and men, and delivers administrative sanction to the parpol violate the minimal quote 40% of the women representation in the potent of the candidate of legislative members by excepting the parpol to participate in the pemilu (the general election). women representation, by considering the more women recruited by parpol (political parties) the more opportunity of women to the members of DPR RI. The placement of the list of candidates uses zipper system so that no vacuum in ordering the list of the candidates by crossing between women and men, and delivers administrative sanction to the parpol violate the minimal quote 40% of the women representation in the potent of the candidate of legislative members by excepting the parpol to participate in the pemilu (the general election). Key word: Representation of women in the Parliament of Republic Indonesia 3 A. Pendahuluan PembukaanUndang-UndangDasar Negara Republik Indonesia Tahun 1945, padaAleniaKeempatdinyatakanbahwa, dibentuknya Negara Republik Indonesia adalahuntukmelindungisegenapbangsa Indonesia, danseluruhtumpahdarah Indonesia. Melindungisegenapbangsa Indonesia berartibaiklaki-lakimaupunperempuan yang menjadibangsa Indonesia harusmendapatkanperlindungandarinegara.Melindungi juga berarti memberikan kesempatan yang sama adilnya bagi laki-laki dan perempuan. Kesejahteraan umum akan tercapai jika perempuan, dan laki-laki mendapatkan peluang yang sama adilnya dalam bidang ekonomi, sosial budaya dan politik. Sehubungan dengan hal tersebut Pasal 27 ayat (1) Undang-Undang Dasar Negara Republik Indonesia Tahun 1945 (selanjutnya disingkat UUD NRI Tahun 1945) dengan tegas menentukan, ” Segala warga negara bersamaan kedudukan di dalam hukum dan pemerintahan dan wajib menjunjung hukum dan pemerintahan itu dengan tidak ada kecualinya. Hal ini dikenal dengan prinsip equal protection before the law (negara dan hukum harus melindungi warga negaranya secara sama). Indonesia sebagai negara demokrasi, harus melibatkan seluruh komponen masyarakat dalam menentukan arah dan kebijakan negara yang dicerminkan dengan keterwakilan mereka di lembaga legislatif. Keterwakilan itu haruslah mencerminkan keterwakilan yang adil dari komposisi penduduk yang ada dalam suatu negara, karena salah satu prasyarat tercapainya pelaksanaan demokrasi adalah terpenuhinya hak rakyat, baik laki-laki maupun perempuan untuk berpartisipasi dalam penyelenggaraan negara. Pada demokrasi modern, hukum menempati posisi yang sangat sentral. Demokrasi harus diletakan dalam koridor hukum. Ada 4 (empat) prinsip pokok dalam demokrasi berdasarkan hukum:2 1. Jaminan persamaan dan kesetaraan dalam kehidupan bersama p p 2. Pengakuan dan penghormatan terhadap perbedaan atau pluralistis 4 3. Adanya aturan yang mengikat dan dijadikan sumber rujukan bersama 4. Adanya mekanisme penyelesaian sengketa berdasarkan mekanisme aturan yang ditaati bersama. 3. Adanya aturan yang mengikat dan dijadikan sumber rujukan bersama 3. Adanya aturan yang mengikat dan dijadikan sumber rujukan bersama 4. Adanya mekanisme penyelesaian sengketa berdasarkan mekanisme aturan yang ditaati bersama. Jaminan persamaan dan kesetaraan dalam kehidupan bersama menempati urutan teratas karena tanpa adanya persamaan dan kesetaraan seseorang tidak dapat berpartisipasi dalam kehidupan bernegara. Demikian pula dengan pengakuan dan penghormatan terhadap perbedaan, sehingga perlu adanya aturan yang mengikat, dan jika terjadi sengketa maka ada mekanisme penyelesaian yang disepakati bersama. Jaminan persamaan dan kesetaraan dalam kehidupan bersama menempati urutan teratas karena tanpa adanya persamaan dan kesetaraan seseorang tidak dapat berpartisipasi dalam kehidupan bernegara. Demikian pula dengan pengakuan dan penghormatan terhadap perbedaan, sehingga perlu adanya aturan yang mengikat, dan jika terjadi sengketa maka ada mekanisme penyelesaian yang disepakati bersama. Makna demokrasi dianggap menjadi tidak demokratis ketika ada sekelompok masyarakat atau golongan tersingkir dan tidak terwakili dalam lembaga perwakilan hasil pemilu. 3 Sekretariat Jenderal Dewan Perwakilan Rakyat Republik Indonesia bekerjasama dengan Proyek PROPER-United Nations Development Programme Indonesia, Buku Kompilasi : Pengarusutamaan Gender Dalam Parlemen, (Jakarta, 2008), hlm.11. 3 Sekretariat Jenderal Dewan Perwakilan Rakyat Republik Indonesia bekerjasama dengan y p j g Proyek PROPER-United Nations Development Programme Indonesia, Buku Kompilasi : Pengarusutamaan Gender Dalam Parlemen, (Jakarta, 2008), hlm.11. 4 Wahida Zein Br Siregar,” Gaining Representation In Parliament: A Stud Of The Strunggle Of Indonesia Women To Increase Their Numbers In The National Provincial And Local parliament In The 2004 Elections”A thesis submitted for the degree of Doctor of Philosophy of The Australian Nation University, May, 2008, p.v. A. Pendahuluan Ada masalah krusial mengenai prinsip keadilan yang menjadi esensi demokrasi yakni ketika ada kelompok tertentu tersingkir dari proses politik, sehingga makna demokrasi dapat dipertanyakan. Jumlah peduduk perempuan hampir setengah dari penduduk Indonesia, merupakan potensi yang sangat besar untuk menunjang pembangunan di Indonesia. Namun dalam kenyataannya, perempuan masih jauh tertinggal dengan laki-laki, di segala bidang kehidupan. Berdasarkan Susenas Tahun 2006,3 rata-rata lama sekolah anak perempuan lebih rendah dibandingkan anak laki-laki (anak perempuan 6,7 tahun, anak laki-laki 9,5 tahun di perkotaan dan 5,7 tahun berbanding 8,5 tahun di perdesaan). Angka buta huruf perempuan lebih tinggi (11,6%) dibandingkan laki-laki (5,4%), khususnya di bidang politik, keterwakilan perempuan di lembaga legislatif sejak pemilu tahun 1955 sampai dengan pemilu tahun 2009, paling tinggi hanya mencapai 18 %. (hasil pemilu tahun 2009). 5 Penjelasan mengapa keterwakilan perempuan masih rendah di Dewan Perwakilan Rakyat Republik Indonesia (DPR RI), dapat dicermati dari hasil penelitian Wahida Zein Br.Siregar4, yang menyatakan: “why the quota failed to deliver the anticipate increase in women’s representation. Significant factors were found to include the non-compulsory status of the quota: the lack of women in political parties, especially in leadership positions; dominant role of political party leaders in nominating candidates for parliament, and the electoral system (the semi-open list of proportional representation)”. (Terjemahanbebas: sebabgagalnyausahameningkatkanjumlahperempuan di parlemen: kurangnyajumlahperempuan di partaipolitik, khususnyapadaposisikepemimpinan; peranpemimpinpartai yang dominandalamnominasikankandidatuntukDewanPerwakilan Rakyat; sistempemilihan (daftarproposionalperwakilan semi-terbuka). (Terjemahanbebas: sebabgagalnyausahameningkatkanjumlahperempuan di parlemen: kurangnyajumlahperempuan di partaipolitik, khususnyapadaposisikepemimpinan; peranpemimpinpartai yang dominandalamnominasikankandidatuntukDewanPerwakilan Rakyat; sistempemilihan (daftarproposionalperwakilan semi-terbuka). Sebagai bagian dari masyarakat internasional Indonesia terikat dengan kesepakatan-kesepakatan internasional, seperti rekomondasi Perserikatan Bangsa-Bangsa (selanjutnya disingkat PBB) Nomor 23 Tahun 1997 tentang pentingnya meningkatkan peran serta perempuan di lembaga-lembaga eksekutif, legislatif, dan yudikatif.Indonesia meratifikasiConvention on the Elimination of All Forms Discrimination Against Women (CEDAW), kedalam UU Nomor 7 Tahun1984 tentangPengesahanKonvensiMengenaiPenghapusanSegalaBentukDiskriminasiTer hadapPerempuan.KonvensiinikhususnyaPasal 4 ayat (1) merupakanlandasanhukumbagibangsa Indonesia tentangPengesahanKonvensiMengenaiPenghapusanSegalaBentukDiskriminasiTer hadapPerempuan.KonvensiinikhususnyaPasal 4 ayat (1) merupakanlandasanhukumbagibangsa Indonesia (1) untukmemberlakukankebijakankhusussementarauntukmeningkatkanjumlahketer wakilanperempuan di legislatif.Tujuankonvensiinidiarahkanpadapersamaande untukmemberlakukankebijakankhusussementarauntukmeningkatkanjumlahketer wakilanperempuan di legislatif.Tujuankonvensiinidiarahkanpadapersamaande untukmemberlakukankebijakankhusussementarauntukmeningkatkanjumlahketer 6 factomelaluijaminansecarakonstitusional, hukumdanregulasi-regulasi, jugamenempuhlangkah-langkahlainnya, termasuklangkah-langkahkhusus, sepertitindakankhusussementaraatauaffirmative action.5 factomelaluijaminansecarakonstitusional, hukumdanregulasi-regulasi, jugamenempuhlangkah-langkahlainnya, termasuklangkah-langkahkhusus, sepertitindakankhusussementaraatauaffirmative action.5 hukumdanregulasi-regulasi, termasuklangkah-langkahkhusus, Di bidang politik, pemberlakuan sistem kuota yang dikenal dengan penetapan angka 30 % merupakan referensi PBB, berdasarkan riset Profesor Drude Dahlerup dari Stockholm University yang menyatakan angka 30 % sebagai critical numbers untuk bisa mempengaruhi kebijakan.6 Selanjutnya penetapan angka ini terus digulirkan. Jaminan hukum dalam bentuk tindakan khusus sementara yang menetapkan kuota minimal 30 % dalam Undang-undang Politik (UU Partai Politik dan UU Pemilu) ini dianggap penting memungkinkan terjadinya suatu perubahan. 5Affirmative action adalah tindakan khusus sementara untuk mengangkat kaum minoritas atau marginal (masyarakat yang terpinggirkan). Misalnya kebijakan tentang kuota 30 % untuk melibatkan perempuan di parlemen, sehingga kebijakan-kebijakan untuk penghapusan diskriminasi terhadap perempuan dapat terakomodasi. p g p p p p p 6 Figur, “Politik Perempuan Habislah Gelap Terbitlah Terang”,( Edisi XXIV/Th.2008), hlm.9. 5Affirmative action adalah tindakan khusus sementara untuk mengangkat kaum minoritas A. Pendahuluan Dengan dicantumkannya jumlah minimal 30 % berarti ada target yang harus dicapai dan bisa diukur sejauhmana terjadi perubahan. Suatu “critical mass” yang akan membawa dampak pada kualitas keputusan yang diambil dalam lembaga- lembaga publik, termasuk di lembaga legislatif. Jumlah 30 % ditetapkan untuk menghindari dominasi dari salah satu jenis kelamin dalam lembaga-lembaga politik yang merumuskan kebijakan publik, dan menyangkut hidup hajat orang banyak. Dengan kata lain jumlah keterwakilan laki-laki dan perempuan tidak boleh ada yang melebihi 70%. Dengan demikian penting untuk dibahas mengenai pengaturan keterwakilan perempuan di DPR RI berdasarkan prinsip keadilan, HAM, dan demokrasi. 7 7 B. Metode Penelitian Jenis penelitian ini merupakan penelitian hukum normatif, yaitu. penelitian yang mengkaji peraturan perundang-undangan, yaitu semua peraturan perundang-undangan yang terkait dengan pemilu dan partai politik terkhusus mengenai keterwakilan perempuan. Pendekatan yang digunakanadalah:pertamapendekatan perundang- undangan (statute approach), yaitu dengan mengkaji dan meneliti peraturan perundang-undangan di bidang politik yang berkaitan dengan Undang-undang Partai Politik, Undang-undang Pemilu, dan undang- undang yang berkaitan dengan peningkatan keterwakilan perempuan di DPR RI.Kedua pendekatan konsep (conceptual approach), dimaksudkan untuk mengkaji konsep pembaharuan hukum di bidang politik sebagai upaya peningkatan keterwakilan perempuan di DPR RI. Pendekatan filsafat (philosophical approach), yaitu mengkaji hakikat keterwakilan perempuan di DPR RI.Ketiga, pendekatan perbandingan (comparativeapproach) untuk membandingkan pengalaman negara majudalam upaya meningkatkan keterwakilan perempuan di legislatif, yaitu Negara Argentina, dengan alasan sistem pemerintahan sama dengan Indonesia yaitu presidensial, dan perjuangan tindakan khusus sementara atau affirmative action keterwakilan perempuan di lembaga legislatif sudah dimulai sejak tahun 1993. bahanhukumsekunder, Pengumpulanterhadapbahanhukumprimer, danbahanhukumtersiermelaluilibrary Pengumpulanterhadapbahanhukumprimer, bahanhukumsekunder danbahanhukumtersiermelaluilibrary researchdilakukanpenelitidenganpencariandanmemfoto copy bahan hukum. 8 Setelah bahan hukum primer, sekunder, dan tersier, serta bahan penunjang, dikumpulkan, selanjutnya diolah dan dianalisis secara kualitatif kemudian dituangkan dalam bentuk deskriptif,7 artinya dipaparkan dalam bentuk uraian dengan penalaran deduktif- induktif untuk menghasilkan proposisi atau konsep sebagai jawaban dari permasalahan atau hasil /berupa temuan penelitian. 9Piagam Perserikatan Bangsa-Bangsa Dan Statuta Mahkamah Pengadilan Internasional, (Bandung, Binacipta, 1982), hlm.3 C. a. PengertianMenataketerwakilan perempuan di DPR RI berdasarkan prinsip keadilan, HAM, dan demokrasi MenatadalamkamusBahasa Indonesia, berasaldarikata “tata” yang berarti: 8 aturan, peraturan, susunan, carasusunan, dan sistem.Pengertianmenatadalamtulisaniniadalahpengaturan.Pengaturanketerwakila nperempuan di DPR RI berdasarkanprinsipkeadilan, HAM, dandemokrasi, haliniberkaitandenganadanyatindakan khusus sementara/affirmative action, hal ini tercantum dalam Undang-undang Partai Politik dan Undang-undang Pemilu. Keterkaitanantaraprinsipkeadilan, HAM, dandemokrasi, dapatdigambarkanpadabagan 1 berikutini: Keterkaitanantaraprinsipkeadilan, HAM, dandemokrasi, dapatdigambarkanpadabagan 1 berikutini: HAM, Bagan 1. Hubungan antara Prinsip Keadilan, HAM, dan Demokrasi 7Ronny Hanitijo Soemitro, Loc. Cit. 8W.J.S Purwadarminta,KamusBahasa Indonesia, PN.BalaiPustaka, Jakarta, 1984. HAM Keadilan Bagan 1. Hubungan antara Prinsip Keadilan, HAM, dan Demokrasi 7Ronny Hanitijo Soemitro, Loc. Cit. 8W.J.S Purwadarminta,KamusBahasa N.BalaiPustaka, Jakarta, 1984. 9 9 Keterangan: Keterangan: Prinsip keadilan menjiwai (merupakan roh dari) prinsip Hak Asasi Manusia, setiap orang mempunyai hak yang sama atas kebebasan dasar yang paling luas, seluas kebebasan yang sama bagi semua orang. Pada pembukaan Piagam Perserikatan Bangsa-Bangsa, pada alenia ketiga dikatakan “ demi membangunkan keadaan dimana keadilan dan penghargaan terhadap kewajiban- kewajiban yang timbul dari perjanjian-perjanjian dan lain-lain sumber hukum internasional dapat dipelihara.9 Nilai-nilai yang ada dalam prinsip keadilan selanjutnya dituangkan dalam prinsip Hak Asasi Manusia. Salah satu prinsip Hak Asasi Manusia adalah prinsip kesetaraan, “ meletakan semua orang terlahir bebas dan memiliki kesetaraan dalam HAM. Kesetaraan mensyaratkan adanya perlakuan yang setara, dimana pada situasi sama harus diperlakukan dengan sama, dan dengan perdebatan, dimana pada 10 situasi yang berbeda diperlakukan berbeda pula “. Nilai-nilai yang ada dalam prinsip Hak Asasi Manusia selanjutnya menjiwai (merupakan roh dari) prinsip demokrasi. Nilai-nilai yang ada dalam prinsip demokrasi, tercantum dalam generasi pertama Hak Asasi Manusia yaitu; kebebasan berfikir, kebebasan untuk berkumpul dan menyatakan pikiran. Ketiga prinsip ini merupakan satu kesatuan yang tidak terpisahkan, yang satu mempengaruhi yang lain. Titik singgung dari ketiga prinsip ini yaitu adanya persamaan atau kesetaraan. Hal ini dapat dilihat dari kalimat pada prinsip keadilan dinyatakan : Setiap orang mempunyai hak yang sama atas kebebasan dasar yang paling luas, seluas kebebasan yang sama bagi semua orang. Pada prinsip HAM, dinyatakan: Prinsip kesetaraan, ide yang meletakan semua orang terlahir bebas dan memiliki kesetaraan dalam HAM. Pada prinsip demokrasi dinyatakan: Jaminan persamaan dan kesetaraan. Jika kesetaran gender dikaitkan dengan keterwakilan perempuan di DPR RI, diketahui jumlah perempuan saat ini mencapai 18 %, belum mencapai minimal 30% seperti yang diharapkan. Pertanyaannya bagaimana mencapai minimal 30% keterwakilan perempuan di DPR RI sehingga diharapkan sesuai dengan prinsip keadilan, HAM, dan demokrasi? Jawabannya adalah dengan memberlakukan tindakan khusus sementara/affirmative action. Pengertian awal tindakan khusus sementara atau affirmative action adalah hukum dan kebijakan yang mensyaratkan dikenakannya kepada kelompok tertentu, pemberian kompensasi dan keistimewaan dalam kasus-kasus tertentu guna mencapai representasi yang lebih proporsional dalam beragam institusi dan okupasi. Ia merupakan diskriminasi positif (positive discrimination) yang dilakukan untuk mempercepat tercapainya keadilan dan 11 kesetaraan. Salah satu sarana terpenting untuk menerapkannya melalui sarana hukum, dimana jaminan pelaksanaannya harus ada dalam Konstitusi dan undang-undang.10 DasarpemberlakuantindakankhusussementarayaituPasal 4 UU Nomor 7 Tahun 1984 tentangPengesahanKonvensiMengenaiPenghapusanSegalaBentukDiskriminasiTer hadapWanita (Convention on the Ellimination of All Forms of Discrimination Against Women), yaitu: Pasal 4,mewajibkannegaramelakukanlangkah- langkahtindakkhusussementara (temporary special measures) untukmempercepatpersamaan de- facto, sertamencapaiperlakuandankesempatan yang samabagiperempuandanlaki-laki (ayat 1). 10 Ani Soetjipto, et.all, Pengarusutamaan Gender di Parlemen:Studi Terhadap DPR dan DPD Periode 2004-2009, ( Jakarta: Program Dukungan Parlemen UNDP, 2010), hlm.xvii. 11 Ani Widyani Soetjipto, “ Demokrasi adalah Kesetaraan keterwakilan dan Keadilan “Affirmative Action” untuk Perempuan di Parlemen”, dalam Politik Perempuan Bukan Gerhana, (Jakarta, Penerbit Buku Kompas, 2005), hlm.98. Keterangan: Peraturan dan tindakan khusus yang ditujukan untuk melindungi kehamilan kehamilan tidak dianggap sebagai diskriminasi (ayat 2). Pasal 7, hak perempuan dalam kehidupan politik dan publik menentukan kewajiban negara untuk menjamin bahwa perempuan mempunyai hak yang sama dengan laki-laki untuk (a) dipilih dan memilih, (b) berpartisipasi dalam perumusan kebijakan pemerintah dan implementasinya, memegang jabatan dalam pemerintahan di semua tingkat, (c) berpartisipasi dalam organisasi dan perkumpulan non pemerintah yang berhubungan dengan kehidupan masyarakat dan politik negara. 12 Tindakan khusus sementara telah dilakukan di negara lain, dan terbukti produktif untuk meningkatkan representasi jumlah perempuan di parlemen. Keterlibatan perempuan dalam kehidupan politik akan mempunyai banyak keuntungan bagi masyarakat. Jumlah perempuan yang cukup dalam institusi pengambilan keputusan akan membuat pergeseran pada cara pandang dalam menyelesaikan masalah-masalah politik dengan mengutamakan perdamaian dan cara-cara anti kekerasan.11 Dengan demikian pengaturan keterwakilan perempuan di DPR RI yang sesuai dengan prinsip keadilan, HAM, dan demokrasi adalah adanya tindakan khusus sementara/affirmative action, dalam hal ini harus tercantum dalam Undang-undang Partai Politik dan Undang-undang Pemilu, dan bagaimana mendesain agar Undang-undang Partai Politik dan Undang-undang Pemilu lebih berperspektif gender. b. Pengaturan Keterwakilan Perempuan dalam Undang-Undang Partai Politik Berdasarkan Prinsip Keadilan, HAM, dan Demokrasi. Penjelasan atas UU Nomor 2 Tahun 2011 tentang Partai Politik, sebagaimana diamanatkan dalam UUD NRI Tahun 1945, kemerdekaan berserikat, berkumpul, dan mengeluarkan pendapat, merupakan hak asasi manusia yang harus dilaksanakan untuk memperkuat semangat kebangsaan dalam Negara Kesatuan Republik Indonesia yang demokratis. Hal tersebut lalu diwujudkan dalam pembentukan parpol, sebagai pilar demokrasi dalam sistem politik di Indonesia. 13 Tujuan Umum Parpol mewujudkan cita-cita nasional bangsa Indonesiasebagaimana yang dimaksud dalam Pembukaan UUD Negara Republik Indonesia Tahun 1945, menjaga, dan memelihara keutuhan Negara Kesatuan Republik Indonesia, mengembangkan kehidupan demokrasi berdasarkan Pancasila dengan menjunjung tinggi kedaulatan rakyat, dan mewujudkan kesejahteraan bagi seluruh rakyat Indonesia. Tujuan khusus parpol adalah meningkatkan partisipasi politik masyarakat dalam rangka penyelenggaraan kegiatan politik, dan pemerintahan, memperjuangkan cita-cita parpol dalam kehidupan berbangsa dan bernegara. Keterangan: Pasal 11 dalam UU Nomor 2 Tahun 2011 tentang Partai Politik, mengatur fungsi parpol sebagai sarana untuk: - Memberikan pendidikan politik bagi anggota dan masyarakat luas, agar menjadi warga negara Indonesia yang sadar akan hak dan kewajibannya dalam kehidupan berbangsa, dan bernegara; - Menciptakan iklim yang kondusif bagi persatuan dan kesatuan bangsa Indonesia untuk kesejahteraan masyarakat; - Menyerap, menghimpun, dan menyalurkan aspirasi politik masyarakat dalam merumuskan, dan menetapkan kebijakan negara; - Partisipasi politik warga negara Indonesia, dan; - Rekrutmen politik dalam proses pengisian jabatan politik melalui mekanisme demokrasi dengan memperhatian kesetaraan dan keadilan gender. Dengan demikian pengaturan keterwakilan perempuan dalam UU Nomor 2 Tahun 2011 tentang Partai Politik penting untuk dibahas, karena parpol 14 sebagai sarana partisipasi politik masyarakat ikut mengusung perubahan yang terjadi dalam masyarakat. Parpol juga harus mempunyai tanggungajwab untuk membawa perubahan terhadap komposisi keanggotaan DPR RI lebih berkesetaran dan berkeadilan gender, sehingga diharapkan kebijakan publik yang dikeluarkan DPR RI lebih berperspektif gender, bermanfaat bagi perempuan dan laki-laki. Selanjutnya desain Undang-Undang Partai Politik yang mengusung keterwakilan perempuan di parpol, dapat dilihat pada tabel 1, berikut ini: Tabel 1. Desain Undang-Undang Partai Politik yang Mengusung Keterwakilan Perempuan Ketentuan UU No2/2011 Desain berikutnya Pembentukan/pendirian partai Pasal 2 ayat (2) menyatakan: pendirian dan pembentukan partai politik sebagaimana dimaksud pada ayat (1) menyertakan 30% keterwakilan perempuan Pembentukan dan pendirian partai politik, wajib menyertakan minimal 40% keterwakilan perempuan Kepengurusan Pasal 20 menyatakan: kepengurusan partai politik tingkat provinsi dan kabupaten/kota sebagaimana dimaksud dalam Pasal 19 ayat (2) dan ayat (3) disusun dengan memperhatikan keterwakilan perempuan paling rendah 30% yang diatur dalam AD/ART partai politik masing- masing. Pasal 2 ayat (5) menyatakan: kepengurusan partai politik tingkat pusat sebagaimana pada ayat (2) disusun dengan Kepengurusan partai politik tingkat pusat, provinsi dan kabupaten/kota, wajibdisusun dengan memperhatikan keterwakilan perempuan minimal 40% yang diatur dalam AD/ART partai politik masing-masing. (Catatan: AD/ART partai politik, harus mencerminkan kesetaraan dan keadilan gender). Tabel 1. Desain Undang-Undang Partai Politik yang Mengusung Tabel 1. Desain Undang-Undang Partai Politik yang Mengusung Keterwakilan Perempuan 15 menyertakan paling sedikit 30 % keterwakilan perempuan. Pengambilan keputusan Pasal 27 dan 28 menyatakan: pengambilan keputusan dilakukan secara demokratis sesuai AD/ART partai politik. Pengambilan keputusan di parpol dilakukan secara demokratis, sesuai AD/ART parpol yang berperspektif gender. Rekrutmen politik Pasal 29 ayat (1a) menyatakan: rekrutmen sebagaimana dimaksud pada ayat (1) huruf b dilaksanakan melalui seleksi kaderisasi secara demokratis sesuai dengan AD/ART dengan mempertimbangkan. paling sedikit 30% keterwakilan perempuan. Keterangan: Rekrutmen politik dilakukan melalui seleksi kaderisasi secara demokratis sesuai dengan AD/ART partai politik, dan wajibmempertimbangkan minimal 40% keterwakilan perempuan. Pendidikan politik Pasal 31 menyatakan: partai politik melakukan pendidikan politik bagi masyarakat sesuai dengan ruang lingkup tanggung jawabnya dengan memperhatikan keadilan dan kesetaraan gender. Partai politik wajib melakukan pendidikan politik bagi masyarakat sesuai dengan ruang lingkup tanggung jawabnya dengan memperhatikan keadilan dan kesetaraan gender, Sumber: Bahan hukum diolah g Sumber: Bahan hukum diolah Pada tabel 1 di atas, diketahui: 1. Pada pembentukan/pendirian parpol, desain berikutnya adalah parpol wajib menyertakan minimal 40% keterwakilan perempuan. Kata wajib, menegaskan agar parpol bersungguh-sungguh menyertakan minimal 40% keterwakilan perempuan, ketentuan ini dicantumkan dalam akta notaris pendirian parpol, kemudian didaftarkan ke Kementerian (Kementerian Hukum dan HAM)untuk memperoleh status badan hukum. Dengan demikian syarat pada pembentukan/pendirian parpol merupakan syarat administrasi yang harus dipenuhi parpol untuk memperoleh status badan hukum. 16 2. Di kepengurusan parpol. Kepengurusan parpol di pusat, provinsi, maupun di kabupaten/kota wajib disusun dengan memperhatikan keterwakilan perempuan minimal 40% yang diatur dalam AD/ART parpol masing-masing. Hal ini dimaksudkan agar parpol mempunyai komitmen yang kuat terhadap keterwakilan perempuan di parpolnya. Komposisi perempuan di kepengurusan parpol sangat strategis karena perempuan dapat untuk mencalonkan diri dalam pemilu atau mempunyai kontribusi dalam menyusun daftar caleg yang akan berlaga pada pemilu. 3. Pada pengambilan keputusan. Pengambilan keputusan di parpol dilakukan secara demokratis sesuai dengan AD/ART parpol yang berperspektif gender. Dari hasil kajian sebelumnya AD/ART parpol, dari 8 (delapan) parpol yang memperoleh suara yang besar, hanya 3 (tiga) parpol yang AD/ARTnya sudah mencantumkan kebijakan afirmatif, artinya masih banyak parpol yang belum mencantumkan kebijakan afirmatif. Kata berperspektif gender menekankan pada pengambilan keputusan di parpol perlu mengakomodasi suara perempuan sebagai bahan pertimbangan di dalam pengambilan keputusan, termasuk pengambilan keputusan dalam menyusun daftar caleg untuk mengikuti pemilu. 4. Mengenai Rekrutmen politik. Rekrutmen politik dilaksanakan melalui seleksi kaderisasi secara demokratis sesuai AD/ART parpol, dan wajib mempertimbangkan minimal 40% keterwakilan perempuan. Bagi parpol rekrutmen politik sangat strategis, karena parpol mulai membuka kesempatan bagi masyarakat untuk bergabung, parpol mulai memilah 17 dan memilih yang akan menjadi kader partai sesuai dengan AD/ARTnya. Kata wajib dalam mempertimbangkan minimal 40% keterwakilan perempuan, menunjukan adanya komitmen yang kuat dari parpol untuk ikut memperjuangkan peningkatkan keterwakilan perempuan di lembaga legislatif. 5. Pada Pendidikan politik. Parpol wajib melakukan pendidikan politik bagi masyarakat sesuai dengan ruang lingkup tanggungjawabnya dengan memperhatikan keadilan dan kesetaraan gender. / p p p , perempuan di lembaga legislatif-belum tercapai, down load 10 Mei 2011. 12Http://www.madina- sk.com/index.php?option=com,content&view=articel&id=1720:keterwakilan 12Http://www.madina- sk.com/index.php?option=com,content&view=articel&id=1720:keterwakilan perempuan di lembaga legislatif-belum tercapai, down load 10 Mei 2011. 12Http://www.madina- Keterangan: Kata wajib disini bermakna adanya komitmen parpol untuk melakukan pendidikan politik, baik kepada masyarakat maupun kadernya. Materi yang dapat diberikan pada pendidikan politik adalah, bagaimana menjadi warga negara Indonesia yang sadar akan hak dan kewajibannya dalam kehidupan bermasyarakat, berbangsa, dan bernegara. Ketrampilan yang juga penting diberikan adalah ketrampilan berorasi, berargumentasi, bernegosiasi, kiat-kiat memenangkan pemilu secara santun dan bermartabat, mengenali dan mengidentifikasi masalah yang ada di dalam masyarakat dan mengemasnya sebagai isu yang ditawarkan pada saat pemilu. Materi ini diberikan baik kepada laki-laki dan perempuan, sehingga di dalam proses pencalonan, dan kampanye, calon anggota legislatif perempuan sama terampilnya dengan calon anggota legislatif laki-laki. Dengan demikian, parpol seyogyanyamempunyai komitmen yang kuat untuk ikut memperjuangkan keadilan dan kesetaraan gender di bidang politik, mulai dari pembentukan/pendirian partai, kepengurusan, pengambilan keputusan, rekrutmen politik, dan pendidikan politik, dan memberlakukan 18 sanksiadministratif, misalnya parpol tidak boleh mengikuti pemilu atau tidak memperoleh subsidi dari pemerintah, seperti yang terjadi di Perancis: “ Apabila kuota perempuan sebagai calon peserta politik tidak tercapai, subsidi bagi parpol dikurangi sampai 50%.”12 Sanksi ini juga dapat dicantumkan dalam Undang- undang Pemilu. Pembelajaran juga diperoleh dari perbandingan politik hukum keterwakilan perempuan di Parlemen Argentina, yaitu adanya konsensus antara perempuan dan parpol dalam mendukung proyek Quota Law(Undang-undang Kuota), yang akhirnya disetujui oleh Konggres pada tahun 1991. Quota law mengatur, di dalam menyusun daftar calon untuk pemilihan pada jabatan- jabatan publik, harus menyertakan sedikitnya 30% calon perempuan, yang harus diletakan dalam proporsi yang sedemikian rupa sehingga memungkinkan mereka untuk terpilih . Diharapkan pemerintah Indonesia, dan parpol juga mempunyai komitmen yang kuat untuk meningkatkan keterwakilan perempuan di lembaga legislatif, c. Pengaturan Keterwakilan Perempuan dalam Undang-Undang Pemilu Berdasarkan Prinsip Keadilan, HAM, dan Demokrasi. Pasal 2 ayat (1) UUD NRI Tahun 1945, menyatakan bahwa “ Kedaulatan berada di tangan rakyat dan dilaksanakan menurut Undang-undang Dasar”. Makna “ Kedaulatan berada di tangan rakyat “, bahwa rakyat memiliki kedaulatan, tanggungjawab, hak dan kewajiban untuk secara demokratis memilih pemimpin yang akan membentuk pemerintahan guna mengurus dan melayani 19 seluruh lapisan masyarakat, serta memilih wakil-wakil rakyat untuk mengawasi jalannya pemerintahan.13 Perwujudan kedaulatan dilaksanakan melalui pemilihan umum secara langsung sebagai sarana bagi rakyat untuk memilih wakil-wakilnya yang akan menjalankan fungsi melakukan pengawasan, menyalurkan aspirasi politik rakyat, membuat undang-undang, serta merumuskan anggaran pendapatan dan belanja negara. Tabel 2. 13 Penjelasan Umum UU Nomor 10 Tahun 2008 Tentang Pemilihan Umum Anggota DPR, DPD, DPRD. Keterangan: Desain UU Pemilu yang Mengusung Keterwakilan Perempuan Ketentuan UU Pemilu No.10/2008 RUU Pemilu yang akan datang Undang-Undang Pemilu selanjutnya Kebijakan afirmatif dalam pencalonan Pasal 53 menyatakan: daftar bakal calon sebagaimana dimaksud dalam Pasal 52 memuat paling sedikit 30% keterwakilan perempuan Pasal 53 menyatakan: daftar bakal calon sebagaimana dimaksud dalam Pasal 52 memuat paling sedikit 30% keterwakilan perempuan Daftar bakal calon wajib memuat minimal 40% keterwakilan perempuan Penempatan perempuan dalam daftar calon Pasal 55 menyatakan: di dalam bakal calon sebagaimana dimaksud pada ayat (1) dalam setiap 3 orang bakal calon terdapat sekurang- kurangnya satu orang perempuan bakal calon. Pasal 55 ayat (2) menyatakan: di dalam bakal calon sebagaimana dimaksud pada ayat (1) dalam setiap 3 orang bakal calon terdapat sekurang- kurangnya satu orang perempuan bakal calon. Partai politik wajib menempatan perempuan dalam daftar calon, dan menyusun daftar nama bakal calon, dengan cara silang menyilang antara laki-laki dan perempuan. Penentuan calon terpilih Pasal 214 menyatakan: penentuan bakal calon terpilih harus memenuhi 30% BPP. Jika tidak bisa memenuhi ditentukan berdasarkan Pasal 214 menyatakan: calon terpilih anggota DPR, DPRD provinsi, DPRD kab/kota ditetapkan berdasarkan suara terbanyak. Sistem pemilu yang digunakan adalah proposional daftar Penentuan calon terpilih anggota DPR, DPRD provinsi, DPRD kab/kota ditetapkan berdasarkan suara terbanyak. Sistem pemilu yang digunakan adalah proposional daftar terbuka. 13 Penjelasan Umum UU Nomor 10 Tahun 2008 Tentang Pemilihan Umum Anggota DPR Tabel 2. Desain UU Pemilu yang Mengusung Keterwakilan Perempuan Penentuan calon terpilih anggota DPR, DPRD provinsi, DPRD kab/kota ditetapkan berdasarkan suara terbanyak. Sistem pemilu yang digunakan adalah proposional daftar terbuka. 20 nomor urut. Sistem pemilu yang digunakan adalah proposional daftar terbuka. Kemudian pasal ini dibatalkan oleh keputusan MK Penentuan bakal calon terpilih dengan suara terbanyak. terbuka. Sumber: Bahan hukum diolah y Sumber: Bahan hukum diolah Penjelasan dari tabel 2. di atas, desain Undang-undang Pemilu yang akan datang, yaitu : 1. Kebijakan afirmatif dalam pencalonan, desain untuk Undang-undang Pemilu yang akan datang yaitu daftar bakal calon wajib memuat minimal 40% keterwakilan perempuan. Kata wajib dimaksudkan agar parpol sebagai peserta pemilu bersungguh-sungguh memperjuangkan keterwakilan perempuan sekurang-kurangnya 40% di lembaga legislatif. Mengapa keterwakilan perempuan yang akan datang, desainnya minimal 40% dalam pencalonan baik dalam Undang- undang Partai Politik maupun Undang-undang Pemilu? 14 Risalah Rapat Panitia Khusus RUU tentang Pemilu Anggota DPR, DPD,dan DPRD dan RUU tentang Pemilu Presiden dan Wakil Presiden, Tahun Sidang 2006-2007, Rabu 18 Juli 2007. 15Joko Widarto, “ Implikasi Hukum Putusan MK No.22-24/PUU-VI/2008 Bagi Anggota DPR”, Tesis, Program Pascasarjana, Fakultas Hukum Universitas Brawijaya, Malang, 2009, hlm.130. Keterangan: Alasanya karena ada kesepakatan internasional yang menyatakan tidak boleh di dalam lembaga –lembaga pengambilan keputusan yang menyangkut hidup orang banyak, salah satu jenis kelamin melebihi 70%.14 Jika perempuan dicalonkan minimal 40%, dan caleg disusun berdasarkan zipper system yang dilakukan berdasarkan silang menyilang antara laki-laki dan perempuan, dan parpol telah melakukan rekrutmen dan 21 21 pendidikan politik kepada masyarakat, pemerintah dan dan Lembaga Sosial Masyarakat aktif melakukan pemberdayaan terhadap masyarakat dengan melibatkan khususnya perempuan dalam setiap proses pembangunan, maka dapat dipastikan akan terjadi peningkatkan cukup signifikan terhadap keterwakilan perempuan di lembaga legislatif. Sebagai bahan perbandingan pada periode 1990 di Amerika Latin, 11 negara meloloskan undang-undang nasional yang menuntut sedikitnya 20 sampai 40% di pemilihan nasional. Argentina adalah adalah negara pertama yang memperkenalkan kuota 30% di tingkat wilayah, dan menjadi salah satu negara yang paling berhasil meningkatkan keterwakilan perempuan lebih dari 30%. Di Perancis, amandemen konstitusi tahun 1999 mengukuhkan akses yang setara bagi perempuan dan laki-laki , dimana 50% dari calon-calon yang ada di dalam daftar yang diajukan untuk pemilihan haruslah perempuan. 2. Penempatan perempuan dalam daftar calon. Partai politik wajib menempatkan perempuan dalam daftar calon, dan menyusun daftar nama bakal calon, dengan cara silang menyilang antara laki-laki dan perempuan. Hal ini dikenal sebagai zipper system, namun zipper system yang tidak bolong, yaitu urutan pertama laki-laki/perempuan, kedua perempuan/laki-laki, dan seterusnya sampai caleg perempuan habis. Hal ini juga dimaksudkan agar peluang perempuan terpilih lebih besar, sekalipun penentuan calon terpilih berdasarkan suara terbanyak. Desain ini merupakan koreksi dari Pasal 55 ayat (2) UU Nomor 10 Tahun 2008, karena pasal ini hanya mengatur: di dalam 2. 22 menyusun bakal calon setiap 3 (tiga) orang bakal calon terdapat sekurang-kurangnya 1 (satu) orang perempuan bakal calon. Hasil penelitian pada pemilu tahun 2009, mengenai penyusunan daftar caleg perempuan sebagai berikut: 15 menyusun bakal calon setiap 3 (tiga) orang bakal calon terdapat sekurang-kurangnya 1 (satu) orang perempuan bakal calon. menyusun bakal calon setiap 3 (tiga) orang bakal calon terdapat sekurang-kurangnya 1 (satu) orang perempuan bakal calon. Hasil penelitian pada pemilu tahun 2009, mengenai penyusunan daftar caleg perempuan sebagai berikut: 15 1) Caleg perempuan pada nomor urut 1 sebanyak 19%, yang terpilih 12%, 2) Caleg perempuan pada nomor urut 2 sebanyak 35 %, yang terpilih 29%, dan 3) Caleg perempuan pada nomor urut 3 sebanyak 61%, yang terpilih 54%. Keterangan: Dari hasil penelitian ini diketahui, bahwa parpol lebih banyak menempatkan perempuan pada nomor urut 3,dan parpol tidak merasa bersalah karena apa yang dilakukan sudah sesuai dengan Pasal 55 ayat (2). Pada waktu pembahasan UU Nomor 10 Tahun 2008 Dari hasil penelitian ini diketahui, bahwa parpol lebih banyak menempatkan perempuan pada nomor urut 3,dan parpol tidak merasa bersalah karena apa yang dilakukan sudah sesuai dengan Pasal 55 ayat (2). Pada waktu pembahasan UU Nomor 10 Tahun 2008 tentang Pemilu, para aktifis perempuan memberikan saran agar di dalam menyusun daftar calon dilakukan secara zig-zag/silang menyilang, dengan alasan supaya perempuan mempunyai peluang untuk terpilih lebih besar, hal ini dapat dibaca pada risalah sidang UU Nomor 10 Tahun 2008 tentang Pemilu. Dalam pemilu tahun 2009, nomor urut kecil masih mempunyai pengaruh yang besar untuk dipilih, sekalipun Putusan Mahkamah Konstitusi Nomor 22-24/PUU- 23 23 VI/2008 menetapkan suara terbanyak dalam pemilu. Dengan alasan:16 VI/2008 menetapkan suara terbanyak dalam pemilu. Dengan alasan:16 - Asumsi pertama mengapa pemilih, memilih nomor urut kecil di pemilu alasannya karena di urutan atas/ nomor kecil adalah kader-kader partai yang terbaik. - Asumsi kedua, pemilih tidak mau repot mencari nomor di bawah, mengingat kertas suara pemilu lebih besar dari biasanya, sehingga menyulitkan pemilih untuk berlama-lama di bilik suara.17 - Asumsi kedua, pemilih tidak mau repot mencari nomor di bawah, mengingat kertas suara pemilu lebih besar dari biasanya, sehingga menyulitkan pemilih untuk berlama-lama di bilik suara.17 Dengan demikian, nomor urut masih berpengaruh terhadap perolehan suara caleg perempuan. - Asumsi kedua, pemilih tidak mau repot mencari nomor di bawah, mengingat kertas suara pemilu lebih besar dari biasanya, sehingga menyulitkan pemilih untuk berlama-lama di bilik suara.17 Dengan demikian, nomor urut masih berpengaruh terhadap perolehan suara caleg perempuan. 3. Penentuan calon terpilih, masih sama seperti yang diatur dalam. pemilu tahun 2009 yaitu penentuan calon terpilih anggota DPR, DPRD provinsi, DPRD kab/kota ditetapkan berdasarkan suara terbanyak. (merupakan Keputusan MK atas yudicial review atas Pasal 214 UU Nomor 10 Tahun 2008 tentang Pemilu).Sistem pemilu yang digunakan adalah proposional daftar terbuka. 3. Penentuan calon terpilih, masih sama seperti yang diatur dalam. pemilu tahun 2009 yaitu penentuan calon terpilih anggota DPR, DPRD provinsi, DPRD kab/kota ditetapkan berdasarkan suara terbanyak. (merupakan Keputusan MK atas yudicial review atas Pasal 214 UU Nomor 10 Tahun 2008 tentang Pemilu).Sistem pemilu yang digunakan adalah proposional daftar terbuka. 4. 16Wawancara dengan Prof.Ramlan Surbakti, Mei 2011. D. Penutup Pengaturan keterwakilan perempuan di DR RI, berdasarkan prinsip keadilan, HAM,dan demokrasi, yaitu dengan merumuskan kembali tindakan khusus sementara/affirmative action dalam Undang-undang Partai Politik, dan Undang-undang Pemilu, wajib memperhatikan keterwakilan perempuan minimal 40%, dengan pertimbangan semakin banyak perempuan yang direkrut oleh parpol maka peluang perempuan lebih besar untuk masuk di lembaga legislatif. Dalam penempatan daftar calon menggunakan zipper system tidak bolong- menyusun caleg secara silang menyilang antara laki-laki dan perempuan, secara bergantian,dan adanya sanksi administratif bagi parpol yang tidak memenuhi kuota sekurang-kurangnya 40% perempuan dalam daftar bakal calon legislatif, sehingga parpol tidak berhak mengikuti pemilu. Keterangan: Adanya sanksi administratif bagi parpol yang tidak memenuhi kuota sekurang-kurangnya 40% perempuan dalam daftar bakal calon legislatif, yaitu pertama dengan mengembalikan daftar nama tersebut untuk diperbaiki, kedua jika masih belum benar maka dikembalikan lagi untuk diperbaiki, ketiga jika masih belum benar maka partai politik tersebut tidak berhak mengikuti pemilu. 24 Buku Ani Widyani Soetjipto, 2005, “ Demokrasi adalah Kesetaraan keterwakilan dan Keadilan “Affirmative Action” untuk Perempuan di Parlemen”, dalam Politik Perempuan Bukan Gerhana, Jakarta, Penerbit Buku Kompas. _________________, 2010, et.all, Pengarusutamaan Gender di Parlemen:Studi Terhadap DPR dan DPD Periode 2004-2009, Jakarta: Program Dukungan Parlemen UNDP. Jimly Asshidiqie, 2005, Demokrasi dan Nomokrasi Prasyarat Menuju Indonesia Baru, dalam Hukum Tata Negara dan Pilar-pilar Demokrasi Serpihan Pemikiran Hukum Media dan HAM, Jakarta, Konstitusi Press 25 25 Sekretariat Jenderal Dewan Perwakilan Rakyat Republik Indonesia bekerjasama dengan Proyek PROPER-United Nations Development Programme Indonesia, 2008, Buku Kompilasi : Pengarusutamaan Gender Dalam Parlemen, Jakarta W.J.S Purwadarminta, 1984, KamusBahasa Indonesia, PN.BalaiPustaka, Jakarta Piagam Perserikatan Bangsa-Bangsa Dan Statuta Mahkamah Pengadilan Internasional, 1982, Bandung, Binacipta. Ronny Hanitijo Soemitro, Loc. Cit. Ronny Hanitijo Soemitro, Loc. Cit. Majalah Figur, 2008, “Politik Perempuan Habislah Gelap Terbitlah Terang”,( Edisi XXIV/Th.2008). HasilPenelitian Wahida Zein Br Siregar,2008, ” Gaining Representation In Parliament: A Stud Of The Strunggle Of Indonesia Women To Increase Their Numbers In The National Provincial And Local parliament In The 2004 Elections” A thesis submitted for the degree of Doctor of Philosophy of The Australian Nation University, May. Joko Widarto, 2009, “ Implikasi Hukum Putusan MK No.22-24/PUU-VI/2008 Bagi Anggota DPR”, Tesis, Program Pascasarjana, Fakultas Hukum Universitas Brawijaya, Malang. Peraturan RepublikIndonesia, Undang-UndangDasar Negara RepublikIndonesiaTahun 1945. RepublikIndonesia, Undang-UndangRepublik Indonesia Nomor 7 Tahun 1984 TentangPengesahanKonvensiMengenaiPenghapusanSegalaBentukDiskriminasiTer hadapWanita (Convention on the Ellimination all forms of Discrimination Against Women). Republik Indonesia, Undang-UndangRepublik Indonesia Nomor 31 Tahun 2002 TentangPartaiPolitik. RepublikIndonesia, Undang-UndangDasar Negara RepublikIndonesiaTahun 1945. Undang-UndangDasar RepublikIndonesia, RepublikIndonesiaTahun 1945. Negara Republik Indonesia, Putusan Mahkamah Konstitusi Nomor 22-24/PUU- VI/2008 tentang Penetapan Caleg Terpilih Berdasarkan Suara Terbanyak. , Republik Indonesia, Putusan Mahkamah Konstitusi Nomor 22-24/PUU- VI/2008 tentang Penetapan Caleg Terpilih Berdasarkan Suara Terbanyak. Republik Indonesia, Putusan Mahkamah Konstitusi Nomor 22-24/PUU- VI/2008 tentang Penetapan Caleg Terpilih Berdasarkan Suara Terbanyak. Negara RepublikIndonesia, Undang-UndangDasar Negara RepublikIndonesiaTahun 1945. RepublikIndonesia, Undang-UndangRepublik Indonesia Nomor 7 Tahun 1984 TentangPengesahanKonvensiMengenaiPenghapusanSegalaBentukDiskriminasiTer hadapWanita (Convention on the Ellimination all forms of Discrimination Against Women). RepublikIndonesia, Undang-UndangRepublik Indonesia Nomor 7 Tahun 1984 TentangPengesahanKonvensiMengenaiPenghapusanSegalaBentukDiskriminasiTer hadapWanita (Convention on the Ellimination all forms of Discrimination Against Women). Republik Indonesia, Undang-UndangRepublik Indonesia Nomor 31 Tahun 2002 TentangPartaiPolitik. 26 Republik Indonesia, Undang-UndangRepublik Indonesia Nomor 12 Tahun 2003 TentangPemilihanUmumAnggotaDewanPerwakilan Rakyat, DewanPerwakilan Daerah, danDewanPerwakilan Rakyat Daerah, Lembaran Negara Republik Indonesia Tahun 2003 Nomor 37. g gg y , DewanPerwakilan Daerah, danDewanPerwakilan Rakyat Daerah, Lembaran Negara Republik Indonesia Tahun 2003 Nomor 37. Republik Indonesia, Undang-UndangRepublik Indonesia Nomor 2 Tahun 2008 TentangPartaiPolitik, Lembaran Negara Republik Indonesia Tahun 2008 Nomor 2. Republik Indonesia, Undang-UndangRepublik Indonesia Nomor 10 Tahun 2008 TentangPemilihanUmumAnggotaDewanPerwakilan Rakyat, DewanPerwakilan Daerah, danDewanPerwakilan Rakyat Daerah, Lembaran Negara Republik Indonesia NomorTahun 2008 Nomor 51. Republik Indonesia, Undang-UndangRepublik Indonesia Nomor 39 Tahun 1999 TentangHakAsasiManusia. Republik Indonesia, KetetapanMajelisPermusyawaratan Rakyat Republik Indonesia Nomor VI/MPR/2002 TentangRekomondasiAtasLaporanPelaksanaanPutusanMajelisPermusyawaratan Rakyat Republik Indonesia OlehPresiden, DPA, DPR, BPK, MA PadaSidangTahunanMajelisPermusyawaratan Rakyat Republik Indonesia Tahun 2002. Republik Indonesia, KetetapanMajelisPermusyawaratan Rakyat Republik Indonesia Nomor VI/MPR/2002 TentangRekomondasiAtasLaporanPelaksanaanPutusanMajelisPermusyawaratan Rakyat Republik Indonesia OlehPresiden, DPA, DPR, BPK, MA PadaSidangTahunanMajelisPermusyawaratan Rakyat Republik Indonesia Tahun 2002. Republik Indonesia, KetetapanMajelisPermusyawaratan Rakyat Republik Indonesia Nomor I/MPR/2003 TentangPeninjauanTerhadapMateri Dan Status HukumKetetapanMajelisPermusyawaratan Rakyat Sementara Dan KetetapanMajelisPermusyawaratan Rakyat Republik Indonesia Tahun 1960 SampaiDenganTahun 2002. Republik Indonesia, Undang-Undang Republik Indonesia Nomor 27 Tahun 2009 tentang MPR, DPR, DPD, dan DPRD. Republik Indonesia, Undang-Undang Republik Indonesia Nomor 11 Tahun 2011 tentang Partai Politik. Republik Indonesia, Undang-Undang Republik Indonesia Nomor 11 Tahun 2011 tentang Partai Politik. Penjelasan Umum UU Nomor 10 Tahun 2008 Tentang Pemilihan Umum Anggota DPR, Risalah Rapat Panitia Khusus RUU tentang Pemilu Anggota DPR, DPD,dan DPRD dan RUU tentang Pemilu Presiden dan Wakil Presiden, Tahun Sidang 2006-2007, Rabu 18 Juli 2007. DPD, DPRD. Republik Indonesia, Putusan Mahkamah Konstitusi Nomor 22-24/PUU- VI/2008 tentang Penetapan Caleg Terpilih Berdasarkan Suara Terbanyak. 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https://openalex.org/W4320167825	https://jneuroinflammation.biomedcentral.com/counter/pdf/10.1186/s12974-023-02723-y	English	null	The reciprocal interactions between microglia and T cells in Parkinson’s disease: a double-edged sword	Journal of neuroinflammation	2,023	cc-by	13,719	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract In Parkinson’s disease (PD), neurotoxic microglia, Th1 cells, and Th17 cells are overactivated. Overactivation of these immune cells exacerbates the disease process and leads to the pathological development of pro-inflammatory cytokines, chemokines, and contact-killing compounds, causing the loss of dopaminergic neurons. So far, we have mainly focused on the role of the specific class of immune cells in PD while neglecting the impact of interactions among immune cells on the disease. Therefore, this review demonstrates the reciprocal interplays between micro- glia and T cells and the associated subpopulations through cytokine and chemokine production that impair and/ or protect the pathological process of PD. Furthermore, potential targets and models of PD neuroinflammation are highlighted to provide the new ideas/directions for future research. Keywords Microglia, T cell, Cell–cell interactions, Chemokines, Parkinson’s disease, Cytokines appearance of Lewy bodies in the nerves and axons [1, 2]. PD patients may exhibit a range of motor symptoms (e.g., resting tremor, bradykinesia, shuffling gait, dysto- nia) and non-motor symptoms (e.g., cognitive impair- ment, anxiety, autonomic dysfunction, sleep disorders) [2, 3]. Epidemiological studies of PD have shown that the characteristics of PD are independently associated with age and gender, and age is one of the essential factors in its development [4]. A report of an epidemiological study published in 2018 on a North American population of patients with PD showed that the prevalence ranged from less than 1% of patients between 45 and 54 years old [5, 6]. In comparison, the number of patients aged 85 years was 4%, and the number of male patients was twice as high as that of female patients [4–8]. The number of PD patients is expected to increase by more than 50% by 2030 due to the aging of the population and increasing life expectancy. Therefore, it is urgent to determine how to effectively treat PD [9]. Journal of Neuroinfammation Journal of Neuroinfammation Xu et al. Journal of Neuroinflammation (2023) 20:33 https://doi.org/10.1186/s12974-023-02723-y Open Access Yuxiang Xu1,2†, Yongjie Li3†, Changqing Wang1, Tingting Han1, Haixuan Liu1, Lin Sun4, Jun Hong1, Makoto Hashimoto5 and Jianshe Wei1,2* Yuxiang Xu1,2†, Yongjie Li3†, Changqing Wang1, Tingting Han1, Haixuan Liu1, Lin Sun4, Jun Hong1, Makoto Hashimoto5 and Jianshe Wei1,2* Introduction Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer’s disease and is characterized by the loss of dopaminergic neurons in the substantia nigra, which leads to the reduction of dopamine influx in the nigrostriatal pathway and the †Yuxiang Xu and Yongjie Li have contributed equally to this work. *Correspondence: Jianshe Wei jswei@henu.edu.cn 1 Institute for Brain Sciences Research, School of Life Sciences, Henan University, Kaifeng 475004, China 2 Henan International Joint Laboratory for Nuclear Protein Regulation, Henan Medical School, Henan University, Kaifeng 475004, China 3 Department of Rehabilitation Medicine, Beijing Jishuitan Hospital Guizhou Hospital, Guizhou Provincial Orthopedics Hospital, Guiyang, China 4 Henan Key Laboratory of Polyoxometalate Chemistry, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, Henan, China 5 Tokyo Metropolitan Institute of Medical Science, Tokyo 156‑8506, Japan †Yuxiang Xu and Yongjie Li have contributed equally to this work. 2 Henan International Joint Laboratory for Nuclear Protein Regulation, Henan Medical School, Henan University, Kaifeng 475004, China 3 Department of Rehabilitation Medicine, Beijing Jishuitan Hospital Guizhou Hospital, Guizhou Provincial Orthopedics Hospital, Guiyang, China 4 Henan Key Laboratory of Polyoxometalate Chemistry, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, Henan, China 5 Tokyo Metropolitan Institute of Medical Science Tokyo 156 8506 Japa Role of microglia and related mechanisms in PD Microglia are macrophages residing in the central nerv- ous system (CNS). As critical immune cells in the brain, they perform an irreplaceable role in brain health. Under normal physiological conditions, microglia are in a rela- tively resting state, monitoring the brain microenviron- ment and protecting the brain homeostasis by secreting neurotrophic factors to the corresponding neurons [28, 37, 38]. First, microglia can remove invading pathogens, abnormal metabolic cells, and protein fragments from the brain through phagocytosis, thus actively remov- ing potential threats to brain homeostasis [28, 39]. Con- comitantly, altered microglia further remove potentially threatening substances by producing cytokines and, chemokines, and through other ways. Currently, most studies focus on the role of a single cell (microglia, T cells and their subtypes) in the pathological process of PD, which helps researchers to clearly articu- late the specific roles of these cells in the pathological process and associated mechanisms of PD. However, var- ious immune cells with mutual regulatory influences are required to prevent the onset of the disease. As a result, studying the reciprocal cooperative regulation of micro- glia and T cells in PD is necessary. Current reports show minocycline reduces the inflammatory response and decreases microglia proliferation and IL-1β production [29–31]. However, the effect of minocycline on T cells has a different phenomenon between species [32, 33]. Treatment with minocycline in rodents had no impact on T cells and IFN-γ production [32]. Still, human-derived T cells treated with minocycline reduced the prolifera- tion of cells and the ability to release pro-inflammatory cytokines [31, 33]. In addition, minocycline treatment reduced inflammation by decreasing the expression of adhesion molecules and reducing the interaction between T cells and microglia [33]. Notably, increased IL-10 production was only found in microglia and T-cell co-cultures after higher concentrations of minocycline treatment [33]. However, one of the possible reasons for the failure of minocycline to effectively treat amyotrophic lateral sclerosis in the clinic is that its concentration did not reach the concentration used in in vitro cellu- lar assays [34]. It suggests that the interactions between immune cells are essential in treating various diseases. It is believed that microglia have different cellular phe- notypic profiles, which may be influenced by the local microenvironment and other factors [40, 41]. Microglia have a complex "sensome" for sensing changes in the brain environment [40]. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 14 Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation (2023) 20:33 Recent studies suggest that PD is associated with a variety of factors, including family genetics [10], disor- ders of the gut microbiota [11], pathogenic infections [12], air pollution [13, 14], head trauma [15], making the understanding of the exact pathogenesis of PD more challenging. The pathogenesis of PD has been explored from various perspectives, including epidemiology, neu- ropathology, proteomics, genomics, and immunology, and possible mechanisms that cause PD were proposed, such as mitochondrial dysfunction [16–18], oxidative stress [17–20], protein aggregation [21, 22], and abnor- mal autophagy [23, 24]. Among them, immunological studies suggest that PD results from an imbalance in the immune system homeostasis in pathological processes in PD patients and related animal models [25–27]. Com- pared to controls, immune cells infiltrate the brain paren- chyma and peripheral tissues, causing a release of large amounts of inflammatory and regulatory factors [26, 27]. Furthermore, the activation of pre-existing immune cells in the brain also promotes the damage of dopaminer- gic neurons and exacerbates the progression of the dis- ease [8, 28]. These findings further reveal the possible mechanisms of PD pathogenesis from an immunological perspective. In particular, the phenomenon of interactions between microglia and T cells in PD was previously demonstrated and played a vital role in the pathological changes of the disease [35, 36].h Therefore, this review demonstrates the impact of the reciprocal effects between T cells and microglia in PD on the pathological process of PD through cytokine and chemokine production. Also, potential targets and mod- els in PD neuroinflammation are highlighted to provide new ideas/directions for future research. Role of microglia and related mechanisms in PD The role of microglia in PD may be neuroprotective and neurotoxic, and their cellular state is altered depending on the external environment. In PD, neurotoxic microglia are hyperactivated and increase inflammation in the microenvironment by releasing pro-inflammatory cytokines that are toxic to neuronal cells and lead to their death. Conversely, anti-inflammatory cytokines released by neuroprotective microglia reduce the number and function of neurotoxic microglia. In addition, neuroprotective microglia interact with neurons to protect neuronal survival, thereby alleviating neuronal death caused by the storm of inflammatory factors in PD experiments revealed that aberrant α-synuclein in PD mediates the transition of microglia to a neurotoxic state through TLR2, TLR4-NK-κB, and LC3-related phago- cytosis, thus exhibiting increased phagocytosis and production of pro-inflammatory cytokines [48–52]. In addition, impaired autophagy of microglia in PD resulted in increased intracellular NLRP3 (NOD-like receptor family, pyrin domain containing 3) activity, which also facilitated the transition to a neurotoxic state in micro- glia [53]. Co-culture of neurotoxic microglia with dopa- minergic neurons promoted the death of dopaminergic neurons [54]. In contrast, culture of neurotoxic microglia with neuroprotective microglia and then co-culturing the fraction with dopaminergic neurons reversed the phenomenon of dopaminergic neuron death caused by neurotoxic microglia [54]. This may be due to the pro- inflammatory cytokines (IL-1β, TNF-α, chemokines) released by overactivated neurotoxic microglia that alter the brain homeostasis, making it more favorable for microglia conversion to neurotoxic, thus causing neuro- toxic microglia proliferation and release of inflammatory cytokines. In addition, TNF-α released from neurotoxic microglia can directly induce apoptosis by binding to the tumor necrosis factor receptor-1 (TNFR1) of dopa- minergic neurons, a process that may be related to the inhibition of c-Rel with anti-apoptotic function by tumor necrosis factor-alpha (TNF-α) in dopaminergic neurons [55]. C-Rel, one of the five DNA-binding proteins that make up the nuclear factor-kappa B (NF-κB) complex, and its competition with Rel A can downregulate the transcription of apoptotic genes such as Bim and Noxa while initiating the transcription of the anti-apoptotic gene Bcl-2 in cells to maintain neuronal survival [56–59]. In addition to causing direct neuronal damage, neuro- toxic microglia can amplify inflammatory phenomena in the PD brain by altering astrocyte status [52, 60]. In the physiological state, astrocytes protect neuronal survival by secreting neurotrophic factors required by neurons. Role of microglia and related mechanisms in PD Alterations in the microglia’s epigenome, transcriptome, proteome, and metabolome can affect their morphology, ultrastructure, or cellu- lar function [40, 42]. The state of microglia is associated with their unique functions, and the clearance of poten- tially threatening substances from the brain by microglia involves the interrelation of different states of microglia [40]. In the PD environment, microglia may be neuropro- tective and neurotoxic, and the balance between these changes depends on time and environmental changes [43–45]. When microglia recognize a potentially threat- ening substance, their cellular state changes, and they kill the threatening substance by releasing pro-inflammatory cytokines and chemokines. In contrast, the excessive release of inflammatory factors causes neurotoxicity, described as "neurotoxic microglia". Moreover, microglia regulate the function of neurotoxic microglia by releas- ing anti-inflammatory cytokines and chemokines, thus restoring the homeostasis of the brain microenviron- ment, described as "neuroprotective microglia" [46, 47] (Fig. 1). However, the hyperactivation of microglia in PD may be caused by multiple factors. In vitro, cellular Xu et al. Journal of Neuroinflammation (2023) 20:33 Page 3 of 14 Xu et al. Journal of Neuroinflammation Fig. 1 Role of microglia on neurons in Parkinson’s disease (PD). The role of microglia in PD may be neuroprotective and neurotoxic, and their cellular state is altered depending on the external environment. In PD, neurotoxic microglia are hyperactivated and increase inflammation in the microenvironment by releasing pro-inflammatory cytokines that are toxic to neuronal cells and lead to their death. Conversely, anti-inflammatory cytokines released by neuroprotective microglia reduce the number and function of neurotoxic microglia. In addition, neuroprotective microglia interact with neurons to protect neuronal survival, thereby alleviating neuronal death caused by the storm of inflammatory factors in PD Fig. 1 Role of microglia on neurons in Parkinson’s disease (PD). The role of microglia in PD may be neuroprotective and neurotoxic, and their cellular state is altered depending on the external environment. In PD, neurotoxic microglia are hyperactivated and increase inflammation in the microenvironment by releasing pro-inflammatory cytokines that are toxic to neuronal cells and lead to their death. Conversely, anti-inflammatory cytokines released by neuroprotective microglia reduce the number and function of neurotoxic microglia. In addition, neuroprotective microglia interact with neurons to protect neuronal survival, thereby alleviating neuronal death caused by the storm of inflammatory factors in PD Fig. 1 Role of microglia on neurons in Parkinson’s disease (PD). Role of microglia and related mechanisms in PD In contrast, neurotoxic microglia in PD cause further neu- ronal damage by releasing pro-inflammatory cytokines that convert astrocyte phenotype to the neurotoxic A1 type [52, 60].h experiments revealed that aberrant α-synuclein in PD mediates the transition of microglia to a neurotoxic state through TLR2, TLR4-NK-κB, and LC3-related phago- cytosis, thus exhibiting increased phagocytosis and production of pro-inflammatory cytokines [48–52]. In addition, impaired autophagy of microglia in PD resulted in increased intracellular NLRP3 (NOD-like receptor family, pyrin domain containing 3) activity, which also facilitated the transition to a neurotoxic state in micro- glia [53]. Co-culture of neurotoxic microglia with dopa- minergic neurons promoted the death of dopaminergic neurons [54]. In contrast, culture of neurotoxic microglia with neuroprotective microglia and then co-culturing the fraction with dopaminergic neurons reversed the phenomenon of dopaminergic neuron death caused by neurotoxic microglia [54]. This may be due to the pro- inflammatory cytokines (IL-1β, TNF-α, chemokines) released by overactivated neurotoxic microglia that alter the brain homeostasis, making it more favorable for microglia conversion to neurotoxic, thus causing neuro- toxic microglia proliferation and release of inflammatory cytokines. In addition, TNF-α released from neurotoxic microglia can directly induce apoptosis by binding to the tumor necrosis factor receptor-1 (TNFR1) of dopa- minergic neurons, a process that may be related to the inhibition of c-Rel with anti-apoptotic function by tumor necrosis factor-alpha (TNF-α) in dopaminergic neurons There are three possible reasons for the emergence of the phenomenon of microglia hyperactivation: (1) Micro- glia mitochondrial dysfunction. Normal mitochondria via tunneling nanotubes transfer into microglia with abnormal mitochondrial function, the oxidative stress levels and the release of pro-inflammatory cytokines in microglia were downregulated, and the dysfunction of microglia was restored, which in turn alleviated the loss of dopaminergic neurons in PD [61]. (2) The ratio of neurotoxic microglia to neuroprotective microglia is imbalanced. Inhibiting the expression of Jmjd3 in the mouse midbrain impaired the conversion of microglia to Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation (2023) 20:33 Page 4 of 14 Page 4 of 14 Xu et al. Journal of Neuroinflammation example, tumors, infections, and cardiovascular diseases. In recent years, T cells have also been found to play an essential role in neurodegenerative diseases. Role of microglia and related mechanisms in PD Post-mor- tem examinations of PD patients and some animal mod- els revealed significant T-cell infiltration in the PD brain, altered surface characteristics of T cells in the peripheral circulation, and a marked reduction in cell numbers. This indicates that T cells actively respond to and participate in the onset and progression of PD. The infiltrated T cells in PD brains were classified according to their cell sur- face markers, and they were distinguished into two major types, CD4+ T cells and CD8+ T cells. Further studies focus on the two types of T cells function in PD. It reveals that both types of T cells infiltrate the substantia nigra of the PD brain, with CD8+ T cells being the main type, which was activated by recognizing target cells express- ing major histocompatibility complex (MHC)-I mol- ecules and releasing lymphotoxins such as perforin and granzyme to directly kill the target cells [71, 72] (Fig. 2). neuroprotective microglia and increased dopaminergic neuronal damage caused by the overactivation of neuro- toxic microglia [62]. Furthermore, the transfer of in vitro induced differentiation of neuroprotective microglia to mice at different time points of the disease effectively reduced the inflammatory state in the CNS, thereby pro- tecting neurons [63]. (3) Peripheral immune cell infiltra- tion. Overactivation of neurotoxic microglia causes an increase in the level of pro-inflammatory factors, and microglia remove potentially threatening substances while altering the permeability of the blood–brain barrier (BBB), causing infiltration of peripheral immune cells, which also contributes to the overactivation of neuro- toxic microglia [64, 65]. Neuroprotective microglia are a group of immune cells with the ability to regulate the inflammatory state by releasing corresponding protein molecules (arginase-1, chitinase 3-like 3, cluster of differentiation [CD] 206) and cytokines (insulin-like growth factor-1, transform- ing growth factor-beta [TGF-β], interleukin [IL] -10) as well as phagocytosis to remove cellular debris from the internal environment, promote tissue repair, and regulate neuronal damage caused by the overactivation of neuro- toxic microglia in the brain (Fig. 1). It is instrumental in maintaining the brain microenvironment homeostasis [66–68]. However, in PD, persistent stimulation of micro- glia by endogenous factors leads to a reduced conver- sion of microglia to a neuroprotective state and, thus, to a reduced ability to regulate inflammation, which in turn leads to hyperinflammatory phenomena [69]. Role of microglia and related mechanisms in PD The reason for this phenomenon may be the continuous prolifera- tion of neurotoxic microglia in PD and the correspond- ing release of inflammatory cytokines that maintains the brain microenvironment inflamed. At this time, the rele- vant factors that promote the conversion of neuroprotec- tive microglia are not secreted sufficiently, thus reducing the conversion of microglia to neuroprotective microglia. In addition, due to the increased oxidative stress in the brain environment of PD patients and animals, Rel A is continuously activated in microglia that respond to oxi- dative stress, resulting in the continuous activation of the NF-κB pathway that facilitates the conversion of micro- glia to neurotoxic microglia [57, 58, 70]. However, path- ways such as NF-κB/C-Rel, which induce microglial to neuroprotective microglia conversion, are inhibited [67]. g y y g [ ] ( g ) Meanwhile, other studies targeting T cells in PD have found that CD4+ T cells play an equally important role in the onset and progression of the disease [73, 74]. Increased motor dysfunction in PD patients was associ- ated with a rise in the number of effector memory CD4+ T cells [75]. In addition, the study reveals the effect of CD4+ T cells and CD8+ T cells on the pathological devel- opment and progression of PD by knocking the mice sep- arately [76], and found that CD4+ T cells exacerbated PD pathological progression by possibly increasing the secre- tion of pro-inflammatory factors and promoting dopa- minergic neuronal toxicity by activating the apoptotic signal Fas- Fas Ligand (FasL) [77]. However, the concept that all CD4+ T cells are considered to cause dopamin- ergic neurons damage is inaccurate. Further division of CD4+ T cells by function can be categorized into pro- inflammatory Th1 and Th17 cells and anti-inflammatory Th2 and Treg cells (Fig. 2). Among them, Treg cells are protective factors in the progression of the disease. Increasing the frequency of Treg cells through adoption, induction, and other methods can significantly improve the level of inflammation in the PD brain and alleviate the pathological process of PD [78]. In the study by Wil- liams et al. Role of microglia and related mechanisms in PD [73], when mice were injected with adeno- associated virus 2 (AAV2)-synaptophysin (SYN) 4 weeks after the detection of T cell activation and corresponsive factors expression, it was found that the expression of transcription factors T-bet and Foxp3, cytokines inter- feron gamma (IFN-γ), and IL-10 in each of Th1 and Treg cells showed a significant increase, which indicated that both Th1 and Treg cell activation levels in the early stage of PD development showed an increase. This suggests that Treg cells may regulate the inflammatory response in the early stage of the disease, but the regulatory role of The role of T cells and related mechanisms in PD T cells are one of the most important adaptive immune cells in the body, showing powerful functions in cleaning up abnormal cells, invading foreign pathogens, and play- ing an irreplaceable role in maintaining health. Abnor- malities in T-cell activation and function are closely associated with the development of many diseases, for Interactions between microglia and T cells and the related subtypes in PD Microglia and T cells are linked in terms of activation and function. All of them interact with each other through the secretion of cytokines and chemokines, which have significant effects on the pathological state of PD. Next, we discuss the interactions between microglia and T cells and their related subtypes separately on neuronal damage and protection, and further investigate the interactions between microglia and T cells and their related subtypes in PD. Treg cells on inflammation in the PD brain is gradually reduced/suppressed with the development of the disease, which eventually leads to a change in the ratio of pro- inflammatory cells to anti-inflammatory cells, resulting in an imbalance in the immune balance of the body and exacerbating the development of PD. This phenomenon may be related to the suppression of gamma-delta T (γδ T) cells. The frequency of γδ T cells in peripheral blood of PD patients is significantly reduced, and γδ T activated by IL-23 can directly inhibit the differentiation and func- tion of Treg cells through the induction of heat-sensitive mediators or humoral factors with paracrine activity [79– 81] (Fig. 2).h The role of T cells and related mechanisms in PD In addition, Tc cells can also cause neuronal death by releasing IFN-γ. The Th cells infiltrating the brain can be divided into pro-inflammatory Th1 and Th17 and anti-inflammatory Th2 and Treg. Pro-inflammatory Th cells can cause neuronal death by releasing inflammatory cytokines and exacerbating cytokine storms. In contrast, anti-inflammatory Th cells can protect neurons by reducing the release of inflammatory cytokines through the release of anti-inflammatory cytokines, thereby regulating the activity and function of pro-inflammatory Th cells. However, it is worth noting that γδ T inhibits the activity and function of Treg cells and thus reduces their ability to regulate the inflammation In the pathological process of PD, T cells can be affected by abnormal α-synuclein, DA neurons, and thus immune response, and also interact with other immune cells in the internal environment to further influence the disease progression. Overall, T cells play an irreplaceable role in the pathological development and course of PD. The role of T cells and related mechanisms in PD Page 5 of 14 Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation Fig. 2 The role of peripherally infiltrating T cells on neurons in PD. Since the damage of the BBB in PD allows T cells in the peripheral circulation to infiltrate the brain, Tc cells infiltrating the brain can recognize MHC-I molecules expressed on the surface of neurons and release granzyme and perforin, to directly cause neuronal death. In addition, Tc cells can also cause neuronal death by releasing IFN-γ. The Th cells infiltrating the brain can be divided into pro-inflammatory Th1 and Th17 and anti-inflammatory Th2 and Treg. Pro-inflammatory Th cells can cause neuronal death by releasing inflammatory cytokines and exacerbating cytokine storms. In contrast, anti-inflammatory Th cells can protect neurons by reducing the release of inflammatory cytokines through the release of anti-inflammatory cytokines, thereby regulating the activity and function of pro-inflammatory Th cells. However, it is worth noting that γδ T inhibits the activity and function of Treg cells and thus reduces their ability to regulate the inflammation [82–85]. Dopamine contributes to the differentiation of CD4+ T cells towards Th1 and Th17 by interacting with dopamine D3 receptor (DRD3) on their surface, increasing the number of pro-inflammatory T cells. At the same time, the combination of dopamine and dopa- mine D1 receptor (DRD1) inhibits the function of Treg cells, then causing dysregulation of immune regulation of the infiltrating brain T cell population [86]. In addi- tion, abnormal alpha-synuclein in PD also affects the number and function of T cells, thereby triggering severe neurodegeneration [87]. Nitrated alpha-synuclein (N-α- synuclein) can disrupt immune tolerance and activate T cells in the periphery by diverting lymphoid tissue [88]. It was noted that using N-α-synuclein as an immunogen- induced effector T cell exacerbated microglia activation, thereby amplifying neuroinflammation and neurodegen- eration [87–89]. Further investigation revealed that N-α- synuclein-stimulated T cells tend to differentiate more toward Th1 and Th17 phenotypes with pro-inflammatory cytokine release while suppressing Treg cell function [89]. Fig. 2 The role of peripherally infiltrating T cells on neurons in PD. Since the damage of the BBB in PD allows T cells in the peripheral circulation to infiltrate the brain, Tc cells infiltrating the brain can recognize MHC-I molecules expressed on the surface of neurons and release granzyme and perforin, to directly cause neuronal death. The risk of neuronal damage from interactions between microglia and T cell‑related subtypes in PD Under the normal physiological state, immune cell acti- vation maintains a stable dynamic balance. The immune cells and their related subtypes regulate each other to enable the clearance of abnormal substances to main- tain the microenvironment homeostasis. However, there is the phenomenon of cytokine storm caused by exces- sive activation of immune cells in the PD brain, which indicates that the regulation of homeostasis between immune cells in a normal physiological state is faulty. The BBB is a vital tissue for maintaining the balance of the The cause of T-cell immune dysregulation in PD may also be related to dopamine release from dopaminergic neurons. Dopamine receptors (DR) are expressed not only in neurons but also in immune cells, thus possess- ing the ability to regulate immune functions, for instance, cell differentiation, cytokine release, and cytotoxicity Page 6 of 14 Fig. 3 Microglia and T-cell interactions further amplify the inflammatory response to PD. The interaction of PD microglia and T cells amplifies the inflammatory response. In the presence of TGF-β in the microenvironment, IL-6 and IL-1β released from neurotoxic microglia act on naïve T cells had to induce their differentiation to Th17 cells and inhibit their differentiation to Treg cells, respectively. The chemokines CXCL9, CXCL10, CXCL11, CCL5, and CXCL16 released by neurotoxic microglia can bind to CD8+ T cells and CXCR3, CCR5 and CXCR6 on the surface of Th1 cells to further increase cell activation and release of inflammatory cytokines. Notably, inflammatory cytokines released from T cells can promote microglial activation and function. At the same time, microglia respond to inflammatory cytokines and increase their cellular activation by releasing cytokines and chemokines. Overall, the interaction between neurotoxic microglia and pro-inflammatory types of T cells in PD further amplifies the inflammation of the microenvironment and exacerbates neuronal damage Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation Page 6 of 14 brain microenvironment. It effectively prevents periph- eral immune cells, foreign invasive viruses, and neuro- toxic substances in the blood from entering the brain and expels metabolic waste from the CNS out of the brain. However, the BBB has been shown to be severely dam- aged in PD brains, giving the opportunity for peripheral immune cells to infiltrate the brain parenchyma. The risk of neuronal damage from interactions between microglia and T cell‑related subtypes in PD i y Following the alteration of the BBB permeability, immune cells in the peripheral circulation have the opportunity to infiltrate the brain parenchyma. Inflam- matory factors as TNF-α and IL-1β released by microglia enhance the expression of cell adhesion molecules (inter- cellular adhesion molecule 1) and vascular cell adhesion molecules (vascular cell adhesion molecule 1) on vascu- lar endothelial cells, further promoting the infiltration of peripheral immune cells into the brain parenchyma [38, 90, 91]. T cells infiltrating the brain parenchyma are induced by activated microglia to form pro-inflammatory types of T cell inflammation and through the release of inflammatory factors, exacerbating the extent of the BBB damage in PD and increasing the number of T cells infil- trating the brain [92]. These interactions lead to damage the BBB. In addition, the expression of MHC-II-like mol- ecules on the surface of microglia activate T cells, and the infiltrating T cells simultaneously induce microglia to express MHC-II-like molecules, thus further deepen- ing the interaction between these two cells [93, 94]. This has clarified the damaging behavior of the interactions on this BBB. Fig. 3 Microglia and T-cell interactions further amplify the inflammatory response to PD. The interaction of PD microglia and T cells amplifies the inflammatory response. In the presence of TGF-β in the microenvironment, IL-6 and IL-1β released from neurotoxic microglia act on naïve T cells had to induce their differentiation to Th17 cells and inhibit their differentiation to Treg cells, respectively. The chemokines CXCL9, CXCL10, CXCL11, CCL5, and CXCL16 released by neurotoxic microglia can bind to CD8+ T cells and CXCR3, CCR5 and CXCR6 on the surface of Th1 cells to further increase cell activation and release of inflammatory cytokines. Notably, inflammatory cytokines released from T cells can promote microglial activation and function. At the same time, microglia respond to inflammatory cytokines and increase their cellular activation by releasing cytokines and chemokines. Overall, the interaction between neurotoxic microglia and pro-inflammatory types of T cells in PD further amplifies the inflammation of the microenvironment and exacerbates neuronal damage Neurotoxic microglia, as one of the primary forces for clearing abnormal substances from the brain, actively kill pathogenic substances by releasing various cytokines and chemokines, and recruit other immune cells to join the process. Protection of neurons by interactions between microglia and T cell‑related subtypes in PD Concerning PD treatment, inducing microglia to convert to a neuroprotective state and increasing their function can effectively slow neuronal death in PD [62, 69, 121, 122]. At the same time, Th2 and Treg cells in the CD4+ T cell subpopulation could also slow down the loss of dopaminergic neurons by increasing their number and functional expression. Further investigation revealed that significant microglia–T cell interactions in PD have an essential role in the remission and treatment of PD. Modulating neuronal damage resulting from imbalances in regulating environmental homeostasis in the brain by targeting the interaction between microglia and T cells could be a new starting point for treating PD. Although CD8+ T cells can directly kill and release IFN-γ inflammatory factors to cause neurons death, CD4+ T cells are considered the major players in accel- erating the PD processes. In particular, Th1 and Th17 cells promote the conversion of microglia to neurotoxic microglia and increase cellular functions (Fig. 3). Th1 cells increase the activity of the NF-κB pathway in micro- glia by releasing IFN-γ and TNF-α, respectively, increas- ing the expression of toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) in microglia and inhibiting the function of the c-Rel mol- ecule. Simultaneously IFN-γ could further enhance the release of neurotoxic microglia chemokines [112–115]. On the other hand, Th17 enhanced the expression of adhesion molecules on microglia and the response to lipopolysaccharide (LPS) stimulation through the release of IL-17 [92, 116]. The effect of these two T cell subtypes on microglia increased the number of neurotoxic micro- glia and the release of pro-inflammatory cytokines (TNF- α, IL-1β, IL-6). It exacerbates oxidative stress and further disrupts the brain microenvironment homeostasis, thus accelerating neuronal loss [117] (Fig. 3). In addition to causing dopaminergic neurons damage by inducing the release of inflammatory factors from microglia, Th1 cells can also enhance the phagocytic capacity of microglia by upregulating the expression of transcripts of mac- rophage c-mer tyrosine kinase (MerTK), a receptor asso- ciated with phagocytosis, in microglia through the release of effector molecules, thus exacerbating the damage to dopaminergic neurons [115]. Enhanced phagocytosis of dopaminergic neurons by microglia during inflammation contributes to the pathological process of PD, which may be one of the reasons for the loss of dopaminergic neu- rons in PD [118–120]. First, how do neuroprotective microglia affect T-related subtypes of cells? The risk of neuronal damage from interactions between microglia and T cell‑related subtypes in PD By comparison, CXCL16 released from microglia through CXCR6 recep- tors can drive Th1 and T-cytotoxic (Tc)1 cell migration and directly activate the NF-κB pathway in Th1 cells, increasing the expression of pro-inflammatory genes and enhancing T cell-mediated inflammatory responses [109–111]. substantia nigra [105, 106]. Besides, inhibition of CXCR3 activation reduced the production of IFN-γ activated T cells while protecting against lethal CD8+ T cell-medi- ated tissue damage [107, 108]. In contrast, inhibition of CCR5 activation polarized activated T cells toward Th2 cells and secreted anti-inflammatory factors such as IL-4 and IL-10, while decreasing IL-17 release from Th17 cells and alleviating neuronal loss. By comparison, CXCL16 released from microglia through CXCR6 recep- tors can drive Th1 and T-cytotoxic (Tc)1 cell migration and directly activate the NF-κB pathway in Th1 cells, increasing the expression of pro-inflammatory genes and enhancing T cell-mediated inflammatory responses [109–111]. Th1, and Th17, and increasing the release of correspond- ing inflammatory cytokines. At the same time, the interaction between microglia and T cells increases the phagocytic capacity of microglia. It allows CD8+ T cells to recognize dopaminergic neurons, thus accelerating the rate and extent of dopamine neuron damage, which in turn exacerbates the pathological state of PD. The risk of neuronal damage from interactions between microglia and T cell‑related subtypes in PD Among the cytokines, neurotoxic microglia promote and regulate the differentiation and function of Th17 cells by releasing inflammatory cytokines, while suppressing the differentiation of Treg cells, and TGF-β is an essential factor in the differentiation of naïve T to Th17 and Treg [95] (Fig. 3). When conditions of TGF-β factors are present in the environment, IL-6 and IL-1β released from neurotoxic microglia induce differentiation of naïve T cells infiltrating into the brain parenchyma to pro-inflammatory Th17 cells [96–99]. This is basically because IL-6 determines the fate of naïve T differentia- tion to Th17 cells by activating the transcription factor STAT3, while IL-1β inhibits the differentiation of naïve T cells to Treg cells. In addition, IL-1β increases the activ- ity of Th17 and the release of the inflammatory cytokine IL-17 and decreases the secretion of the anti-inflamma- tory factor IL-10 [100] (Fig. 3). T cells infiltrating into the brain parenchyma, thereby accelerating the rate of dopaminergic neuron loss in PD (Fig. 2).i Chemokines play a significant role in the communi- cation between neurons and immune cells, and they have chemotactic activity on immune cells and affect immune cell proliferation, cytokine secretion, and phagocytosis [101, 102]. In PD, the chemokines (C-X-C motif) ligand (CXCL) 9, CXCL10, CXCL11, CCL5, and CXCL16 released by neurotoxic microglia can increase their activation and functional expression by binding to C-X-C motif receptor (CXCR) 3, C–C motif receptor (CCR) 5, and CXCR6 chemokine receptors on the sur- face of T cells [103, 104] (Fig. 3). It was noted that inhi- bition of CXCR3 and CCR5 activation on T cells could reduce the number of CD3+ T cell infiltration in the Cytokines released from microglia can also act on dopaminergic neurons, causing their surface MHC-I-like molecule expression to be recognized and killed by CD8+ Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation Page 7 of 14 substantia nigra [105, 106]. Besides, inhibition of CXCR3 activation reduced the production of IFN-γ activated T cells while protecting against lethal CD8+ T cell-medi- ated tissue damage [107, 108]. In contrast, inhibition of CCR5 activation polarized activated T cells toward Th2 cells and secreted anti-inflammatory factors such as IL-4 and IL-10, while decreasing IL-17 release from Th17 cells and alleviating neuronal loss. Protection of neurons by interactions between microglia and T cell‑related subtypes in PD At the same time, cellular interactions reduced the number and function of pro-inflammatory-type cells, thereby regulating the inflammatory state in the microenvironment and further protecting neurons from damage the activity of Janus kinase (JAK) signal transducer and activator of transcription (STAT) 6 pathway and decreasing the activity of JAK–STAT3 pathway in microglia, respectively. This decrease in neurotoxic microglia is due to the inhibition of STAT1 phospho- rylation by IL-10, which in turn leads to a decrease in the activity of the NF-κB pathway [130–135]. Mean- while, IL-4 and IL-13 released from Th2 cells induced IL-1 receptor antagonist (IL-1Ra) expression blocking the induction of IL-1β into microglia, further reduc- ing the number of neurotoxic microglia and the release of inflammatory cytokines and chemokines [136, 137]. In addition, infiltrating Treg cells also inhibit the acti- vation of microglia by nitrocellularized α-synuclein and regulate the state of neurotoxic microglia and the release of inflammatory factors by reducing the migra- tion, phagocytosis, reactive oxygen species (ROS) pro- duction, and NF-κB activation of neurotoxic microglia [87, 89, 138]. Notably, Treg can also reduce the num- ber of neurotoxic microglia by inducing apoptosis, thus protecting neurons from dopaminergic neuronal dam- age caused by excessive activation of neurotoxic micro- glia [117]. Fig. 4 Microglia and T cells reciprocally regulate inflammation in PD. Neuroprotective microglia can induce the differentiation of naïve T cells to Th2 and Treg cells by secreting cytokines IL-4 and TGF-β. Meanwhile, chemokines CCL1, CCL17, CCL22, CCL24, and CCL18 released from neuroprotective microglia can bind to CCR4, CCR8, and PITPNM3 on the surface of Th2 and Treg cells, further increasing cell activation, anti-inflammatory cytokine release, and migration. Notably, anti-inflammatory cytokines released by Th2 and Treg cells can increase the number of neuroprotective microglia and inhibit the function of neurotoxic microglia. In conclusion, the interaction between neuroprotective microglia and anti-inflammatory T cell subtypes in PD increases the number and function of anti-inflammatory-type cells. Protection of neurons by interactions between microglia and T cell‑related subtypes in PD At the same time, cellular interactions reduced the number and function of pro-inflammatory-type cells, thereby regulating the inflammatory state in the microenvironment and further protecting neurons from damage Overall, this evidence suggests that the interaction between microglia and T cells increases the number and function of neuroprotective microglia, Th2, and Treg cells and effectively reduces the conversion of micro- glia to neurotoxic microglia, decreases the activation of pro-inflammatory type T cells, thus decreasing the level of inflammatory cytokine secretion in the brain and regulating the dysregulation of internal environmental homeostasis, which in turn protects against damage to dopaminergic neurons in PD and slows down the patho- logical symptoms and disease process. Protection of neurons by interactions between microglia and T cell‑related subtypes in PD Among cytokines, inflam- mation-suppressing cytokines such as IL-4, IL-10, IL-13, and TGF-β released from neuroprotective micro- glia in the normal physiological state can inhibit the production of pro-inflammatory cytokines such as IL-6 and TNF-α [123–125]. Meanwhile, IL-4 and TGF-β released from microglia are important cytokines that induce the differentiation of naïve T cells to Th2 and Treg cells, respectively, and have a significant impact on the developmental and functional regulation between T cells and their subpopulations (Fig. 4). Among the chemokines, neuroprotective microglia can induce T cell differentiation of Th2 and Treg cells and enhance cell function by releasing chemokines CCL1, CCL17, CCL22, and CCL24 that bind to T cell surface CCR4 and CCR8, thereby regulating the inflammatory state in the brain to protect neurons from death [103, 104] (Fig. 4). Moreover, CCL18 released from neuroprotec- tive microglia, can block the recruitment of T cells in tumors, reduce the suppressive effect of Treg cells, and regulate the immune response by inhibiting the CCL18- PITPNM3 signaling pathway in tumor-related research [102, 126–129]. The role of CCL18 chemokine in PD is not clear. Considering the co-immunomodulatory role that Treg cells exhibit in PD and neoplastic disease, whether increasing the secretion of neuroprotective microglia CCL18 in PD can increase the number and Collectively, this evidence suggests that interactions between microglia and T cells in PD act synergistically to cause neuronal injury. The interactions between cells alter the brain homeostasis toward a pro-inflammatory state, promoting the number of neurotoxic microglia, Page 8 of 14 Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation Fig. 4 Microglia and T cells reciprocally regulate inflammation in PD. Neuroprotective microglia can induce the differentiation of naïve T cells to Th2 and Treg cells by secreting cytokines IL-4 and TGF-β. Meanwhile, chemokines CCL1, CCL17, CCL22, CCL24, and CCL18 released from neuroprotective microglia can bind to CCR4, CCR8, and PITPNM3 on the surface of Th2 and Treg cells, further increasing cell activation, anti-inflammatory cytokine release, and migration. Notably, anti-inflammatory cytokines released by Th2 and Treg cells can increase the number of neuroprotective microglia and inhibit the function of neurotoxic microglia. In conclusion, the interaction between neuroprotective microglia and anti-inflammatory T cell subtypes in PD increases the number and function of anti-inflammatory-type cells. A preliminary survey of the dysregulation of protective effects by microglia–T cell interactions in PD function of Treg cells at the site of inflammation and thus regulate the inflammatory state needs to be veri- fied by subsequent studies. Previously, we described the relevant pathways of neu- ronal damage by inflammatory factor storms mediated by immune cells in PD. The relevant role of microglia and T cell and their related subtype interactions for damage and protection in PD has also been described. However, the regulatory role of microglia and T cell-related sub- types interactions on inflammation seems to be dimin- ished or to have failed in PD. The possible reasons are: (1) the negative feedback mechanism of age-related neu- roprotective microglia regulating inflammatory state may be defective. The increase in pro-inflammatory levels of microglia with age is accompanied by a "dystrophic" phenomenon of denuclearization and process fragmen- tation [139, 140]. This causes a decrease in receptor expression and cytokine secretion converting microglia i Next, how do infiltrating inflammatory T-cell affect microglia? The T cell that infiltrates the brain also exerts critical regulatory effects on inflammation through interactions with microglia. Among them, Th2 and Treg cells can induce the conversion of microglia to neuroprotective microglia and release anti-inflam- matory cytokines and neurotrophic factors by releas- ing anti-inflammatory cytokines while reducing the number of neurotoxic microglia and the expression of related functions (Fig. 4). It was shown that microglia responding to IL-4 and IL-10 induce microglial con- version to neuroprotective microglia by increasing Page 9 of 14 Xu et al. Journal of Neuroinflammation (2023) 20:33 Page 9 of 14 Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation highly consistent with dopamine neurons and microglia in the brains of human PD patients. This model allows for a better investigation of the pathological effects of genetic mutations and other factors in sporadic PD patients [144, 150]. Currently, iPSC models have been used to study PD pathogenesis. In contrast, in the study on PD neuro- inflammation, it was found that Th17 cells act on iPSC- induced neurons by releasing IL-17 to increase their surface IL-17 receptor expression and NF-κB activation- induced neuronal death [151]. In recent years, as iPSC technology has become more mature, the establishment of organoid models has also been rapidly developed, ena- bling the derivation of in vitro 2D models to 3D models that better assess the spatial effects of neurons and other outcomes. A preliminary survey of the dysregulation of protective effects by microglia–T cell interactions in PD T cells infiltrated into the brain were induced by pro-inflammatory microglia, dopa- mine secreted by neurons, the internal environment of the brain and other factors, and the activation state and function of pro-inflammatory Tc, Th1, and Th17 cells were further enhanced, while the function of anti-inflam- matory Th2 and Treg was suppressed. At the same time, naïve T cells were also induced to polarize toward pro- inflammatory types of Th1 and Th17, which increased the number of pro-inflammatory cell types, further pro- moting inflammation in the cerebral environment and weakening the regulatory effect of anti-inflammatory cell types. In this process, the increased inflammatory state of the brain environment promotes the conversion of microglia to neurotoxic microglia. It releases inflam- matory cytokines and neurotoxic mediators to aggravate neuronal damage further, resulting in a vicious cycle between dying neurons and acute inflammation [142, 143]. A potential new model for studying the cellular interactions in PD In previous studies on the pathogenesis of PD, experi- mental investigations were often conducted using model animals and cells. These models have had significant results, and the pathological mechanisms associated with PD have been further explained. However, some of the limitations of the above models include epigenetic dif- ferences between species, leading to findings that do not accurately describe the pathological process in human PD patients [144]. Although data were obtained in some studies by taking postmortem brain tissue samples from human PD patients, it should be taken into account that this data only reflects the pathological changes in the brains of patients with end-stage PD [145]. The develop- ment of PD is a slow process, and the presence of primary microglia and dopamine neurons that are more difficult to acquire and fail to proliferate has created some diffi- culties in exploring the development of this disease in the brain of PD patients. Along with the rapid development of induced pluripotent stem cells (iPSC) technology, researchers have used a variety of terminally differenti- ated cells from PD patients to induce them to become midbrain dopamine neurons, and microglia, by iPSC technology and further characterized them using gene sequencing, calcium imaging, and electrophysiological methods [146–149]. iPSC-induced cells were found to be A preliminary survey of the dysregulation of protective effects by microglia–T cell interactions in PD However, the current midbrain organoid sys- tem is mainly derived from neuroepithelial stem cells, resulting in a lack of microglia in this model, making its use in PD neuroinflammation problematic [152]. How- ever, a recent study has successfully integrated human functional microglia into the midbrain organoid system and demonstrated the feasibility and stability of this approach by examining the gene expression, functional changes, and communication ability of microglia in this model [152]. The use of this microglia-embedded mid- brain organoid model to study the interaction between microglia and T cells in PD on the pathological develop- ment of PD is expected to provide a more detailed and accurate assessment of the impact of microglia–T cell interaction on the progressive pathological process of human PD patients. to neuroprotective microglia, resulting in a decrease in the number of neuroprotective microglia and, thus, a significant reduction in the regulation of neurotoxic microglia [141]. (2) The microenvironment in the brain promotes the differentiation and functional expression of the inflammatory types of cells, further contributing to immune dysregulation. T cells infiltrated into the brain were induced by pro-inflammatory microglia, dopa- mine secreted by neurons, the internal environment of the brain and other factors, and the activation state and function of pro-inflammatory Tc, Th1, and Th17 cells were further enhanced, while the function of anti-inflam- matory Th2 and Treg was suppressed. At the same time, naïve T cells were also induced to polarize toward pro- inflammatory types of Th1 and Th17, which increased the number of pro-inflammatory cell types, further pro- moting inflammation in the cerebral environment and weakening the regulatory effect of anti-inflammatory cell types. In this process, the increased inflammatory state of the brain environment promotes the conversion of microglia to neurotoxic microglia. It releases inflam- matory cytokines and neurotoxic mediators to aggravate neuronal damage further, resulting in a vicious cycle between dying neurons and acute inflammation [142, 143]. to neuroprotective microglia, resulting in a decrease in the number of neuroprotective microglia and, thus, a significant reduction in the regulation of neurotoxic microglia [141]. (2) The microenvironment in the brain promotes the differentiation and functional expression of the inflammatory types of cells, further contributing to immune dysregulation. Conclusionh This article illustrates that interactions between micro- glia and T cells and their related subtypes exhibit a sig- nificant role in the onset and course of PD. Overall, the interactions among neurotoxic microglia, Tc, Th1, and Th17 cells in PD raise the level of inflammation in the microenvironment and elevate the activation and func- tion of pro-inflammatory cells, amplifying the effects of immune cells on neuronal damage, thus exacerbating the pathological condition of PD and accelerating the disease process. Meanwhile, there is a complex inter- play between neuroprotective microglia, Th2, and Treg cells in regulating the activation and function of pro- inflammatory cells, and controlling the inflammatory state of the internal environment, furthermore reduc- ing neuronal damage. These cells could protect neurons from damage in PD by potentiating the communica- tions between microglia and T cells and their related subtypes in PD to regulate the inflammatory cytokine storm caused by excessive activation of immune cells. This review also focuses on the CCL18–PITPNM3 signaling pathway and γδ T cells, which show great Page 10 of 14 Xu et al. Journal of Neuroinflammation (2023) 20:33 Xu et al. Journal of Neuroinflammation Availability of data and materials Not applicable. 21. Mehra S, Sahay S, Maji SK. alpha-Synuclein misfolding and aggregation: Implications in Parkinson’s disease pathogenesis. Biochim Biophys Acta Proteins Proteom. 2019;1867:890–908. Competing interests Received: 15 July 2022 Accepted: 8 February 2023 Received: 15 July 2022 Accepted: 8 February 2023 Abbreviations PD Parkinson’s disease Th T-helper CCL C–C motif ligand PITPNM3 Phosphatidylinositol transfer protein 3 CNS Central nervous system TNFR Tumor necrosis factor receptor TNF-α Tumor necrosis factor-alpha NF-κB Nuclear factor-kappa B BBB Blood–brain barrier CD Cluster of differentiation TGF-β Transforming growth factor-beta IL Interleukin MHC Major histocompatibility complex FasL Fas Ligand AAV2 Adeno-associated virus 2 SYN Synaptophysin IFN-γ Cytokines interferon gamma γδ T Gamma-delta T DR Dopamine receptors DRD3 Dopamine D3 receptor DRD1 Dopamine D1 receptor CXCL C-X-C motif ligand CXCR C-X-C motif receptor CCR C–C motif receptor Tc T-cytotoxic TLR4 Toll-like receptor 4 MyD88 Myeloid differentiation primary response LPS Lipopolysaccharide MerTK Macrophage c-mer tyrosine kinase JAK Janus kinase STAT Signal transducer and activator of transcription IL-1Ra IL-1 receptor antagonist ROS Reactive oxygen species IPSC Induced pluripotent stem cells NLRP3 NOD-like receptor family, pyrin domain containing 3 N-α-synuclein Nitrated alpha-synuclein Acknowledgements 14. 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https://openalex.org/W4318752905	https://zenodo.org/records/7595442/files/e01022304002.pdf	es		Trombocitopenia Inducida por Heparinas, Revisión de los Mecanismos Implicados, Diagnostico y Manejo de Un Evento Adverso Complejo	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	10,487	I C T U S 2023;04(01):e01022304002 Trombocitopenia Inducida por Heparinas, Revisión de los Mecanismos Implicados, Diagnostico y Manejo de Un Evento Adverso Complejo Artículo de Revisión Luis Andrés Dulcey-Sarmiento1 , Juan Sebastián Theran-Leon2 , Valentina Cabrera-Peña3 , Rafael Guillermo Parales-Strauch4 y Raimondo Caltagirone5 1 Universidad de los Andes Médico Internista. Docente Catedra Medicina de la Universidad Autonoma de Bucaramanga 2 Universidad de Santander. Residente de Medicina Familiar 3 Universidad Autónoma de Bucaramanga. Interno de Pregrado en Medicina 4 Universidad Autónoma de Bucaramanga. Interno de Pregrado en Medicina 5 Universidad de los Andes Mérida Venezuela. Médico Internista Fecha de recepción del manuscrito: 13/Octubre/2022 Fecha de aceptación del manuscrito: 20/Enero/2023 Fecha de publicación: 31/Enero/2023 DOI: 10.5281/zenodo.7595442 Creative Commons: Esta obra está bajo una Licencia Creative Commons Atribución-No Comercial-Sin Derivadas 4.0 Internacional. Resumen—La trombocitopenia inducida por heparina (TIH) es un trastorno mediado por el sistema inmunitario causado por anticuerpos que reconocen complejos del factor plaquetario 4 y la heparina. La trombosis es una característica central e impredecible de este síndrome. A pesar del manejo óptimo, la morbilidad y la mortalidad de la enfermedad por trombosis siguen siendo altas. El estado hipercoagulable en TIH es biológicamente distinto de otros trastornos trombofílicos en que las complicaciones clínicas son directamente atribuibles a los complejos inmunes ultra grandes (CIUG) circulantes. En algunos individuos, los CIUG provocan respuestas procoagulantes celulares no controladas que culminan en trombosis. Hasta la fecha, los factores de riesgo clínicos y biológicos asociados con el riesgo trombótico en TIH siguen siendo esquivos. Esta revisión resumirá nuestra comprensión actual de la trombosis en TIH con atención a sus características clínicas, mecanismos celulares. ICTUS 2023;4(1):e01022304002 Palabras clave—Heparina, Trombocitopenia, Trombosis, Plaquetas Abstract—Heparin-induced thrombocytopenia, review of mechanisms involved, diagnosis and management of a complex adverse event Heparin induced thrombocytopenia (HIT) is an immune mediated disorder caused by antibodies that recognize complexes of platelet factor 4 and heparin. Thrombosis is a central and unpredictable feature of this syndrome. Despite optimal management, disease morbidity and mortality from thrombosis remain high. The hypercoagulable state in HIT is biologically distinct from other thrombophilic disorders in that clinical complications are directly attributable to circulating ultra-large immune complexes (ULIC). In some individuals, ULIC elicit unchecked cellular procoagulant responses that culminate in thrombosis. To date, the clinical and biologic risk factors associated with thrombotic risk in HIT remain elusive. This review will summarize our current understanding of thrombosis in HIT with attention to its clinical features, cellular mechanisms, and its management. ICTUS 2023;4(1):e01022304002 Keywords—Heparin, Thrombocytopenia, Thrombosis, Platelets 1 TROMBOCITOPENIA INDUCIDA POR HEPARINAS DULCEY-SARMIENTO, LA. et al. incidencia es probablemente rara y significativamente menor que la provocada por la exposición a la heparina. I NTRODUCCIÓN Características clínicas de la trombosis a heparina no fraccionada (HNF) sigue siendo el pilar de la anticoagulación para indicaciones para las que no existen sustitutos efectivos, como la anticoagulación para cirugía de derivación cardiopulmonar (CDC), circuitos de oxigenador de membrana extracorpórea (ECMO), diálisis y válvulas protésicas mecánicas. El uso continuado de HNF en la práctica clínica pone a los pacientes en riesgo de desarrollar trombocitopenia inducida por heparina (TIH), un trastorno inmunitario potencialmente devastador causado por anticuerpos contra complejos del factor plaquetario 4 (FP4) y heparina (anti-FP4/H Abs). L La trombosis es la complicación más grave y potencialmente mortal de la TIH. La trombosis ocurre en la presentación o complica el curso de la enfermedad en 20 a 64 %.1, 2 La morbilidad de la enfermedad se ve agravada por las altas tasas de hemorragia (≈40 %) asociadas con el uso de potentes anticoagulantes distintos de la heparina para la prevención o el tratamiento de la trombosis.2 Incluso con el uso de anticoagulantes alternativos, la trombosis contribuye a los desenlaces fatales en ≈6–26 % de los pacientes con TIH.2–5 Dado que la trombosis es fundamental para la morbilidad y mortalidad de la enfermedad en TIH, esta revisión resumirá nuestra comprensión actual de sus factores de riesgo, mecanismos y tratamiento. Se desarrollarán aspectos de la enfermedad, como la epidemiología,3, 6 el diagnóstico7 o las pruebas de laboratorio8, 9 en TIH a lo largo de la presente revisión. C ARACTERÍSTICAS CLÍNICAS Y FACTORES DE RIESGO DE TROMBOSIS La TIH es principalmente una enfermedad de pacientes hospitalizados o dados de alta recientemente. La TIH ocurre en 0.5 a 1 % de los pacientes expuestos a HNF por indicaciones médicas o quirúrgicas, con una incidencia notablemente menor (0.1 a 0.5 %) en pacientes que reciben HBPM.3, 6, 10, 11 Si bien aún no se ha investigado directamente el impacto de los anticoagulantes orales directos (ACOD), los datos observacionales sugieren que la incidencia de TIH y su carga de morbilidad en los últimos años no se ha visto significativamente afectada. Los datos de una base de datos estadounidense disponible públicamente indican que la incidencia de TIH se ha mantenido bastante estable entre los años 2009 y 2013, con una prevalencia diagnóstica entre el año inicial y el último del período de estudio (2013) que muestra solo una disminución modesta.3 De varias poblaciones expuestas a la heparina, las que se someten a cirugía cardíaca parecen tener un riesgo particularmente alto de TIH, a pesar de que solo un subconjunto de los anticuerpos TIH generados son activadores de plaquetas.12, 13 Si bien se han informado casos aislados de TIH espontánea en los últimos años,14–18 su Datos de contacto: Luis Andrés Dulcey-Sarmiento, ucaramanga,Santander, Colombia, Tel: (601) 339 49 49, luismedintcol@gmail.com Para la mayoría de los pacientes, TIH se presenta en el contexto de una exposición reciente como una caída en el recuento de plaquetas, ya sea como una trombocitopenia absoluta (<150 000/µL) o relativa (>30 % de caída del recuento de plaquetas inicial). A diferencia de otros trastornos trombocitopénicos, los recuentos bajos de plaquetas en TIH no aumentan el riesgo de hemorragia, sino que sirven como marcador de riesgo trombótico.19 La verdadera incidencia de trombosis en TIH es incierta debido a los pocos estudios prospectivos de esta enfermedad y los desafíos para determinar las complicaciones relacionadas con TIH en poblaciones de pacientes en estado crítico con factores de riesgo de confusión. Sin embargo, estudios retrospectivos recientes documentan la aparición de trombosis en ≈20-64 % de los pacientes con TIH (Tabla 1).1, 2 Se desarrollan trombosis nuevas o progresivas en ≈19 a 40 % de los pacientes tratados con anticoagulantes alternativos, mientras que la mortalidad varía según el estudio. Los pacientes que desarrollan trombocitopenia aislada como presentación inicial de la enfermedad siguen teniendo un riesgo significativo de trombosis posterior que ocurre en 18 a 50 %.5, 20 Momento de la trombosis Mientras que la trombocitopenia sigue un curso predecible después de la seroconversión, con recuentos de plaquetas que generalmente disminuyen de 2 a 4 días después de la seroconversión, la trombosis es menos predecible y puede ocurrir en cualquier momento después de la detección de anticuerpos anti-FP4/H circulantes.21 En una gran revisión retrospectiva de 408 pacientes con TIH, aproximadamente dos tercios de los pacientes desarrollaron trombosis simultáneamente (26 %) o varios días después de la trombocitopenia (40 %), mientras que un tercio experimentó trombosis antes de una caída en los recuentos de plaquetas.1 9 El riesgo trombótico en TIH se extiende durante varias semanas más allá de la exposición a la heparina debido a la presencia de Abs antiFP4/H circulantes que reconocen el FP4 unido a glicosamginoglicanos (GAG) endógenos u otros componentes plaquetarios.22–24 La reactividad cruzada de los anticuerpos TIH con FP4 unido a los GAG de la superficie celular también explica por qué el momento de la interrupción de la heparina no tiene un impacto medible en la progresión de la enfermedad en algunos pacientes. Tipos de trombosis La trombosis en TIH afecta tanto a los lechos arteriales como venosos, y la trombosis venosa ocurre de tres a cuatro veces más comúnmente que la trombosis arterial (Tabla 1).19 La trombosis venosa profunda (TVP) y la embolia pulmonar (EP) son los sitios más comunes de trombosis venosa, mientras que la trombosis arterial se manifiesta con mayor frecuencia como embolia arterial periférica, seguida de accidente cerebrovascular e infarto de miocardio. La trombosis 2 I C T U S 2023;04(01):e01022304002 Tipo de estudio (n.º total de pacientes /n.º de pacientes con TIH) Prevalencia de trombosis en el momento del diagnóstico Eventos de trombosis arterial versus venosa en la presentación Desarrollo de trombosis nueva/progresiva durante el tratamiento Amputación Muerte Warkentin et al, 1996.20 Centro Único Retrospectivo (127/127) 51 % 1:4 76 % NR 20 % Elalamy et al, 2009.5 Multicéntrico Retrospectivo (114/49) 59 % 1:1 43 % 8% 53 % Kuter et al, 2017.1 Multicéntrico Retrospectivo (442/355) 20 % NR 19 % 3% 22 % Pishko et al, 2019.2 Centro Único Retrospectivo (310/42) 64 % 1:4 36 % NR 26 % Gachas et al, 2020.4 Multicéntrico Retrospectivo (144/144) 40 % 1:3 NR 4% 6% Autor TABLA 1: C ARACTERÍSTICAS CLÍNICAS DE LA TROMBOSIS EN TIH TOMADO DE LAS REFERENCIAS 1,2,4,5,20. 1, 2, 4, 5, 20 N OTA : NR= N O R EGISTRADO arterial puede ser más común después de una cirugía cardíaca,4 y es más probable que la TIH espontánea sea precipitada por una cirugía ortopédica reciente.30 Las presentaciones distintivas, pero poco comunes, de trombosis incluyen necrosis de la piel, gangrena venosa de las extremidades, hemorragia suprarrenal bilateral y trombosis de las venas cerebrales (TVC).31–35 La necrosis de la piel en TIH ocurre en los sitios de inyección de heparina y es uno de los pocos entornos clínicos donde TIH puede presentarse sin trombocitopenia acompañante.31 La gangrena venosa de las extremidades a menudo se precipita por el uso concomitante de warfarina y es causada por una oclusión venosa severa que se extiende a los lechos venulares.33, 36, 37 La hemorragia suprarrenal es una manifestación trombótica de TIH causada por la oclusión de la única vena central suprarrenal que irriga la glándula suprarrenal. Esta complicación se observa con mayor frecuencia en el posoperatorio y, cuando es bilateral, el paciente presenta síntomas y signos de insuficiencia suprarrenal (hipotensión, náuseas, vómitos e hiponatremia), si no se reconoce, puede progresar hasta la muerte por insuficiencia suprarrenal.32, 38 Raras presentaciones de TVC han sido reportadas en casos espontáneos34, 35 así como TIH inducida por fármacos.39,40,41,42 Factores de riesgo de trombosis Numerosos estudios han examinado los factores de riesgo biológicos y clínicos que predisponen a las personas a complicaciones trombóticas en TIH. Hasta la fecha, no hay evidencia que respalde el papel de las anomalías trombofílicas convencionales, como el factor V Leiden o las deficiencias de antitrombina, proteína C o proteína S.43, 44 Como se analiza a continuación, la variación polimórfica en el dominio extracelular del receptor FcγIIA (H131R; histidina a arginina en la posición del aminoácido 131) y su regulación funcional por las tirosinas fosfatasas, CD148 y el T-Cell Ubiquitin Ligand-2 (TULA-2), se han identificado como posibles causas genéticas que influyen en la predisposición a la trombosis en TIH.45, 46 Si bien los estudios de asociación del genoma completo (GWAS) han identificado varios polimorfismos de un solo nucleótido (SNP) y asociaciones HLA en pequeñas cohortes de TIH en comparación con los pacientes de control,47–50 estos hallazgos pueden ser más relevantes para el desarrollo de la respuesta inmunitaria dado el diseño del estudio. de comparar pacientes con TIH con los que no lo tienen. Otras deficiencias de los estudios GWAS, incluidas las diferencias en la determinación de casos, la falta de validación y la ausencia de estudios funcionales, limitan actualmente la generalización de estos estudios para comprender la patogenia de TIH. Otros factores de riesgo clínico aparecen relacionados con la presentación de la enfermedad. Varios grupos han identificado la enfermedad cardiovascular y/o la cirugía como los principales factores de riesgo de trombosis arterial.3, 4, 43, 51 La lesión endotelial asociada con los catéteres venosos centrales coloca a los pacientes en mayor riesgo de trombosis venosa.51, 52 Otros predictores clínicos de riesgo trombótico incluyen trombocitopenia severa, con una caída >70 % en el recuento de plaquetas que aumenta el riesgo trombótico en 8 veces, niveles altos de anticuerpos o títulos en inmunoensayos o ensayos de activación plaquetaria y activación plaquetaria independiente de heparina observada en un subconjunto de pacientes con TIH autoinmune que explica las manifestaciones de la enfermedad que ocurren después de la interrupción de la heparina.53 Algunos de estos anticuerpos evaluables o de las pruebas funcionales son las aquí 3 TROMBOCITOPENIA INDUCIDA POR HEPARINAS señaladas en la tabla 2. No se recomienda realizar sistemáticamente pruebas de detección de anticuerpos a todos los pacientes en tratamiento con heparina, dado que su sensibilidad y su especificidad para predecir la aparición de TIH es baja.54 La realización de pruebas de detección de anticuerpos debe basarse en una sospecha clínica y no debe retrasar la instauración del tratamiento apropiado cuando la clínica lo indica. Se recomienda realizar las pruebas de detección de anticuerpos heparina-FP4 en todos los pacientes tratados con heparina en los que se sospecha TIH por el patrón temporal del descenso plaquetario o por la aparición de trombosis. Existen diversos tipos de pruebas para la detección de anticuerpos TIH, pero no existe una prueba definitiva con sensibilidad y especificidad del 100 %. Los métodos inmunológicos que detectan IgG, IgA e IgM circulantes tienen una sensibilidad cercana al 97 %, a costa de una baja especificidad (un 74-86 %, más alta en los métodos que sólo detectan IgG) especialmente en pacientes sometidos a cirugía cardiaca,55 lo que resulta en un alto valor predictivo negativo (>95 %). Los métodos funcionales como la medición de la agregación plaquetaria o la liberación de serotonina de plaquetas activadas aumentan la especificidad y el valor predictivo positivo (89100 %). La utilización de ambos métodos de detección puede ser complementaria; dado su alto valor predictivo negativo, se recomienda la realización de una prueba serológica en los casos de sospecha clínica intermedia o alta: si el resultado es negativo, se invita a pensar en diagnósticos alternativos.56 En los pacientes con una sospecha intermedia y prueba serológica positiva se recomienda confirmarlo con una prueba funcional si está disponible. Patogenia de la trombosis en TIH El intenso estado de hipercoagulabilidad en TIH es desencadenado por la activación celular de Abs anti-FP4/H que se involucran principalmente en el receptor celular Fcγ, FcγRIIA (en plaquetas, monocitos y neutrófilos)57, 58 o activan indirectamente las células endoteliales a través de mecanismos no FcγR.59 El FcγRIIA humano (CD32A) es un receptor de baja afinidad de glicoproteína transmembrana tipo 1 de 40 kDa para IgG que se une preferentemente a IgG en complejos inmunes sobre IgG monomérica.60–62 La agrupación de los receptores FcγRIIA por inmunocomplejos inicia la fosforilación del motivo de activación basado en tirosina del inmunorreceptor (ITAM) contenido en la cola citoplásmica del receptor y activa la señalización aguas abajo a través de la tirosina quinasa del bazo (Syk), lo que da como resultado la liberación de reservas intracelulares de Ca 2+, desgranulación, producción de citoquinas y activación celular.60 Aunque FcγRIIA estuvo implicado en la activación celular por anticuerpos TIH en la década de 1980,57 su papel esencial en la patogénesis de la enfermedad no se demostró hasta el desarrollo del modelo TIH murino en 2001. Como los ratones carecen del equivalente humano de FcγRIIA63 y el FP4 murino no cruza al reaccionar con anticuerpos de tipo TIH, DULCEY-SARMIENTO, LA. et al. se generaron 64 ratones transgénicos dobles, que expresaban hFP4 y hFcγRIIA, para caracterizar los requisitos in vivo para la trombosis. Los ratones transgénicos únicos, que expresan los transgenes hFP4 o hFcγRIIA, no desarrollaron trombocitopenia o trombosis cuando se les inyectó un anticuerpo monoclonal similar a TIH (KKO)64 pero los ratones transgénicos dobles desarrollaron trombocitopenia severa (>80 % de caída en el recuento de plaquetas) y trombosis en múltiples lechos vasculares (corazón, hígado, riñones y pulmones) en respuesta al anticuerpo monoclonal.65 Además de establecer los requisitos para FcγRIIA celular en la trombosis TIH, estos estudios murinos también fueron los primeros en demostrar la patogenicidad in vivo de los Abs anti-FP4/H circulantes. Contribuciones celulares a la Trombosis en TIH Las siguientes secciones describirán las contribuciones celulares individuales así como las interacciones celulares que promueven y propagan la trombosis en TIH. Plaquetas Las plaquetas desempeñan una función destacada en prácticamente todos los aspectos de la TIH, desde su evidente participación en la expresión de la enfermedad (la trombocitopenia ocurre en >94 % de los pacientes con TIH)19 hasta su función central en los ensayos funcionales que a menudo se utilizan para la confirmación diagnóstica.56 Los primeros estudios de TIH se centraron en los efectos de la activación del complemento en las respuestas plaquetarias.66, 67 Estos estudios demostraron un aumento del depósito de complemento en las plaquetas circulantes de pacientes con TIH,66 la fijación del complemento por TIH IgG,66, 67 y los requisitos de los componentes de la vía clásica en la activación plaquetaria.66 Una vez que se identificaron los FcγR plaquetarios en 198768 el campo cambió su enfoque a las investigaciones de este receptor celular como un objetivo para los complejos inmunes por TIH. Kelton et al.57 y Chong et al.58 demostraron que la activación de plaquetas por anticuerpos en TIH, medida por la reacción de liberación, requería porciones tanto F(ab’) 2 como Fc de TIH IgG, estaba bloqueada por anticuerpos contra FcγRII y no involucraba a los receptores de glicoproteína (GP), Ib/ V/ y IIb/IIIa IX .57, 58 Otros estudios demostraron que, si bien los inhibidores de GP IIb/IIIa podían prevenir las respuestas de agregación plaquetaria por parte de los anticuerpos TIH,69, 70 no impedían la liberación de 14 C-serotonina radiomarcada67, 71 que requería vías de señalización dependientes de ADP y P2Y12 o Gi.71, 72 Los estudios in vivo que utilizan el modelo TIH murino han validado la importancia de las vías de señalización de FcγRIIA al demostrar la eficacia del inhibidor de la cinasa Syk, PRT-060318 (PRT318), en la prevención de la trombocitopenia y la trombosis inducidas en TIH.73 Las plaquetas de donantes sanos muestran una variación interindividual considerable en las respuestas a TIH CIUG; las plaquetas de algunos donantes sanos se activan constantemente en respuesta a TIH CIUG, mientras que las plaquetas de otros donantes responden mal.74 Se presume que esta va4 I C T U S 2023;04(01):e01022304002 Métodos Técnica Ventajas Desventajas Funcionales Liberación de serotonina Cuantificación de serotonina liberada por granulos plaquetarios mediante radiomarcadores o deteccion quimica deteccion quimica deteccion quimica Sensibilidad más alta (>95 %) Se requiere donantes de plaquetas Método radiactivo mediante radiomarcadores Uso limitado a laboratorios de investigación Activación Plaquetaria Visualización directa de la agregación plaquetaria Fácil disponibilidad Especificidad baja Micropartículas plaquetarias Detección mediante citometría de flujo Alta sensibilidad y especificidad Escasa disponibilidad Liberación de adenosintrifosfato Detección mediante luminografía Alta sensibilidad y especificidad Escasa disponibilidad Test de agregación Medición de la agregación plaquetaria mediante un agregómetro convencional Disponibilidad presente Escasa sensibilidad y especificidad, requiriendo donantes de plaquetas Unión a anexina V Cuantificación mediante citometría de la anexina V unida a plaquetas activadas Alta sensibilidad y especificidad Escasa disponibilidad Inmunológicos Electroinmunoanálisis PF4/polianión Detecta PF4 polivinil sulfonado Disponibilidad Alta sensibilidad Especificidad baja Electroinmunoanálisis PF4/heparina Detecta complejos PF4/heparina/IgG Alta sensibilidad y mejor especificidad (dececta IgG) Escasa disponibilidad solo laboratorios de investigación TABLA 2: M ÉTODOS DE LABORATORIO PARA LA DETECCIÓN DE ANTICUERPOS ANTICOMPLEJO HEPARINA - FACTOR 4 PLAQUETARIO TOMADO DE (53). 53 riabilidad del donante en la respuesta de activación plaquetaria contribuye a la heterogeneidad observada en la enfermedad clínica. Los mecanismos que subyacen a esta variabilidad en las respuestas de las plaquetas no se comprenden por completo, pero varios estudios recientes han demostrado los efectos de las variantes genéticas en las respuestas funcionales de FcγRIIA a los complejos inmunitarios TIH. De estos determinantes genéticos, el polimorfismo FcγRIIA H131R ha atraído la mayor atención en el campo. El polimorfismo H131R muestra afinidad diferencial para unirse a la IgG subclase 2; el alotipo 131R se une mínimamente a la IgG 2 humana mientras que el 131H se une a la IgG 2 así como a otras subclases. Los primeros estudios clínicos no fueron en gran medida concluyentes en cuanto al riesgo trombótico conferido por el polimorfismo H131R.75–77 Sin embargo, un estudio reciente ha revisado las consecuencias funcionales de la expresión alélica de 131RR en el contexto de otras IgG endógenas.4, 78 En estos estudios, Rollin y sus colegas demostraron que las plaquetas de individuos 131RR tienen una mayor reactividad a los anticuerpos TIH (generalmente, de la subclase IgG 1) debido a la incapacidad de la isoforma RR para unirse a la IgG 2 endógena. El alotipo 131HH, por otro lado, se une a la IgG 2 nativa y, por lo tanto, ofrece menos sitios de unión para la TIH IgG 1 activadora de plaquetas.78 En consecuencia, las plaquetas de individuos que expresan el alelo 131RR tienen mayor reactividad a TIH IgG que las plaque- tas que expresan otras isoformas. En un estudio prospectivo de 144 pacientes con TIH cuyo genotipo se caracterizó por varias variantes genéticas, incluidas FcγRIIA H131R, dimorfismo HPA-1a/b que afecta a la glicoproteína IIIa y molécula de adhesión de células endoteliales de plaquetas-1 (L125V), el riesgo trombótico aumentó solo en individuos que expresaban el polimorfismo 131RR, que estaba presente en el 38 % de los pacientes con trombosis frente al 18 % sin trombosis (OR 2,9).4 Otros estudios también han examinado variantes genéticas que regulan la señalización de FcγRIIA. Se demostró que dos polimorfismos en CD148 (276QQ y 326RR), una tirosina fosfatasa que puede modular la señalización de FcγRIIA, protegen de la trombosis.79 Se observaron hallazgos similares en estudios que investigaron TULA-2, una tirosina fosfatasa que desfosforila Syk.46, 80 En humanos, la expresión de TULA-2 se correlacionó inversamente con la respuesta plaquetaria a los anticuerpos TIH,80 mientras que la deficiencia murina de TULA-2 se asoció con una mayor reactividad plaquetaria y trombocitopenia grave en respuesta a los complejos inmunitarios.46 Una vez activadas por TIH CIUG, las plaquetas propagan la trombosis a través de la generación de micropartículas procoagulantes,81 la regulación positiva de la P-selectina que media la formación de agregados de leucocitos plaquetarios82, 83 y la liberación de FP4 y polifosfatos intracelula5 TROMBOCITOPENIA INDUCIDA POR HEPARINAS res.84 El FP4 liberado se une a los GAG celulares en las plaquetas y/o al factor de von Willebrand extruido en el endotelio para formar sitios antigénicos para los anticuerpos TIH circulantes.85 Monocitos Los hallazgos de la participación de monocitos y neutrófilos en la patogenia de la TIH se remontan a los primeros estudios realizados por Kelton y colegas,57 quienes demostraron que la adición de monocitos y neutrófilos a las plaquetas, en proporciones fisiológicas, inhibía la activación de las plaquetas por los anticuerpos de la TIH. Estas observaciones sugirieron la unión competitiva de anticuerpos TIH a GAG o FcγR celular. De hecho, estudios posteriores confirmaron la unión diferencial en TIH CIUG a los monocitos en comparación con las plaquetas. Esta mayor unión sirve como un reservorio fisiológico importante para los anticuerpos TIH y tiene un efecto moderador sobre la trombocitopenia in vivo. En el modelo TIH murino, el agotamiento de los monocitos usando liposomas de clodronato o cloruro de gadolinio exacerba notablemente la trombocitopenia, ya que los monocitos no están disponibles para unirse a los anticuerpos TIH.86 El aumento de la unión de CIUG a los monocitos da como resultado una mayor actividad procoagulante, como lo indica la regulación positiva del ARNm de FT,87, 88 la liberación de micropartículas que contienen FT89 y el aumento de la expresión de FT en la superficie celular en respuesta a KKO o TIH IgG.87–90 La participación de los monocitos en la trombosis también fue evidente en el tamaño de los trombos formados después del agotamiento celular. En el modelo TIH murino, la depleción de monocitos inhibió notablemente la formación de trombos después de la infusión de KKO,86 mientras que la depleción ex vivo de monocitos de sangre humana atenuó la deposición de plaquetas y la generación de fibrina.91 Los efectos mediados por CIUG sobre la activación de los monocitos dependían de FcγRIIA y de la señalización posterior a través de las quinasas Syk, ya que la inhibición de esta vía redujo notablemente la formación de trombina y fibrina. Neutrófilos Al igual que otras células que expresan FcγR, los neutrófilos se activan fácilmente mediante TIH CIUG. La activación celular se acompaña de una mayor expresión de CD11b,83, 93 desgranulación83–95 y una mayor adhesión celular a través de L-selectina.93 Estos eventos de activación, sin embargo, no parecen tan esenciales para la formación de trombos como la capacidad de las células activadas para liberar trampas extracelulares de neutrófilos (NET). Los NET son hebras extruidas de ácido desoxirribonucleico (ADN) y material granular que cumplen funciones importantes en la inmunidad innata, al atrapar patógenos.96 Los NET también son críticos para la formación de trombos,97 ya que se involucran directamente con plaquetas, glóbulos rojos y proteínas hemostáticas circulantes como FP4,98 fibronectina y factor de von Willebrand.99 Dos estudios recientes de Gollomp et al.98 y Perdomo et al.95 demostraron que los neutrófilos promueven la trombosis en TIH a través de la formación de NETS. Estos investigadores demostraron que los pacientes con TIH, en comparación con los sujetos de control, tienen evidencia DULCEY-SARMIENTO, LA. et al. de NETosis en plasma, como lo indican los niveles elevados de ADN libre de células, histonas citrulinadas y mieloperoxidasa.95, 98 Además, en estudios de microfluidos que utilizan sangre humana, plasma TIH o IgG se une a NET,98 protege el ADN de la degradación por desoxirribonucleasas95, 98 e induce la formación de trombos dependientes de neutrófilos95 Utilizando un modelo de lesión cremáster, Gollomp y sus colegas demostraron que había una acumulación significativa de neutrófilos y plaquetas en los trombos venosos, pero no en los arteriales, hallazgos que se reforzaron en ratones TIH que carecían de peptidil arginina deiminasa 4, una enzima esencial para la NETosis.98 Usando un enfoque diferente para evaluar la trombosis, al evaluar la embolia pulmonar espontánea en lugar de la oclusión de la lesión inducida por láser, Perdomo y sus colegas encontraron que la inhibición de la NETosis a través de enfoques químicos o genéticos anuló la trombosis en ratones TIH, un proceso dependiente de FcγRIIA de neutrófilos, pero parecía relativamente independiente de las plaquetas.95 Cabe señalar que, si bien las plaquetas no se consideraron esenciales para la NETosis en ambos estudios murinos, su presencia fue un determinante crítico del tamaño del trombo.95, 98 Estos estudios murinos no solo demuestran un papel fundamental para la participación de los neutrófilos en la TIH, sino que también proporcionan un mecanismo importante (NET) mediante el cual estas células promueven la trombosis. Células endoteliales Si bien hay datos clínicos y de laboratorio persuasivos que indican la participación endotelial en TIH, los mecanismos por los cuales TIH CIUG activan estas células son menos claros. De las células endoteliales, solo las células microvasculares dérmicas expresan el FcγRIIA activador,100 mientras que un número limitado de otros lechos endoteliales (células endoteliales sinusoidales hepáticas, placentarias y aórticas) expresan el receptor inhibidor FcγRIIB.101 Si bien no hay evidencia de activación endotelial directa, existe un amplio respaldo para la lesión del espectador mediada por CIUG. Los estudios histológicos de la microvasculatura de pacientes con TIH sometidos a amputaciones de extremidades indican múltiples trombos plaquetarios, células endoteliales infiltrantes e hiperplasia intraluminal de células endoteliales (CE).102 Los anticuerpos de los pacientes se unen directamente a las CE a través de los GAG celulares,59, 64, 103 promueven la deposición del complemento en las superficies celulares e inducen la actividad procoagulante.59 La activación de CE por suero en TIH o IgG se ve marcadamente potenciada por las plaquetas, lo que conduce a una mayor expresión de proteínas de adhesión celular (E-selectina, ICAM1, VCAM), liberación de citocinas (IL-1 β , IL6, PAI-1 y TNFα) y depósito de plaquetas.104 Estos efectos celulares de las plaquetas son atenuados por la inhibición selectiva de los receptores plaquetarios GPIIb/IIIa o ADP.104 En estudios de microfluidos, los anticuerpos de pacientes con KKO y/o TIH se unen fácilmente a los complejos FP4-VWF, promoviendo la adhesión plaquetaria y el agrandamiento de los trombos dentro de los canales de microfluidos.85 En cámaras microfluídicas recubiertas con células endoteliales de vena umbilical humana (HUVEC) o células endoteliales de aortas huma6 I C T U S 2023;04(01):e01022304002 nas, la lesión química de las CE va acompañada de la liberación de Factor de vW al glucocáliz de las CE105 y un aumento de la unión de FP4 a las cadenas de Factor de vW, lo que conduce a la expresión de sitios antigénicos para anticuerpos TIH.105 En ratones transgénicos TIH, los anticuerpos TIH exacerban la formación de trombos en los sitios perilesionales del endotelio donde el FP4 liberado se une al endotelio y la oclusión del trombo puede modularse mediante el uso de ADAMTS13 o N-acetilcisteína.85 Estos últimos estudios están de acuerdo con los hallazgos clínicos que muestran que los sitios de lesión endotelial, como los vasos que contienen catéteres venosos centrales, son particularmente propensos a la trombosis.51 Tomados en conjunto, estos estudios sugieren que el endotelio promueve la trombosis en TIH a través de respuestas a lesiones celulares directas o indirectas. La lesión física directa, tal vez a través de cateterismo, cirugía o enfermedad aterosclerótica subyacente, libera multímeros de vWF hemostáticos que favorecen la trombosis al unirse a las plaquetas y proporcionar sitios de unión adicionales para los anticuerpos TIH. El endotelio intacto podría activarse indirectamente mediante la activación del complemento y/o productos secretados de células portadoras de FcγRIIA, lo que conduce a la expresión de marcadores de adhesión y/o expresión de FT. T RATAMIENTO El tratamiento en TIH está dirigido a disminuir la intensa generación de trombina que acompaña a la enfermedad.106 En el tratamiento de la TIH se han utilizado una serie de terapias aprobadas y no autorizadas que se analizan brevemente a continuación. Para obtener información detallada relacionada con la farmacología, la dosificación y/o el control de las terapias, se remite al lector a las guías y revisiones recientes.107–109 Inhibidores directos de trombina (IDT) El argatrobán y la bivalirudina son medicamentos aprobados por la FDA para el tratamiento de TIH, el último en el contexto de intervenciones coronarias percutáneas en pacientes con TIH. En los ensayos clínicos que condujeron a la aprobación del fármaco, el argatrobán redujo el riesgo de un resultado compuesto (nueva trombosis, muerte por trombosis, amputación por trombosis) en comparación con los controles históricos.110 Debido al aclaramiento hepático, el argatrobán tiene un papel limitado en pacientes con enfermedad hepática grave. La bivalirudina tiene una vida media más corta de aproximadamente 25 min en pacientes con función renal normal. Este agente se ha estudiado en detalle como una alternativa sin heparina para el bypass cardiopulmonar en pacientes con TIH aguda/subaguda que requieren cirugía cardíaca de emergencia111, 112 pero no se usa comúnmente para esta indicación en la mayoría de los centros médicos debido a las altas tasas de sangrado. Una consideración importante cuando se usan IDT es el concepto de confusión de la anticoagulación debido a las elevaciones iniciales del tiempo de tromboplastina parcial activada (aPTT) o la razón internacional normalizada (INR). La elevación inicial de aPTT o INR puede ser causada por IDT o complicaciones asociadas con TIH de la coagulación intravascular diseminada y contribuir a la infradosificación de la terapia con IDT, lo que lleva al fracaso del tratamiento posterior debido a la anticoagulación subterapéutica.113 Cada vez se reconoce más que las complicaciones hemorrágicas por el uso de anticoagulantes alternativos también contribuyen a la morbilidad de la enfermedad en TIH. De hecho, estudios recientes indican que se producen complicaciones hemorrágicas en el 38-44 % de los pacientes tratados con anticoagulantes distintos de la heparina.1, 2 El ambiente protrombótico de TIH no mitiga el riesgo de sangrado, ya que varios estudios muestran que los pacientes con TIH parecen tan susceptibles a complicaciones hemorrágicas como aquellos sin enfermedad. En un gran estudio retrospectivo de 300 pacientes tratados con anticoagulación alternativa a heparinoides por TIH sospechada o confirmada, las tasas de hemorragia fueron similares en pacientes con o sin enfermedad, mientras que la enfermedad crítica, la disfunción renal y el recuento de plaquetas <25 × 10 9/L fueron más predictivos de riesgo de sangrado.2 Derivados de heparina La HBPM está contraindicada en TIH debido a las altas tasas de reactividad cruzada con anticuerpos TIH.114 Fondaparinux, un pentasacárido sintético que contiene la secuencia de unión a heparina de la antitrombina, muestra una reactividad cruzada mínima con los anticuerpos TIH in vivo115 y por sí mismo, es una causa rara de TIH.116 Fondaparinux a menudo se usa fuera de etiqueta en pacientes con TIH,117 pero tiene una utilidad limitada en pacientes con enfermedad renal debido a la depuración renal y una vida media prolongada (15 a 20 horas).118 Danaparoid es un heparinoide que se compone principalmente de sulfato de heparán, pero no está disponible en los Estados Unidos.119 Además de su efecto anticoagulante, el fármaco inhibe la formación de complejos FP4-heparina que pueden contribuir adicionalmente a su eficacia.120 Anticoagulantes orales directos (ACOD) Los ACOD se utilizan cada vez más fuera de etiqueta para el tratamiento de TIH. De estos fármacos, rivaroxabán tiene la experiencia más publicada. Un estudio observacional prospectivo realizado por Linkins y colegas sobre el uso de rivaroxabán para el tratamiento de la TIH confirmada serológicamente, aunque alentador, tuvo que cerrarse prematuramente debido a la baja acumulación de pacientes.121 Una revisión bibliográfica reciente de la experiencia publicada y posterior al ensayo de los ACOD por el mismo grupo de Hamilton indica que estos agentes son seguros y efectivos para su uso en la TIH aguda, con resultados de ausencia de complicaciones de hemorragia y trombosis que ocurren en solo 1/46 (2.2 %) pacientes tratados con rivaroxabán.122 Se observaron hallazgos comparables en pacientes tratados con apixabán y dabigatrán. Con base en este y otros informes similares en la literatura123 el panel de directrices de la Sociedad Estadounidense de Hematología sobre TIH proporcionó recomendaciones condicionales para el uso de ACOD en TIH aguda en pacientes clínicamente estables que se consideran de riesgo 7 TROMBOCITOPENIA INDUCIDA POR HEPARINAS Aspecto evaluado DULCEY-SARMIENTO, LA. et al. 2 Puntos 1 Punto 0 Puntos Disminución del recuento de plaquetas >50 % ≥20 y nadir de plaquetas Recuento de plaquetas cae 30-50 % o nadir de plaquetas 10-19 Disminución del recuento de plaquetas <30 % o nadir de plaquetas <10 Momento de la caída del recuento de plaquetas Aparición clara entre los días 5 y 10 o caída de plaquetas ≤ 1 día (exposición previa a heparina dentro de los 30 días) Consistente con los días 5 a 10 de otoño, pero no claro (p. ej., falta de recuento de plaquetas); inicio después del día 10‡; o caída ≤1 día (exposición previa a heparina (hace 30 a 100 días) Disminución del recuento de plaquetas <4 días sin exposición reciente Trombosis y otras secuelas Trombosis nueva (confirmada); necrosis de la piel; reacción sistémica aguda posterior al bolo de heparina no fraccionada (HNF) intravenosa Trombosis progresiva o recurrente; Lesiones cutáneas no necrosantes (eritematosas); Sospecha de trombosis (no comprobada)** Ninguna Otras causas de trombocitopenia Ninguno aparente Posible Definitivo Trombocitopenia TABLA 3: Í NDICE DE LAS 4T PARA TROMBOCITOPENIA INDUCIDA POR HEPARINA (HIT) TOMADO DE LA REFERENCIA 140. 140 promedio de hemorragia.107 realización de pruebas de detección de anticuerpos tomado de (Tabla 3).140 Terapias no anticoagulantes A pesar de la máxima anticoagulación, algunos pacientes con TIH desarrollan enfermedad refractaria, caracterizada por trombocitopenia grave y persistente, trombosis nueva y/o progresiva. Algunos de estos pacientes responden a terapias adicionales dirigidas a la respuesta inmune a través del tratamiento con inmunoglobulina intravenosa (IVIG)124 o recambio plasmático terapéutico (RPT). IVIG se informó por primera vez como una terapia complementaria en 1989 para la trombocitopenia.125 Informes posteriores han demostrado que la IVIG interrumpe la activación plaquetaria por los anticuerpos TIH al interferir con la activación plaquetaria dependiente de FcγRIIA (126, 127) y es eficaz para el tratamiento de la trombosis124, 127, 128 y/o enfermedad refractaria.127, 129, 130 Si bien existen preocupaciones sobre el riesgo trombótico con IVIG,131, 132 un informe reciente que utiliza una gran base de datos de atención médica para pacientes hospitalizados sugiere que el tratamiento puede ser seguro.133 El RPT es otra modalidad que a menudo se emplea como terapia complementaria para el tratamiento de la TIH aguda o subaguda, en particular para el tratamiento de cirugía cardíaca de emergencia134 o como terapia de rescate para la enfermedad refractaria.135 Se supone que la eficacia de la RPT en TIH es secundaria a la eliminación de Abs antiFP4/H,136 pero los inconvenientes incluyen solo efectos transitorios en la eliminación de anticuerpos y disponibilidad limitada en el entorno comunitario. Las terapias emergentes en la etapa preclínica incluyen proteasas bacterianas para escindir IgG (IdeS o enzima degradante de IgG de Streptococcus pyogenes 137 Syk 73 e inhibidores de la tirosina quinasa,6 2 así como inhibidores de la tetramerización de FP4.138, 139 Ante la sospecha clínica, se debe establecer el tratamiento; se ha descrito un sistema de puntuación (sistema de puntuación de las 4 t) para establecer la probabilidad de TIH antes de la C ONCLUSIONES Los fenómenos tromboticos en TIH representa una respuesta multicelular orquestada. La TIH desencadena respuestas procoagulantes específicas de las células que se refuerzan mutuamente para generar un estado profundamente hipercoagulable. En la actualidad, las razones por las que algunos pacientes seropositivos desarrollan esta potente respuesta celular, mientras que la mayoría de los pacientes no la desarrollan, siguen siendo esquivas. Si bien algunas de estas diferencias pueden explicarse por la variación genética en varios mecanismos efectores,45, 46, 78, 80 no explican completamente la heterogeneidad de la expresión de la enfermedad. Se necesitan estudios adicionales centrados en aspectos menos caracterizados de la enfermedad, como la activación del complemento, estudios estructurales de complejos antigénicos y caracterización de anticuerpos patógenos y no patógenos. Los nuevos conocimientos sobre los mecanismos de la trombosis en TIH deberían traducirse en una mejor comprensión de otros trastornos trombóticos inmunomediados, como el síndrome de anticuerpos antifosfolípidos y/o el lupus eritematoso sistémico. F INANCIAMIENTO No se obtuvo ningún financiamiento externo para la realización de este trabajo. 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https://openalex.org/W2967728202	https://europepmc.org/articles/pmc6719917?pdf=render	English	null	Probiotics Upregulate Trefoil Factors and Downregulate Pepsinogen in the Mouse Stomach	International journal of molecular sciences	2,019	cc-by	16,081	Received: 16 July 2019; Accepted: 5 August 2019; Published: 10 August 2019 Abstract: Probiotics are used in the management of some gastrointestinal diseases. However, little is known about their eﬀects on normal gastric epithelial biology. The aim of this study was to explore how the probiotic mixture VSL#3 aﬀects gastric cell lineages in mice with a special focus on protective and aggressive factors. Weight-matching littermate male mice (n = 14) were divided into treated and control pairs. The treated mice received VSL#3 (5 mg/day/mouse) by gastric gavage for 10 days. Control mice received only the vehicle. Food consumption and bodyweight were monitored. All mice were injected intraperitoneally with bromodeoxyuridine (120 mg/Kg bodyweight) two hours before sacriﬁced to label S-phase cells. Stomach tissues were processed for lectin- and immunohistochemical examination. ImageJ software was used to quantify immunolabeled gastric epithelial cells. Real-time quantitative polymerase chain reaction was used to provide relative changes in expression of gastric cell lineages speciﬁc genes. Results revealed that treated mice acquired (i) increased production of mucus, trefoil factor (TFF) 1 and TFF2, (ii) decreased production of pepsinogen, and (iii) increased ghrelin-secreting cells. No signiﬁcant changes were observed in bodyweight, food consumption, cell proliferation, or parietal cells. Therefore, VSL#3 administration ampliﬁes speciﬁc cell types specialized in the protection of the gastric epithelium. Keywords: probiotics; VSL#3; trefoil factor; mucin; ghrelin; pepsinogen; gastric epithelium International Journal of Molecular Sciences International Journal of Molecular Sciences Probiotics Upregulate Trefoil Factors and Downregulate Pepsinogen in the Mouse Stomach Ghalia Khoder 1,† , Farah Al-Yassir 2,3,† , Asma Al Menhali 4, Prashanth Saseedharan 2, Subi Sugathan 2, Catherine Tomasetto 5 and Sherif M. Karam 2,* 1 Department of Pharmaceutics and Pharmaceuticals Technology, College of Pharmacy, Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, UAE 1 Department of Pharmaceutics and Pharmaceuticals Technology, College of Pharmacy, Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, UAE j , y j , j , 2 Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates Universit AlAin 17666, UAE 3 Department of Biological Sciences, Faculty of Science, Beirut Arab University, Debbieh Campus PO Box 11-50-20 Riad El Solh, Beirut 11072809, Lebanon 5 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM), U1258, Centre National de la Recherche Scientiﬁque (CNRS), UMR7104, Université de Strasbourg, F-67404 Illkirch, France g * Correspondence: skaram@uaeu.ac.ae; Tel.: +971-3-713-749 † These authors contributed equally to this work. 1. Introduction Probiotics are microorganisms that exert beneﬁcial eﬀects on their hosts when ingested in suﬃcient amounts [1]. The most prevalent probiotics used are lactic acid bacteria and biﬁdobacteria, although other bacteria and yeasts are also used [2]. Ingestion of probiotics helps in maintaining the balance of intestinal microﬂora and in reducing the side eﬀects of antibiotics [3]. Some studies have shown that probiotics are safe and their use has no side eﬀects [4]. Therefore, probiotics are now incorporated into an expanding array of food products, nutritional supplements, and pharmaceutical compounds [5]. In the last decades and with the great discovery of the next generation sequencing technique, several studies have emerged to explore the beneﬁcial eﬀects of probiotics on human diseases [5,6], Int. J. Mol. Sci. 2019, 20, 3901; doi:10.3390/ijms20163901 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 3901 2 of 20 including cancer [7–9]. In the gastrointestinal tract, it has been shown that probiotics have potential roles in the management of inﬂammatory, ulcerative, and neoplastic diseases [10–12]. When administered, probiotics compete with pathogenic bacteria for nutrients [13], and contribute to the stability of the intestinal epithelial barrier including tight junctions [14]. Probiotics are provided as mono-strain [15,16] or multi-strain, such as the product called VSL#3 or simply VSL, which is a mixture of highly concentrated lyophilized living bacteria [17]. Studies have shown that multi-strain probiotics are more eﬀective than mono-strains and have more successful chances of colonization [18] due to the ability to overcome adverse physiological conditions of the gut such as acidic pH, digestive enzymes, bile acid, and mechanical stress. VSL has been extensively investigated in the last few years [9,19–21]. Currently, VSL is recommended for the prevention and treatment of inﬂammatory bowel diseases [22], such as pouchitis [23], Crohn’s disease [24], and ulcerative colitis [21,25,26]. A recent study has also revealed the preventive eﬀect of VSL in ulcerative colitis-associated carcinogenesis in mice [9]. With all these advances in the basic knowledge and clinical applications of probiotics in gastrointestinal disorders, little is known about their mechanism of action in the lower gastrointestinal tract [27]. Modulation of host-microbe interactions, pathogen exclusion, stimulation of goblet cells to produce mucus, enhancing intestinal epithelial barrier formation, production of antibacterial factors, and activation of host adaptive immune response have been proposed [28,29]. 1. Introduction Several studies, about the impact of probiotic bacteria to health, notably host mucosal defense responses, have shown potentials in reinforcing epithelial integrity, modulation of barrier function, and upregulation of adaptive defense responses such as secretion of mucins and intestinal trefoil factor (TFF3) [30]. It was also reported that under inﬂammatory conditions, probiotic administration normalized the abnormal stimulation of mucus secretion [31,32]. Several studies have demonstrated the potent eﬀect of probiotics in mucin gene expression in colonic and intestinal epithelial cell lines [33,34] and in animal models [35]. In addition, an interesting study on the eﬀect of VSL administration on acetic acid-induced gastric ulcer in rats reported that the main impact for VSL was on the upregulation of vascular endothelial growth factor [19]. However, the eﬀects of VSL on the gastric epithelial cell lineages of the healthy stomach are not yet explored. The stomach is lined by an epithelial cell layer organized to form numerous pits lined by mucous cells, and continuous with tubular glands populated with multiple cell lineages producing mucus, acid, pepsinogen, and several hormones. These cell lineages originate from proliferating epithelial stem cells [36]. This study aims to investigate whether VSL administration into mice aﬀects gastric epithelial cell biology with speciﬁc reference to the defensive and aggressive factors of the gastric epithelium. 2. Results All seven pairs of young male control mice and their littermates exposed to VSL were physically active during the 10 days of gastric gavages. However, measurements of various body and gastric tissue parameters revealed some important observations. 2.2. VSL Enhances Mucous Production in the Mouse Gastric Mucosa 2.2. VSL Enhances Mucous Production in the Mouse Gastric Mucosa Tissue sections of the gastric mucosae of control and VSL-treated mice stained with conventional hematoxylin and eosin showed no apparent cellular differences. Both groups of tissues had long oxyntic glands with large scattered eosinophilic parietal cells. The VSL-treated tissues had a tendency to have some increase in mucosal thickness and glandular length. Measurements of the glandular mucosal thickness in a comparable region (close to the fundus) of tissue sections of control and VSL- exposed stomachs showed values that varied from 312 to 423 µm in control and 401 to 412 µm in VSL-treated tissues. This difference was not statistically significant (p > 0.05). Similar insignificant differences were also found for two other comparable regions of the corpus mucosa. While conventional microscopy did not reveal significant differences, systematic molecular examination using a variety of microscopy approaches demonstrated several differences related to the epithelial cell lineages. T i h f i b h i d k ll li d bl l i bi di Tissue sections of the gastric mucosae of control and VSL-treated mice stained with conventional hematoxylin and eosin showed no apparent cellular diﬀerences. Both groups of tissues had long oxyntic glands with large scattered eosinophilic parietal cells. The VSL-treated tissues had a tendency to have some increase in mucosal thickness and glandular length. Measurements of the glandular mucosal thickness in a comparable region (close to the fundus) of tissue sections of control and VSL-exposed stomachs showed values that varied from 312 to 423 µm in control and 401 to 412 µm in VSL-treated tissues. This diﬀerence was not statistically signiﬁcant (p > 0.05). Similar insigniﬁcant diﬀerences were also found for two other comparable regions of the corpus mucosa. While conventional microscopy did not reveal signiﬁcant diﬀerences, systematic molecular examination using a variety of microscopy approaches demonstrated several diﬀerences related to the epithelial cell lineages. To examine the amount of mucus in both pit and neck cell lineages, double lectin binding was employed, and revealed the presence of more mucus in the VSL-treated tissues than in control (Figure 2A,B). Measurements of UEA-lectin labeling in each pair of control and VSL treated mice showed an increased labeling in VSL treated tissues by 1.84 ± 0.23 folds (p < 0.05) (Figure 2C). Similarly, there was intensified GSII-lectin labeling of neck cells in VSL-treated mice when compared with control tissues and the fold increase was 1.58 ± 0.12 (p < 0.01; Figure 2C). 2.2. VSL Enhances Mucous Production in the Mouse Gastric Mucosa 2.2. VSL Enhances Mucous Production in the Mouse Gastric Mucosa To eliminate possible processing variations, tissue sections of control and VSL-mice were processed together for embedded in the same paraffin block, simultaneously exposed to the same immunolabeleing solutions, and photgraphed with the same microscopy settings. To examine the amount of mucus in both pit and neck cell lineages, double lectin binding was employed, and revealed the presence of more mucus in the VSL-treated tissues than in control (Figure 2A,B). Measurements of UEA-lectin labeling in each pair of control and VSL treated mice showed an increased labeling in VSL treated tissues by 1.84 ± 0.23 folds (p < 0.05) (Figure 2C). Similarly, there was intensiﬁed GSII-lectin labeling of neck cells in VSL-treated mice when compared with control tissues and the fold increase was 1.58 ± 0.12 (p < 0.01; Figure 2C). To eliminate possible processing variations, tissue sections of control and VSL-mice were processed together for embedded in the same paraﬃn block, simultaneously exposed to the same immunolabeleing solutions, and photgraphed with the same microscopy settings. 2.1. VSL Administration Has No Signiﬁcant Eﬀect on Mouse Bodyweight and Food Intake The bodyweight of VSL-treated mice showed slightly higher values as compared to control mice. After ﬁve days of VSL administration, the bodyweight was 28.7 ± 0.32 gm on the average as compared to 26.6 ± 0.31 gm in control mice. By day 10, the bodyweight averaged 29.3 ± 0.25 gm in VSL-mice and 26.9 ± 0.29 gm in control mice. This increase in weight gain of VSL-mice was not statistically signiﬁcant. Estimation of bodyweight gain after 10 days in each pair of control and VSL-treated mice showed that the percentages of increase in weight gain were 5.2% and 8.3%, respectively (Figure 1). Estimating the weight of food consumed by mice after ﬁve and 10 days revealed slight increase (1.2-fold) in the cumulative food intake that was not statistically signiﬁcant. 3 of 20 21 Int. J. Mol. Sci. 2019, 20, 3901 Int. J. Mol. Sci. 2019, 20, x F Figure 1. Effect of VSL on mice bodyweight gain in grams after five and 10 days of administration. VSL treatment shows an increasing trend in bodyweight gain after treatment but was not statistically significant when compared to control. Data from seven control and seven VSL-mice are presented as mean ± SD. Figure 1. Eﬀect of VSL on mice bodyweight gain in grams after ﬁve and 10 days of administration. VSL treatment shows an increasing trend in bodyweight gain after treatment but was not statistically signiﬁcant when compared to control. Data from seven control and seven VSL-mice are presented as mean ± SD. Figure 1. Effect of VSL on mice bodyweight gain in grams after five and 10 days of administration. VSL treatment shows an increasing trend in bodyweight gain after treatment but was not statistically significant when compared to control. Data from seven control and seven VSL-mice are presented as mean ± SD. Figure 1. Eﬀect of VSL on mice bodyweight gain in grams after ﬁve and 10 days of administration. VSL treatment shows an increasing trend in bodyweight gain after treatment but was not statistically signiﬁcant when compared to control. Data from seven control and seven VSL-mice are presented as mean ± SD. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells 2019, 20, x FOR PEER REVIEW 5 of 21 Figure 2. Double lectin histochemistry of the gastric mucosae of control and VSL-treated tissues. Stomach tissue sections of control (A) and VSL-treated (B) mice were incubated with UEA-I (red) and GS-II (green). Images obtained with 20× objective lense, scale bar: 100 µm. Fold changes in the quantification of the UEA-I and GS-II labeling intensity of control (n = 7) and VSL-treated (n = 7) tissues (C) are presented as mean ± SE. The asterisks indicate significant differences from the control group. p <0.05, p < 0.01. Figure 2. Double lectin histochemistry of the gastric mucosae of control and VSL-treated tissues. Stomach tissue sections of control (A) and VSL-treated (B) mice were incubated with UEA-I (red) and GS-II (green). Images obtained with 20× objective lense, scale bar: 100 µm. Fold changes in the quantiﬁcation of the UEA-I and GS-II labeling intensity of control (n = 7) and VSL-treated (n = 7) tissues (C) are presented as mean ± SE. The asterisks indicate signiﬁcant diﬀerences from the control group. p <0.05, ** p < 0.01. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 21 To test whether increased mucus was associated with a change in the production of trefoil factors, tissues were probed with antibodies specific for TFF1 and TFF2. The labeling for TFF1 occurred in the pit cells lining the luminal surface and along the pit regions (Figure 3A,B). Measurements revealed increased labeling for TFF1 in VSL-tissues when compared to the control (Figure 3C). In addition, counts of nuclei of TFF1-labeled cells indicated the presence of 12.4 ± 0.19 and 14.4 ± 0.14 cells per gland in the control and VSL-treated mice, respectively (p < 0.01, Figure 3C). Quantification of the percentage of pixels in labeled areas in images obtained with 20× magnification also revealed a significant increase in the intensity of TFF1-labeling of VSL-treated tissues (Figure 3D). To confirm the increase of the TFF1 level, images obtained from the same region of the corpus of both control and VSL tissues were compared at higher magnification (Figure 4A,B). Interestingly, the increased level of TFF1-labeling was not only demonstrated in surface and pit cells of VSL-treated tissue, but also dividing progenitor cells of the isthmus region showed some cytoplasmic staining for TFF1 (Figure 4C). Figure 3. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells To test whether increased mucus was associated with a change in the production of trefoil factors, tissues were probed with antibodies speciﬁc for TFF1 and TFF2. The labeling for TFF1 occurred in the pit cells lining the luminal surface and along the pit regions (Figure 3A,B). Measurements revealed increased labeling for TFF1 in VSL-tissues when compared to the control (Figure 3C). In addition, counts of nuclei of TFF1-labeled cells indicated the presence of 12.4 ± 0.19 and 14.4 ± 0.14 cells per gland in the control and VSL-treated mice, respectively (p < 0.01, Figure 3C). Quantiﬁcation of the percentage of pixels in labeled areas in images obtained with 20× magniﬁcation also revealed a signiﬁcant increase in the intensity of TFF1-labeling of VSL-treated tissues (Figure 3D). To conﬁrm the increase of the TFF1 level, images obtained from the same region of the corpus of both control and VSL tissues were compared at higher magniﬁcation (Figure 4A,B). Interestingly, the increased level of TFF1-labeling was not only demonstrated in surface and pit cells of VSL-treated tissue, but also dividing progenitor cells of the isthmus region showed some cytoplasmic staining for TFF1 (Figure 4C). 4 of 20 Int. J. Mol. Sci. 2019, 20, 3901 Int. J. Mol. Sci. 2019, 2 Figure 2. Double lectin histochemistry of the gastric mucosae of control and VSL-treated tissues. Stomach tissue sections of control (A) and VSL-treated (B) mice were incubated with UEA-I (red) and GS-II (green). Images obtained with 20× objective lense, scale bar: 100 µm. Fold changes in the quantification of the UEA-I and GS-II labeling intensity of control (n = 7) and VSL-treated (n = 7) tissues (C) are presented as mean ± SE. The asterisks indicate significant differences from the control group. p <0.05, p < 0.01. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells Figure 2. Double lectin histochemistry of the gastric mucosae of control and VSL-treated tissues. Stomach tissue sections of control (A) and VSL-treated (B) mice were incubated with UEA-I (red) and GS-II (green). Images obtained with 20× objective lense, scale bar: 100 µm. Fold changes in the quantiﬁcation of the UEA-I and GS-II labeling intensity of control (n = 7) and VSL-treated (n = 7) tissues (C) are presented as mean ± SE. The asterisks indicate signiﬁcant diﬀerences from the control group. p <0.05, p < 0.01. Int. J. Mol. Sci. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells Interestingly, the increased level of TFF1-labeling was not only demonstrated in surface and pit cells of VSL-treated tissue, but also dividing progenitor cells of the isthmus region showed some cytoplasmic staining for TFF1 (Fi 4C) ciated with a change in the production of trefoil pecific for TFF1 and TFF2. The labeling for TFF1 urface and along the pit regions (Figure 3A,B). FF1 in VSL-tissues when compared to the control 1-labeled cells indicated the presence of 12.4 ± 0.19 VSL-treated mice, respectively (p < 0.01, Figure 3C). ed areas in images obtained with 20× magnification ity of TFF1-labeling of VSL-treated tissues (Figure mages obtained from the same region of the corpus t higher magnification (Figure 4A,B). Interestingly, demonstrated in surface and pit cells of VSL-treated hmus region showed some cytoplasmic staining for To test whether increased mucus was assoc factors, tissues were probed with antibodies spe occurred in the pit cells lining the luminal su Measurements revealed increased labeling for TF (Figure 3C). In addition, counts of nuclei of TFF1 and 14.4 ± 0.14 cells per gland in the control and V Quantification of the percentage of pixels in labele also revealed a significant increase in the intensit 3D). To confirm the increase of the TFF1 level, im of both control and VSL tissues were compared at the increased level of TFF1-labeling was not only d tissue, but also dividing progenitor cells of the isth TFF1 (Figure 4C). Figure 3. Immunohistochemical analysis of TFF1 in gastric mucosal tissue sections obtained from control and VSL-treated mice. Note that TFF1-labeled epithelial cells (brown cytoplasm) of VSL- treated tissues in (B) appear more expanded at the pit region of gastric gland when compared with control tissues in (A). Magnification, 20×, scale bar: 200 µm. Analysis of TFF1-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF1-labeling intensity per field in the oxyntic glands of control and VSL treated mice (D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisk indicates significant differences from the control group. p < 0.01. Figure 3. Immunohistochemical analysis of TFF1 in gastric mucosal tissue sections obtained from control and VSL-treated mice. Note that TFF1-labeled epithelial cells (brown cytoplasm) of VSL-treated tissues in (B) appear more expanded at the pit region of gastric gland when compared with control tissues in (A). Magniﬁcation, 20×, scale bar: 200 µm. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells Immunohistochemical analysis of TFF1 in gastric mucosal tissue sections obtained from control and VSL-treated mice. Note that TFF1-labeled epithelial cells (brown cytoplasm) of VSL- treated tissues in (B) appear more expanded at the pit region of gastric gland when compared with control tissues in (A). Magnification, 20×, scale bar: 200 µm. Analysis of TFF1-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF1-labeling intensity per field in the oxyntic glands of control and VSL treated mice (D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisk indicates significant differences from the control group. p < 0.01. Figure 3. Immunohistochemical analysis of TFF1 in gastric mucosal tissue sections obtained from control and VSL-treated mice. Note that TFF1-labeled epithelial cells (brown cytoplasm) of VSL-treated tissues in (B) appear more expanded at the pit region of gastric gland when compared with control tissues in (A). Magniﬁcation, 20×, scale bar: 200 µm. Analysis of TFF1-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF1-labeling intensity per ﬁeld in the oxyntic glands of control and VSL treated mice (D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisk indicates signiﬁcant diﬀerences from the control group. p < 0.01. To test whether increased mucus was associated with a change in the production of trefoil factors, tissues were probed with antibodies specific for TFF1 and TFF2. The labeling for TFF1 occurred in the pit cells lining the luminal surface and along the pit regions (Figure 3A,B). Measurements revealed increased labeling for TFF1 in VSL-tissues when compared to the control (Figure 3C). In addition, counts of nuclei of TFF1-labeled cells indicated the presence of 12.4 ± 0.19 and 14.4 ± 0.14 cells per gland in the control and VSL-treated mice, respectively (p < 0.01, Figure 3C). Quantification of the percentage of pixels in labeled areas in images obtained with 20× magnification also revealed a significant increase in the intensity of TFF1-labeling of VSL-treated tissues (Figure 3D). To confirm the increase of the TFF1 level, images obtained from the same region of the corpus of both control and VSL tissues were compared at higher magnification (Figure 4A,B). 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells Both A and B are taken from the same region at the junction with the stratiﬁed epithelium of the fundus (lower left corner). Note the higher intensity of the brown color of TFF1-labeling in VSL-treated tissue (B) compared to control (A). Magniﬁcation bar = 50 µm. Panel (C) shows a high magniﬁcation of the area within the red square of the VSL-tissue. Note the TFF1-labeled mitotic progenitor cells at the arrows. Magniﬁcation bar = 20 µm. To determine whether the level of TFF2 produced by mucous neck cells was also affected by VSL administration, mucosal sections were probed with anti-TFF2 antibodies. Sections of both control (Figure 5A) and VSL-treated tissues (Figure 5B) demonstrated the presence of TFF2 in the neck region. To provide quantitative data for the number of TFF2-labeled neck cells, three different images from four different control and VSL-mice were examined. For each animal, counts in longitudinally cut glands were averaged and expressed as cells per gland. Data showed a significant difference (p < 0.001) in the number of TFF2-labeled cells in VSL-treated mice (6.5 ± 0.5) when compared to controls (3.09 ± 0.2; Figure 5C). Quantification of the percentage of pixels in labeled areas in images obtained with 20× magnification also revealed a significant increase in the intensity of TFF2 labeling of VSL-treated tissues (Figure 5D). In addition, the labeling for TFF2 extended deep in the gastric glands and in some glands reached to the bottom where chief cells are located (Figure 6A,B). To determine whether the level of TFF2 produced by mucous neck cells was also aﬀected by VSL administration, mucosal sections were probed with anti-TFF2 antibodies. Sections of both control (Figure 5A) and VSL-treated tissues (Figure 5B) demonstrated the presence of TFF2 in the neck region. To provide quantitative data for the number of TFF2-labeled neck cells, three diﬀerent images from four diﬀerent control and VSL-mice were examined. For each animal, counts in longitudinally cut glands were averaged and expressed as cells per gland. Data showed a signiﬁcant diﬀerence (p < 0.001) in the number of TFF2-labeled cells in VSL-treated mice (6.5 ± 0.5) when compared to controls (3.09 ± 0.2; Figure 5C). Quantiﬁcation of the percentage of pixels in labeled areas in images obtained with 20× magniﬁcation also revealed a signiﬁcant increase in the intensity of TFF2 labeling of VSL-treated tissues (Figure 5D). 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells Analysis of TFF1-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF1-labeling intensity per ﬁeld in the oxyntic glands of control and VSL treated mice (D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisk indicates signiﬁcant diﬀerences from the control group. p < 0.01. 5 of 20 21 Int. J. Mol. Sci. 2019, 20, 3901 Int. J. Mol. Sci. 2019, 20, x F Figure 4. TFF1-labeled epithelial cells in corresponding regions of control (A) and VSL-treated (B) gastric mucosae at high magnifications. Both A and B are taken from the same region at the junction with the stratified epithelium of the fundus (lower left corner). Note the higher intensity of the brown color of TFF1-labeling in VSL-treated tissue (B) compared to control (A). Magnification bar = 50 µm. Panel C shows a high magnification of the area within the red square of the VSL-tissue. Note the TFF1- labeled mitotic progenitor cells at the arrows. Magnification bar = 20 µm. Figure 4. TFF1-labeled epithelial cells in corresponding regions of control (A) and VSL-treated (B) gastric mucosae at high magniﬁcations. Both A and B are taken from the same region at the junction with the stratiﬁed epithelium of the fundus (lower left corner). Note the higher intensity of the brown color of TFF1-labeling in VSL-treated tissue (B) compared to control (A). Magniﬁcation bar = 50 µm. Panel (C) shows a high magniﬁcation of the area within the red square of the VSL-tissue. Note the TFF1-labeled mitotic progenitor cells at the arrows. Magniﬁcation bar = 20 µm. Figure 4. TFF1-labeled epithelial cells in corresponding regions of control (A) and VSL-treated (B) gastric mucosae at high magnifications. Both A and B are taken from the same region at the junction with the stratified epithelium of the fundus (lower left corner). Note the higher intensity of the brown color of TFF1-labeling in VSL-treated tissue (B) compared to control (A). Magnification bar = 50 µm. Panel C shows a high magnification of the area within the red square of the VSL-tissue. Note the TFF1- labeled mitotic progenitor cells at the arrows. Magnification bar = 20 µm. Figure 4. TFF1-labeled epithelial cells in corresponding regions of control (A) and VSL-treated (B) gastric mucosae at high magniﬁcations. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells In addition, the labeling for TFF2 extended deep in the gastric glands and in some glands reached to the bottom where chief cells are located (Figure 6A,B). Int. J. Mol. Sci. 2019, 20, 3901 Int. J. Mol. Sci. 2019, 20, x F J , , 6 of 20 21 6 of 20 21 Figure 5. Immunohistochemical analysis of TFF2-labeled epithelial cells in tissue sections obtained from the gastric mucosa of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of TFF2-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF2-labeling intensity per field in the oxyntic glands of control and VSL treated mice(D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisks indicate significant differences from the control group. p < 0.001. Figure 5. Immunohistochemical analysis of TFF2-labeled epithelial cells in tissue sections obtained from the gastric mucosa of control (A) and VSL-treated (B) mice. Magniﬁcation bar = 100 µm. Analysis of TFF2-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF2-labeling intensity per ﬁeld in the oxyntic glands of control and VSL treated mice(D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisks indicate signiﬁcant diﬀerences from the control group. p < 0.001. Figure 5. Immunohistochemical analysis of TFF2-labeled epithelial cells in tissue sections obtained from the gastric mucosa of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of TFF2-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF2-labeling intensity per field in the oxyntic glands of control and VSL treated mice(D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisks indicate significant differences from the control group. p < 0.001. Figure 5. Immunohistochemical analysis of TFF2-labeled epithelial cells in tissue sections obtained from the gastric mucosa of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of TFF2-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF2-labeling intensity per field in the oxyntic glands of control and VSL treated mice(D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisks indicate significant differences from the control group. p < 0.001. Figure 5. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells TFF2-labeled epithelial cells (arrows) in tissue sections obtained from the middle and basal portions of the gastric mucosae of control (A) and VSL-treated (B) mice. Note the extension of TFF2 labeling to the bottom of the gastric glands where chief cells (lower three arrows) are located. Magnification bar = 100 µm. Figure 6. TFF2-labeled epithelial cells (arrows) in tissue sections obtained from the middle and basal portions of the gastric mucosae of control (A) and VSL-treated (B) mice. Note the extension of TFF2 labeling to the bottom of the gastric glands where chief cells (lower three arrows) are located. Magniﬁcation bar = 100 µm. 2.3. VSL Enhances TFF1 and TFF2 Production by Gastric Mucous Cells Immunohistochemical analysis of TFF2-labeled epithelial cells in tissue sections obtained from the gastric mucosa of control (A) and VSL-treated (B) mice. Magniﬁcation bar = 100 µm. Analysis of TFF2-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF2-labeling intensity per ﬁeld in the oxyntic glands of control and VSL treated mice(D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisks indicate signiﬁcant diﬀerences from the control group. * p < 0.001. Figure 5. Immunohistochemical analysis of TFF2-labeled epithelial cells in tissue sections obtained from the gastric mucosa of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of TFF2-labeled cell counts per gland in the gastric corpus of control and VSL-treated mice (C), and TFF2-labeling intensity per field in the oxyntic glands of control and VSL treated mice(D). Data from four control and four VSL-mice are presented as mean ± SE. The asterisks indicate significant differences from the control group. **p < 0.001. Figure 6. TFF2-labeled epithelial cells (arrows) in tissue sections obtained from the middle and basal portions of the gastric mucosae of control (A) and VSL-treated (B) mice. Note the extension of TFF2 labeling to the bottom of the gastric glands where chief cells (lower three arrows) are located. Magnification bar = 100 µm. Figure 6. TFF2-labeled epithelial cells (arrows) in tissue sections obtained from the middle and basal portions of the gastric mucosae of control (A) and VSL-treated (B) mice. Note the extension of TFF2 labeling to the bottom of the gastric glands where chief cells (lower three arrows) are located. Magnification bar = 100 µm. Figure 6. TFF2-labeled epithelial cells (arrows) in tissue sections obtained from the middle and basal portions of the gastric mucosae of control (A) and VSL-treated (B) mice. Note the extension of TFF2 labeling to the bottom of the gastric glands where chief cells (lower three arrows) are located. Magniﬁcation bar = 100 µm. Figure 6. TFF2-labeled epithelial cells (arrows) in tissue sections obtained from the middle and basal portions of the gastric mucosae of control (A) and VSL-treated (B) mice. Note the extension of TFF2 labeling to the bottom of the gastric glands where chief cells (lower three arrows) are located. Magnification bar = 100 µm. Figure 6. 2 4 VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller Figure 8. A high magnification micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller Figure 8. A high magniﬁcation micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller than those of control with a less amount of secretory granules (arrows in A vs. B). Magniﬁcation bar = 50 µm. Figure 8. A high magnification micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller Figure 8. A high magnification micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller Figure 8. A high magniﬁcation micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller than those of control with a less amount of secretory granules (arrows in A vs. B). Magniﬁcation bar = 50 µm. 2.5. VSL Treatment neither Aﬀect Cell Proliferation nor H+,K+-ATPase Immunolabeling 2.5. VSL Treatment neither Aﬀect Cell Proliferation nor H+,K+-ATPase Immunolabeling The gastric mucosa of control mice showed a few BrdU-labeled cells at the pit-gland junctions close to the luminal surface (Figure 9A). However, in the VSL-treated mice, the area of pit-gland junction tended to show more dividing cells (Figure 9B). Quantiﬁcation of BrdU labeled cells in three diﬀerent equivalent images of three control and three VSL mice revealed the presence of 1.13± 0.18 and 1.7 ± 0.31 cells per gland, respectively (Figure 9C). 2 4 VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells I i i ep i oge o u io y ief e To test whether increased TFF2 production was associated with alteration in chief cells, tissues were probed with antibodies specific for pepsinogen (Figure 7A,B). It was interesting to find out that To test whether increased TFF2 production was associated with alteration in chief cells, tissues were probed with antibodies specific for pepsinogen (Figure 7A,B). It was interesting to find out that To test whether increased TFF2 production was associated with alteration in chief cells, tissues were probed with antibodies speciﬁc for pepsinogen (Figure 7A,B). It was interesting to ﬁnd out that the intensity of pepsinogen labeling was signiﬁcantly reduced after the 10-day treatment with VSL (Figure 7C). In addition, a close-up examination of tissue sections showed that the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands was associated with a decreased cellular size (Figure 8A,B). 7 of 20 ed ed a Int. J. Mol. Sci. 2019, 20, 3901 (Figure 7C). In add i l b li (Figure 7C). In add pepsinogen labeling decreased cellular size (Figure 8A,B). Figure 7. Immunohistochemical analysis of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of pepsinogen-labeling intensity per field in the oxyntic glands of control and VSL treated mice (C). Data from three control and three VSL-mice are presented as mean ± SE. The asterisk indicates significant differences from the control group. p < 0.05. Figure 7. Immunohistochemical analysis of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Magniﬁcation bar = 100 µm. Analysis of pepsinogen-labeling intensity per ﬁeld in the oxyntic glands of control and VSL treated mice (C). Data from three control and three VSL-mice are presented as mean ± SE. The asterisk indicates signiﬁcant diﬀerences from the control group. * p < 0.05. Figure 7. Immunohistochemical analysis of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of pepsinogen-labeling intensity per field in the oxyntic glands of control and VSL treated mice (C). Data from three control and three VSL-mice are presented as mean ± SE. The asterisk indicates significant differences from the control group. p < 0.05. Figure 7. 2 4 VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells Immunohistochemical analysis of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of pepsinogen-labeling intensity per field in the oxyntic glands of control and VSL treated mice (C). Data from three control and three VSL-mice are presented as mean ± SE. The asterisk indicates significant differences from the control group. p < 0.05. Figure 7. Immunohistochemical analysis of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Magniﬁcation bar = 100 µm. Analysis of pepsinogen-labeling intensity per ﬁeld in the oxyntic glands of control and VSL treated mice (C). Data from three control and three VSL-mice are presented as mean ± SE. The asterisk indicates signiﬁcant diﬀerences from the control group. * p < 0.05. Figure 7. Immunohistochemical analysis of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Magnification bar = 100 µm. Analysis of pepsinogen-labeling intensity per field in the oxyntic glands of control and VSL treated mice (C). Data from three control and three VSL-mice are presented as mean ± SE. The asterisk indicates significant differences from the control group. p < 0.05. Figure 8. A high magnification micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller Figure 8. A high magnification micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller Figure 8. A high magniﬁcation micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. Note the reduced pepsinogen labeling in chief cells of VSL mice at the bottom of gastric glands. These cells tend to appear smaller than those of control with a less amount of secretory granules (arrows in A vs. B). Magniﬁcation bar = 50 µm. Figure 8. A high magnification micrograph of pepsinogen immunolabeling in tissue sections obtained from the gastric mucosae of control (A) and VSL-treated (B) mice. 2 4 VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells The diﬀerence was not statistically signiﬁcant (p > 0.05). Int. J. Mol. Sci. 2019, 20, 3901 different equivalent and 1 7± 0 31 cells pe 8 of 20 18 nt (p > 0.05). Figure 9. Immunohistochemical analysis of S-phase cells using anti-5′-bromo-2′-deoxyuridine (BrdU) antibody in the gastric mucosa of control (A) and VSL-treated (B) mice. Note that BrdU-labelled cells (brown nuclei) of VSL-treated tissues tend to appear more expanded when compared with control tissues and uniformly distributed in most of the glands. Magnification bar = 200 µm. Analysis of BrdU-labeled cell counts per gland in the mucosa of seven control and seven VSL-treated mice are presented as mean ± SE (C). No significant difference was found between VSL and control tissues. Figure 9. Immunohistochemical analysis of S-phase cells using anti-5′-bromo-2′-deoxyuridine (BrdU) antibody in the gastric mucosa of control (A) and VSL-treated (B) mice. Note that BrdU-labelled cells (brown nuclei) of VSL-treated tissues tend to appear more expanded when compared with control tissues and uniformly distributed in most of the glands. Magniﬁcation bar = 200 µm. Analysis of BrdU-labeled cell counts per gland in the mucosa of seven control and seven VSL-treated mice are presented as mean ± SE (C). No signiﬁcant diﬀerence was found between VSL and control tissues. Figure 9. Immunohistochemical analysis of S-phase cells using anti-5′-bromo-2′-deoxyuridine (BrdU) antibody in the gastric mucosa of control (A) and VSL-treated (B) mice. Note that BrdU-labelled cells (brown nuclei) of VSL-treated tissues tend to appear more expanded when compared with control tissues and uniformly distributed in most of the glands. Magnification bar = 200 µm. Analysis of BrdU-labeled cell counts per gland in the mucosa of seven control and seven VSL-treated mice are presented as mean ± SE (C). No significant difference was found between VSL and control tissues. Figure 9. Immunohistochemical analysis of S-phase cells using anti-5′-bromo-2′-deoxyuridine (BrdU) antibody in the gastric mucosa of control (A) and VSL-treated (B) mice. Note that BrdU-labelled cells (brown nuclei) of VSL-treated tissues tend to appear more expanded when compared with control tissues and uniformly distributed in most of the glands. Magniﬁcation bar = 200 µm. Analysis of BrdU-labeled cell counts per gland in the mucosa of seven control and seven VSL-treated mice are presented as mean ± SE (C). No signiﬁcant diﬀerence was found between VSL and control tissues. 2 4 VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells 2.4. VSL Inhibits Pepsinogen Production by Chief Cells As for parietal cells, incubation with antibodies specific for the β-subunit of H+,K+-ATPase demonstrated the scattered distribution of parietal cells in both control (Figure 10A) and VSL-treated tissues (Figure 10B). Counts showed that parietal cells averaged 14.21 ± 0.91 cells per gland in control and 16.17 ± 1.13 cells in the VSL-treated tissues (Figure 10C). When the intensity of the H+,K+-ATPase labeling was compared in both groups of tissues, no apparent difference was noted. Quantification of the percentage of pixels in labeled areas in images obtained with 20× objective revealed no significant difference (p > 0.05). As for parietal cells, incubation with antibodies speciﬁc for the β-subunit of H+,K+-ATPase demonstrated the scattered distribution of parietal cells in both control (Figure 10A) and VSL-treated tissues (Figure 10B). Counts showed that parietal cells averaged 14.21 ± 0.91 cells per gland in control and 16.17 ± 1.13 cells in the VSL-treated tissues (Figure 10C). When the intensity of the H+,K+-ATPase labeling was compared in both groups of tissues, no apparent diﬀerence was noted. Quantiﬁcation of the percentage of pixels in labeled areas in images obtained with 20× objective revealed no signiﬁcant diﬀerence (p > 0.05). 2.6. VSL Treated Tissues Showed an Increase in Ghrelin-Secreting Cells For enteroendocrine cells, ghrelin antibodies were used since they label diﬀerent subtypes of enteroendocrine cells in the gastric mucosa. Immunoprobed tissue sections of control (Figure 11A) and VSL-treated (Figure 11B) mice, revealed that ghrelin secreting cells were scattered along the gastric glands. Surprisingly, tissue sections obtained from the VSL-treated mice showed more ghrelin-labeled cells than in control. Counts revealed the presence of 0.8 ± 0.09 and 1.14 ± 0.07 cells per gland on the average in the gastric mucosa of control and VSL-treated mice, respectively (Figure 11C). This diﬀerence was statistically signiﬁcant (p < 0.05). Int. J. Mol. Sci. 2019, 20, 3901 Int. J. Mol. Sci. 2019, 20, x F 9 of 20 21 9 of 20 21 Figure 10. Immunohistochemical labeling of parietal cells in the gastric mucosa of control (A) and VSL-treated (B) mice with antibodies specific for H+,K+-ATPase β-subunit. Labeled parietal cells are distributed throughout the gastric glands. Magnification bar = 200 µm. Quantification of H+,K+- ATPase-labeled cells per gland (C) and the labeling intensity per field (D) in seven control and seven VSL-mice are presented as mean ± SE. No significant differences were observed. Figure 10. Immunohistochemical labeling of parietal cells in the gastric mucosa of control (A) and VSL-treated (B) mice with antibodies speciﬁc for H+,K+-ATPase β-subunit. Labeled parietal cells are distributed throughout the gastric glands. Magniﬁcation bar = 200 µm. Quantiﬁcation of H+,K+-ATPase-labeled cells per gland (C) and the labeling intensity per ﬁeld (D) in seven control and seven VSL-mice are presented as mean ± SE. No signiﬁcant diﬀerences were observed. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 11 of 21 Figure 10. Immunohistochemical labeling of parietal cells in the gastric mucosa of control (A) and VSL-treated (B) mice with antibodies specific for H+,K+-ATPase β-subunit. Labeled parietal cells are distributed throughout the gastric glands. Magnification bar = 200 µm. Quantification of H+,K+- ATPase-labeled cells per gland (C) and the labeling intensity per field (D) in seven control and seven VSL-mice are presented as mean ± SE. No significant differences were observed. Figure 10. Immunohistochemical labeling of parietal cells in the gastric mucosa of control (A) and VSL-treated (B) mice with antibodies speciﬁc for H+,K+-ATPase β-subunit. Labeled parietal cells are distributed throughout the gastric glands. Magniﬁcation bar = 200 µm. 2.6. VSL Treated Tissues Showed an Increase in Ghrelin-Secreting Cells Quantiﬁcation of H+,K+-ATPase-labeled cells per gland (C) and the labeling intensity per ﬁeld (D) in seven control and seven VSL-mice are presented as mean ± SE. No signiﬁcant diﬀerences were observed. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 11 of 21 2.6. VSL Treated Tissues Showed an Increase in Ghrelin-Secreting Cells For enteroendocrine cells, ghrelin antibodies were used since they label different subtypes of enteroendocrine cells in the gastric mucosa. Immunoprobed tissue sections of control (Figure 11A) and VSL-treated (Figure 11B) mice, revealed that ghrelin secreting cells were scattered along the gastric glands. Surprisingly, tissue sections obtained from the VSL-treated mice showed more ghrelin-labeled cells than in control. Counts revealed the presence of 0.8 ± 0.09 and 1.14 ± 0.07 cells per gland on the average in the gastric mucosa of control and VSL-treated mice, respectively (Figure 11C). This difference was statistically significant (p < 0.05). Figure 11. Immunohistochemical analysis of ghrelin-secreting cells in the gastric mucosa of control (A) and VSL-treated (B) mice. Note the labeled cells scattered along the gastric glands. Magnification bar = 200 µm. Counts of ghrelin-labeled cells per gland in six control and six VSL-mice are presented as mean ± SE (C). The asterisk indicates significant increase in VSL-tissues as compared to control. p < 0.05. Figure 11. Immunohistochemical analysis of ghrelin-secreting cells in the gastric mucosa of control (A) and VSL-treated (B) mice. Note the labeled cells scattered along the gastric glands. Magniﬁcation bar = 200 µm. Counts of ghrelin-labeled cells per gland in six control and six VSL-mice are presented as mean ± SE (C). The asterisk indicates signiﬁcant increase in VSL-tissues as compared to control. * p < 0.05. -Secreting Cells were used since they label different subtypes of noprobed tissue sections of control (Figure 11A) ghrelin secreting cells were scattered along the ined from the VSL-treated mice showed more ed the presence of 0.8 ± 0.09 and 1.14 ± 0.07 cells ontrol and VSL-treated mice, respectively (Figure < 0.05). wer Figure 11. Immunohistochemical analysis of ghrelin-secreting cells in the gastric mucosa of control (A) and VSL-treated (B) mice. Note the labeled cells scattered along the gastric glands. Magnification bar = 200 µm. Counts of ghrelin-labeled cells per gland in six control and six VSL-mice are presented as mean ± SE (C). The asterisk indicates significant increase in VSL-tissues as compared to control. p < 0.05. Figure 11. 2.6. VSL Treated Tissues Showed an Increase in Ghrelin-Secreting Cells GAPDH Reverse TATTATGGGGGTCTGGGATGG Muc5 Forward AGG GCC CAG TGA GCA TCT CCTA Reverse CAT CAT CGC AGC GCA GAG TCA TFF2 Forward GCA GTG CTT TGA TCT TGG ATG C Reverse TCA GGT TGG AAA AGC AGC AGTT F d AAC AGA ATT GTC AAG TTC CTC To test whether VSL administration would inﬂuence its eﬀect on ghrelin expression, we measured the ghrelin gene expression in control as well as VSL groups. VSL group demonstrated ~2.2-fold enrichment in ghrelin mRNA expression. A 10-day period of VSL administration exhibited an abundant expression of ghrelin mRNA when compared to control, this upregulation was statistically signiﬁcant (p < 0.01, Figure 12). Moreover, to conﬁrm that VSL has an eﬀect on gastric enteroendocrine cells, chromogranin A (CgA), a general marker for diﬀerentiated endocrine cells, was also tested. This analysis revealed approximately a 3.5-fold change in CgA gene expression on average in all VSL-treated mice when compared to controls. This upregulation in CgA gene expression in VSL-treated mice was signiﬁcant (p < 0.05, Figure 12). Taken together, these results point to a clear eﬀect for VSL administration on the gastric epithelium. VSL enhances gastric epithelial protection through upregulation of TFF1, TFF2, Muc5ac, Muc6, and ghrelin and downregulation of PgC. These ﬁndings will highlight the preventive and therapeutic values of probiotics in gastric diseases. HK-β Forward AAC AGA ATT GTC AAG TTC CTC Reverse AGA CTG AAG GTG CCA TTG Ghrl Forward AGGAATCCAAGAAGCCACCAGCTA Reverse ATGCCAACATCGAAGGGAGCATTG Muc6 Forward CTC ACC TTC TAC CCC AGT ATC A Reverse GGC AAC GAG TTA GAG TCA CAT T TFF1 Forward GGCCCAGGAAGAAACATGTATC Reverse ACTGCTGGGCGGTGACA PgC Forward AAACCGGCATCATGAAGTGGATGG Reverse TTGTTCCTTCATGGTCTCCCGGAT CgA Forward GCA GCA TCC AGT TCC CAC TTC C Reverse TCC CCA TCT TCC TCC TGC TGA G GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Muc5: Mucin 5ac; TFF2: trefoil factor 2; HK- β; HK-ATPase β; Ghrl: Ghrelin; Muc6: Mucin 6; PgC: Pepsinogen C; CgA: Chromogranin A. β β g p g g g Figure 12. Quantitative RT-PCR analysis of mRNA expression of VSL–treated tissues versus control tissues. Expression of TFF1, TFF2, Muc5ac, Muc6, H+,K+-ATPase β subunit, ghrelin, CgA and PgC in VSL and control tissues were compared. Data from three different pairs of control and VSL-treated mice were presented as mean ± SE and normalized to GAPDH. The asterisks indicate significant differences from the control group. p < 0.05; p < 0.01; p < 0.001. Figure 12. 2.6. VSL Treated Tissues Showed an Increase in Ghrelin-Secreting Cells Immunohistochemical analysis of ghrelin-secreting cells in the gastric mucosa of control (A) and VSL-treated (B) mice. Note the labeled cells scattered along the gastric glands. Magniﬁcation bar = 200 µm. Counts of ghrelin-labeled cells per gland in six control and six VSL-mice are presented as mean ± SE (C). The asterisk indicates signiﬁcant increase in VSL-tissues as compared to control. p < 0.05. Int. J. Mol. Sci. 2019, 20, 3901 Int. J. Mol. Sci. 2019, 20, x F 10 of 20 21 2.7. Eﬀects of VSL Treatment on the Expression of Various Gastric Cell Lineage-Speciﬁc Genes expression in VSL-treated mice was significant (p < 0.05, Figure 12). Taken together, thes Eﬀects of VSL Treatment on the Expression of Various Gastric Cell Lineage-Speciﬁc Genes expression in VSL-treated mice was significant (p < 0.05, Figure 12). Taken together, these To substantiate these ﬁndings using another method, gene expression levels of diﬀerent gastric proteins/peptides including Muc5AC, Muc6, TFF1, TFF2, PgC, H+,K+-ATPase β-subunit, ghrelin, and CgA were measured by real-time quantitative polymerase chain reaction (RT-qPCR). The primers used are listed in Table 1. Analysis of the expression of Muc5ac gene-speciﬁc for mucus-secreting pit cells in VSL-treated mice showed a 2.5-fold increase when compared to control (p < 0.05, Figure 12). Treatment with VSL also showed 5.5-fold induction of Muc6 gene expression (p < 0.05, Figure 12). TFF1 and TFF2 expression analysis revealed a 3.5- and 7.5-fold increase in VSL-treated tissues (p < 0.05, p < 0.01, respectively, Figure 12). point to a clear effect for VSL administration on the gastric epithelium. VSL enhances gastric epithelial protection through upregulation of TFF1, TFF2, Muc5ac, Muc6, and ghrelin and downregulation of PgC. These findings will highlight the preventive and therapeutic values of probiotics in gastric diseases. Table 1. List of primers used to quantify the expression of eight gastric cell lineage markers in VSL- treated versus control tissues. Gene Primer Sequence (5′-3′) GAPDH Forward TCAAGAAGGTGGTGAAGCAGG To further assess the eﬀect of VSL on other gastric cell lineages, a signiﬁcant increase (2.9-fold) in H+,K+-ATPase β-subunit mRNA expression was found when compared to control (p < 0.05). This ﬁnding was not consistent with immunohistochemical analysis. However, consistent with immunohistochemistry, analysis of PgC expression of chief cells in VSL mice showed a signiﬁcant reduction in mRNA abundance compared to control (p < 0.001, Figure 12). 2.6. VSL Treated Tissues Showed an Increase in Ghrelin-Secreting Cells Quantitative RT-PCR analysis of mRNA expression of VSL–treated tissues versus control tissues. Expression of TFF1, TFF2, Muc5ac, Muc6, H+,K+-ATPase β subunit, ghrelin, CgA and PgC in VSL and control tissues were compared. Data from three diﬀerent pairs of control and VSL-treated mice were presented as mean ± SE and normalized to GAPDH. The asterisks indicate signiﬁcant diﬀerences from the control group. * p < 0.05; p < 0.01; * p < 0.001. Figure 12. Quantitative RT-PCR analysis of mRNA expression of VSL–treated tissues versus control tissues. Expression of TFF1, TFF2, Muc5ac, Muc6, H+,K+-ATPase β subunit, ghrelin, CgA and PgC in VSL and control tissues were compared. Data from three different pairs of control and VSL-treated Figure 12. Quantitative RT-PCR analysis of mRNA expression of VSL–treated tissues versus control tissues. Expression of TFF1, TFF2, Muc5ac, Muc6, H+,K+-ATPase β subunit, ghrelin, CgA and PgC in VSL and control tissues were compared. Data from three diﬀerent pairs of control and VSL-treated mice d SE d li d GAPDH Th i k i di i iﬁ diﬀ Figure 12. Quantitative RT-PCR analysis of mRNA expression of VSL–treated tissues versus control tissues. Expression of TFF1, TFF2, Muc5ac, Muc6, H+,K+-ATPase β subunit, ghrelin, CgA and PgC in VSL and control tissues were compared. Data from three different pairs of control and VSL-treated mice were presented as mean ± SE and normalized to GAPDH. The asterisks indicate significant differences from the control group. p < 0.05; p < 0.01; *p < 0.001. Figure 12. Quantitative RT-PCR analysis of mRNA expression of VSL–treated tissues versus control tissues. Expression of TFF1, TFF2, Muc5ac, Muc6, H+,K+-ATPase β subunit, ghrelin, CgA and PgC in VSL and control tissues were compared. Data from three diﬀerent pairs of control and VSL-treated mice were presented as mean ± SE and normalized to GAPDH. The asterisks indicate signiﬁcant diﬀerences from the control group. p < 0.05; p < 0.01; * p < 0.001. 11 of 20 Int. J. Mol. Sci. 2019, 20, 3901 Table 1. List of primers used to quantify the expression of eight gastric cell lineage markers in VSL-treated versus control tissues. 2.6. VSL Treated Tissues Showed an Increase in Ghrelin-Secreting Cells Gene Primer Sequence (5′-3′) GAPDH Forward TCAAGAAGGTGGTGAAGCAGG Reverse TATTATGGGGGTCTGGGATGG Muc5 Forward AGG GCC CAG TGA GCA TCT CCTA Reverse CAT CAT CGC AGC GCA GAG TCA TFF2 Forward GCA GTG CTT TGA TCT TGG ATG C Reverse TCA GGT TGG AAA AGC AGC AGTT HK-β Forward AAC AGA ATT GTC AAG TTC CTC Reverse AGA CTG AAG GTG CCA TTG Ghrl Forward AGGAATCCAAGAAGCCACCAGCTA Reverse ATGCCAACATCGAAGGGAGCATTG Muc6 Forward CTC ACC TTC TAC CCC AGT ATC A Reverse GGC AAC GAG TTA GAG TCA CAT T TFF1 Forward GGCCCAGGAAGAAACATGTATC Reverse ACTGCTGGGCGGTGACA PgC Forward AAACCGGCATCATGAAGTGGATGG Reverse TTGTTCCTTCATGGTCTCCCGGAT CgA Forward GCA GCA TCC AGT TCC CAC TTC C Reverse TCC CCA TCT TCC TCC TGC TGA G GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Muc5: Mucin 5ac; TFF2: trefoil factor 2; HK-β; HK-ATPase β; Ghrl: Ghrelin; Muc6: Mucin 6; PgC: Pepsinogen C; CgA: Chromogranin A. Table 1. List of primers used to quantify the expression of eight gastric cell lineage markers in VSL-treated versus control tissues. Gene Primer Sequence (5′-3′) GAPDH Forward TCAAGAAGGTGGTGAAGCAGG Reverse TATTATGGGGGTCTGGGATGG Muc5 Forward AGG GCC CAG TGA GCA TCT CCTA Reverse CAT CAT CGC AGC GCA GAG TCA TFF2 Forward GCA GTG CTT TGA TCT TGG ATG C Reverse TCA GGT TGG AAA AGC AGC AGTT HK-β Forward AAC AGA ATT GTC AAG TTC CTC Reverse AGA CTG AAG GTG CCA TTG Ghrl Forward AGGAATCCAAGAAGCCACCAGCTA Reverse ATGCCAACATCGAAGGGAGCATTG Muc6 Forward CTC ACC TTC TAC CCC AGT ATC A Reverse GGC AAC GAG TTA GAG TCA CAT T TFF1 Forward GGCCCAGGAAGAAACATGTATC Reverse ACTGCTGGGCGGTGACA PgC Forward AAACCGGCATCATGAAGTGGATGG Reverse TTGTTCCTTCATGGTCTCCCGGAT CgA Forward GCA GCA TCC AGT TCC CAC TTC C Reverse TCC CCA TCT TCC TCC TGC TGA G GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Muc5: Mucin 5ac; TFF2: trefoil factor 2; HK-β; HK-ATPase β; Ghrl: Ghrelin; Muc6: Mucin 6; PgC: Pepsinogen C; CgA: Chromogranin A. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Muc5: Mucin 5ac; TFF2: trefoil factor 2; HK-β; HK-ATPase β; Ghrl: Ghrelin; Muc6: Mucin 6; PgC: Pepsinogen C; CgA: Chromogranin A. 3. Discussion Recently, much attention has been paid to the impact of probiotics in the gastrointestinal tract. However, the mechanisms through which probiotics exert their eﬀect are still unclear. Several clinical studies have shown that probiotics modulate gut microﬂora [37,38] and protect the gastric mucosa through the regulation of cellular proliferation and gastric mucin production [33,35,39–42]. In our current study, the multi-strain probiotic mixture VSL was found to mainly upregulate expression and production of TFF1, TFF2, and mucins in the mouse stomach. Further investigations have shown that VSL administration also enhances the expression of ghrelin and chromogranin and production of enteroendocrine ghrelin-secreting cells. Interestingly, upregulation of TFF2 was associated with their production in some chief cells located at the bottom of the gastric glands. These chief cells not only expressed a lower level of pepsinogen gene and smaller amount of pepsinogen protein, but they appeared smaller in size as compared to those of control mice. It should be stated that conventional histological examination of hematoxylin and eosin-stained stomach tissue samples from VSL-treated mice has not revealed morphological changes when compared to control mice. It was also reported previously that administration of Lactobacillus acidophilus and Biﬁdobacterium spp. had no eﬀect on the morphology of the stomach and intestine of piglets [43]. However, molecular analysis using detailed immuno- and lectin-histochemical methods, as well as RT-qPCR, has revealed several changes. The mucus gel layer is a structural component of the gut that lubricates and protects the gastrointestinal tract against various harmful agents. In the present study, compelling evidence shows that VSL enhances mucus production. Both lectin staining and RT-qPCR for the expression of mucin genes (Muc5ac and Muc6) has supported this observation. The increased gastric mucous secretion is not surprising and coincides with other studies which demonstrated an increase in the thickness of the mucus gel layer of the gastric mucosa when treated with exopolysaccharide producing Streptococcus thermophilus CRL 1190 [44] or Biﬁdobacterium biﬁdum BF-1 [45] to alleviate the chronic gastritis in Int. J. Mol. Sci. 2019, 20, 3901 12 of 20 mice or acute gastric injury in rats. Similarly, it was demonstrated that treatment of ethanol-induced gastric mucosal lesions with Lactobacillus rhamnosus GG indirectly stimulated the mucous secretion and transmucosal resistance through the up-regulation of prostaglandin [40]. 3. Discussion One study aimed to investigate the eﬀect of the supernatant of milk fermented by Lactobacillus helveticus R239 on gut physiology and demonstrated an increase in the number of goblet cells, suggesting an increase in mucus secretion [46]. Moreover, several studies have demonstrated the potent eﬀect of probiotics in mucin gene expression in colonic and intestinal epithelial cell lines [33,34] and in animal models [35]. On the other hand, contradictory studies were observed when VSL probiotic mixture was used in acetic acid induced gastric ulcer in rats [19] or in dextran-sodium sulfate-induced chronic colitis in mice [47]. These studies showed that VSL has either a modest increase in the expression of Muc5ac in a rat model of the acetic acid-induced gastric ulcer [19] or no potential role for mucin in dextran-sodium sulfate-induced chronic colitis in mice [47]. These ﬁndings would rather suggest the beneﬁcial usage of VSL as a preventive agent more than a therapeutic agent. TFFs are typical constituents of mucus-secreting epithelia. The increase in mucous secretion found in the present study coincided with an increase in the number of cells expressing TFF1 and TFF2. This result is not unexpected since TFFs co-localized and co-secreted with mucins [48,49]. Individual TFFs have also been noted to interact with speciﬁc mucins preferentially, and such interaction is important to strengthen the mucous barrier [50]. Accordingly, TFF peptides are considered as constituents of the mucus barrier, where they display lectin-like behavior [51]. TFF3 interacts with speciﬁc carbohydrate moieties and stabilizes the gastric mucous barrier [48,52,53]. TFF2 was found to protect the mucosa from insults by stabilizing the mucous layer and inhibiting acid secretion [48,54]. In normal conditions, TFFs are expressed in the gastrointestinal tract in a tissue-speciﬁc manner. In humans, TFF1 and TFF2 are expressed in the stomach and duodenum [55,56]. TFFs are considered to play critical roles in maintaining mucosal integrity and defense [57,58], promoting cell migration [59,60] and enhancing cell proliferation and diﬀerentiation [61,62]. This raises the proposal that TFFs are good therapeutic candidates for the treatment of several gastrointestinal diseases. While probiotics are considered as a promising therapy against gastrointestinal inﬂammation, only a few studies have addressed how probiotics associate with TFFs in the host defense. One of the most interesting ﬁndings in our study was the increase in the expression of TFF1 and TFF2 in the stomach of VSL-treated mice. 3. Discussion Several studies have pointed to TFF peptides as crucial players in mucosal protection, repair, reconstitution, and diﬀerentiation [49,51,63]. In the present study, we demonstrated a proportional upregulation in TFF1 and TFF2 of all VSL-treated mice. These ﬁndings are coherent with other studies which demonstrated that oral administration of genetically recombinant TFF-secreting Lactococcus lactis exerted both prophylactic and therapeutic eﬀects in the mouse model of acute colitis [64]. Another study has also demonstrated that supplementation of Lactobacillus rhamnosus GG supernatant increased the TFF3 in alcohol damaged Caco-2 cell and also in a mouse model of alcohol-induced liver disease [65,66]. Conversely, another study indicated that LGG supplementation in the diet did not show signiﬁcant regulatory eﬀects in the gene expression of TFFs in mice with induced colitis [67]. Additionally, in a rat model of neonatal necrotizing enterocolitis, the numbers of TFF3 positive cells were reduced to normal level after feeding with live Biﬁdobacterium biﬁdum [31]. Other studies have also demonstrated that supplementation of Enterococcus faecium has no eﬀect on intestinal TFF3 expression in animal models [68,69]. Further studies are needed for better understanding of the role of probiotics and TFFs and how their interactions might aﬀect the host defense. In the present study, the immunolabeling of TFF2 was not only detected in the middle of the gastric glands where mucous neck cells are located but were frequently detected at the bottom of the glands where pepsinogen-secreting chief (or zymogenic) cells dominate. This interesting ﬁnding correlated with the downregulation of pepsinogen gene expression and immunolabeling in the gastric mucosa of VSL mice. Since it has been established that neck and zymogenic cells belong to the same cell lineage [36], this ﬁnding could be explained in diﬀerent ways. As a consequence of VSL administration, either (i) neck cells did not continue to diﬀerentiate into fully mature zymogenic cells Int. J. Mol. Sci. 2019, 20, 3901 13 of 20 13 of 20 and have remained as pre-zymogenic cells producing mucin, TFF2 and pepsinogen, or (ii) zymogenic cells started to dediﬀerentiate into pre-zymogenic cells which acquired some mucin, TFF2, but lost some of its machinery to produce pepsinogen. In either case, the reduction in pepsinogen and increase of mucus and TFF2 indicates a profound gastroprotective eﬀect for VSL on the gastric mucosa. 3. Discussion The decrease in pepsinogen in VSL-treated mice is in agreement with another study that demonstrated a decrease in pepsinogen I/II ratio in humans after 12 weeks of treatment with Biﬁdobacterium biﬁdum fermented milk [70]. By using BrdU labeling method, this study demonstrates that VSL has no eﬀect on gastric epithelial cell proliferation. To eliminate the possible animal variations, VSL-treated and control mice were not only littermates but also sex- and weight-matched. In control and VSL-tissues, a few BrdU-labeled cells were localized in the isthmus regions of the gastric glands. These ﬁndings are in agreement with a previous study of Lam et al., 2007 who demonstrated that treatment with Lactobacillus rhamnosus GG reduced cellular apoptosis in the rat gastric mucosa with ethanol induced lesions but did not report any eﬀect on cellular proliferation [40]. Conversely, VSL-treated rats showed a signiﬁcant increase in the proliferation index in the colon compared to control [27]. In addition, VSL showed antiproliferative eﬀect against cancer cell lines (Jurkat, HT1080, and Caco-2 tumor cell lines) [71]. A recent study using the same VSL probiotic mixture has demonstrated epithelial regenerative eﬀects in the small intestine and colon of mice [72]. Therefore, results on the eﬀects of VSL on cell proliferation from previous studies are contradictory. This could be explained by the fact that the VSL probiotic mixture used in the previous studies might not be the same. Currently, two commercial VSL probiotic mixtures exist in the market. The original VSL form is produced in USA, while the newfound VSL form is produced in Italy and commercialized by European countries (UK and Holland). It was already published that the outcomes of both forms are not similar [71]. The VSL form used in the current study was the original form. As for acid secreting cells, the current ﬁnding indicates that VSL induces about a 3-fold increase in the expression of the regulatory H+,K+ATPase β subunit gene. This may correlate with the previous ﬁndings on the possible use of probiotics in cancer. It is known that gastric cancer is associated with loss of parietal cells, so upregulation of the HK-β subunit gene could be seen as a preventive eﬀect against cancer. However, the immunohistochemical analysis did not show any signiﬁcant diﬀerence in the counts and labeling intensity of parietal cells. Further investigations are needed to clarify this intriguing result. 4.2. Experimental Design Each pair of mice included a treated and a control mouse. The treated mice (n = 7) received, by gastric gavage, the VSL#3 probiotic mixture (Sigma-Tau Pharmaceuticals, Inc., Gaithersburg, MD, USA). The VSL is composed of a mixture of highly concentrated lyophilized living bacteria (450 billion bacteria per sachet) of four species of Lactobacilli: Lactobacillus acidophilus, Lactobacillus parcasei, Lactobacillus plantarum, and Lactobacillus delbrueckii Subsp. Bulgaricus; three species of Biﬁdobacteria: Biﬁdobacterium infantis, Biﬁdobacterium longum, and Biﬁdobactrium breve and one species of Streptococcus salivarius Subsp thermophilus sp. The preparation and dose of VSL were according to previously published information [79]. VSL was suspended in water and given at a dose equivalent to 5 mg/day/mouse for 10 days. The control mice (n = 7) received only the vehicle. The bodyweights of all mice were measured on days 0, 5, and 10 of the experiment. The food intake during the 10-day experimental period was also estimated. To label dividing cells in the S-phase of the cell cycle, all mice received a single intraperitoneal injection of BrdU (120 mg/kg bodyweight) two hours before sacriﬁce. The stomachs were removed under anesthesia and processed for histological, immunohistochemical and RT-qPCR analyses. 4.1. Animals Animal Research Ethics Committee at UAE University approved the protocols used in this study (ERA_2016_5487). Fourteen male C57Bl/6 mice aged from three to ﬁve months and weighing 25 g on average were used in this study. The mice were used in two diﬀerent experiments carried out independently. In the ﬁrst experiment, mice were divided into two groups, three control and three VSL-treated. In the second experiment, each group included four mice. In each experiment, mice were paired, and each pair was a weight matched littermate. All mice were given ad libitum access to laboratory chow and water. Mice were kept under a 12 h light/dark cycle at room temperature (22–24 ◦C). 3. Discussion It is known that gastrointestinal microbiota, probiotics, and heat-killed microbes can regulate intestinal immunity. However, their eﬀect on the secretion of gastrointestinal hormones is not well explored. An important ﬁnding of this study was not only upregulation of ghrelin gene expression in VSL-treated mice, but also the increase in the number of ghrelin-secreting cells. Coherent results were obtained by both immunohistochemistry and RT-qPCR. Only a few studies have investigated the eﬀect of probiotics on ghrelin expression of the gastric mucosa [73–75]. Our results were consistent with the recent study that demonstrated that supplementation of Lactogen microgranules probiotic formula containing Lactobacillus rhamnosus increases signiﬁcantly the ghrelin gene expression in ﬁsh larvae [72]. Interestingly, it was also demonstrated recently that oral administration of heat killed Lactobacillus brevis SBC8803 increased acyl ghrelin serum level in rats. In the same study, in vitro analysis of mouse primary stomach cells treated with the same probiotic also demonstrated induction of ghrelin secretion [74]. Not only ghrelin-secreting cells, but other enteroendocrine cells play essential roles in gut chemo-sensing to orchestrate appropriate functional responses of the host’s physiology and translate signals coming from the gut microbiota through their hormonal secretions [76,77]. In the present study, Quantitative PCR revealed that the VSL-fed mice increased the expression levels of not only ghrelin but also CgA. Recently, another study has also shown that probiotic strain Escherichia coli Nissle 1917 increased CgA mRNA expression of enteroendocrine cells in piglets [78]. Since ghrelin stimulates appetite, this explains the slight increase (even though insigniﬁcant) in the gain of bodyweight in Int. J. Mol. Sci. 2019, 20, 3901 14 of 20 14 of 20 VSL-treated mice. This ﬁnding requires further investigation perhaps by increasing the dose or the duration of VSL administration to ﬁnd out whether a signiﬁcant increase in bodyweight will occur. Although the mechanism of action of probiotics in the mouse stomach tissue appears to be diverse, still there is a need for additional studies to fully understand the mechanistic strategy by which probiotics modulate various gastric cell lineages. This will hopefully provide greater opportunities for improving prevention and therapeutic strategies for gastric disorders. 4.3. Histological, Lectin Histochemistry, and Immunohistochemical Analysis To examine the structure of the gastric mucosa, the posterior walls of the stomachs of the mice in each of the two groups were processed together for routine histological examination. The tissues were immediately immersed overnight in Bouin’s solution and then processed for paraﬃn embedding. To ensure equal conditions, tissues of each group of VSL-treated and their littermate control pairs were embedded together in the same paraﬃn block. Five-µm-thick tissue sections were stained with hematoxylin and eosin or periodic acid Schiﬀ(Abcam, Cambridge, UK) and examined with the Olympus microscope connected to DP70 digital camera. To label surface mucous cells and neck mucous cells, gastric tissues were deparaﬃnized, rehydrated, and incubated with blocking buﬀer (1% BSA, 0.5% Tween-20 in PBS) for 45 min at room temperature, and then incubated in fucose-speciﬁc Ulex europaeus agglutinin I (UEA-I) lectin (Vector Laboratories, United States) conjugated to rhodamine for 1 hr. Following PBS washes, tissues were incubated for 1 hr with N-Acetyl-D-glucosamine-speciﬁc Griﬀonia simplicifolia II (GS-II) lectin (Thermo-Fisher Scientiﬁc, Molecular probes by Life Technologies, Eugene, OR, USA) conjugated to ﬂuorescein isothiocyanate Int. J. Mol. Sci. 2019, 20, 3901 15 of 20 (FITC). The tissue sections were washed in PBS and mounted with ﬂuoro-shield mounting medium with 4’,6-diamidino-2-phenylindole (Abcam, Cambridge, UK). To localize TFF1, TFF2, chief cells, proliferating cells, parietal cells, and ghrelin-secreting cells, tissue sections were deparaﬃnized, rehydrated and washed in PBS. The endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 35 min. All tissue sections on the slide (representing three or four pairs of control and VSL stomachs) were circled with a hydrophobic ﬁlm using a PAP pen (Dako, Glostrup, Denmark). To block non-speciﬁc binding sites, the sections were incubated in 1% BSA containing 0.5% Tween-20 and PBS for 45 min. The sections were then incubated for 1 hr using mouse monoclonal anti-BrdU antibody (Medical and Biological Laboratories Co., Nagoya, Japan) or for overnight at 4 ◦C with mouse monoclonal anti-pepsinogen C (Abcam, Cambridge, UK), or anti-H+,K+-ATPase β-subunit (Medical and Biological Laboratories Co. Woburn, MA, USA), or anti-ghrelin [raised in the IGBMC laboratories, Strasbourg, France] antibodies, or rabbit polyclonal anti-TFF1 and anti-TFF2 antibodies [57]. Following PBS washes, the tissue sections were incubated with biotinylated donkey anti-mouse immunoglobulin G for 1 hr. Tissues were washed in PBS and then incubated in peroxidase-conjugated extravidin (Sigma, St. Louis, MO, USA). The antigen-antibody binding sites were revealed by using 3,3’-diaminobenzidine tetrahydrochloride (Sigma, St. 4.4. Immunohistochemical Quantiﬁcation To estimate the number of cells immunolabeled with antibodies speciﬁc for TFF1, TFF2, BrdU, H+,K+-ATPase, and ghrelin, the Fiji ImageJ software was used. At least three JPEG images obtained at 20× objective lens representing diﬀerent ﬁelds with the best longitudinal orientation of the gastric gland were examined in each control and VSL-treated tissues. In each image, the labeled cells were tracked using the cell counter of the software and the total number of glands was manually counted. The total number of labeled cells was divided by the total number of glands seen in the image. The number of cells per gland was expressed as the mean ± SE. For estimating the labeling intensities of mucus (in pit and neck cells), TFF1 (in pit cells), TFF2 (in neck cells), pepsinogen (in chief cells) and H+,K+-ATPase (in parietal cells), images obtained from tissue sections probed with lectins or antibodies were loaded into the ImageJ densitometric software. In the case of immunoperoxidase labeling, image deconvolution was used to separate the DAB staining from the hematoxylin and/or periodic acid Schiﬀstaining. Images were then converted to 8-bit and pixel density was calculated using the analysis tool. The percentage values obtained from the software were taken to reﬂect the amount of mucus/peptide/protein in the cells analyzed. Data were presented as mean ± SE. 4.3. Histological, Lectin Histochemistry, and Immunohistochemical Analysis Louis, MO, USA). 4.5. RT-qPCR Analysis The total RNA was isolated from gastric mucosal tissues of control and VSL-treated mice using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Then the cDNA was synthesized from 1µg of total RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientiﬁc, Baltics UAB, Vilnius, Lithuania) and Biometra Trio-ThermoblockTM. To study the diﬀerences in gene expression, SyBR green based (Applied Biosystems, Thermo Fisher Scientiﬁc, TX, USA) PCR was performed by keeping cDNA as template and primers speciﬁc for H+,K+-ATPase β-subunit, MUC-5ac, ghrelin, TFF1, TFF2, pepsinogen C (PgC), MUC6 and chromogranin A (CgA) were used as shown in Table 1. The QuantiStudio® 7 Flex Real-Time PCR instrument (Applied Biosystems by Life Technologies) was used for ampliﬁcation and quantiﬁcation of dsDNA. Each sample was performed in triplicates and normalized with the housekeeping gene GAPDH. The Gene expression levels were calculated using the comparative cycle threshold method (∆∆C). Values were presented as mean ± SEM. p < 0.05 was considered signiﬁcant. Int. J. Mol. Sci. 2019, 20, 3901 16 of 20 16 of 20 4.6. Statistical Analysis Results were presented as mean ± SE. Signiﬁcance diﬀerences between control and VSL-treated groups were determined using the Student’s t test. A p < 0.05 was considered statistically signiﬁcant. 5 C l i Results were presented as mean ± SE. Signiﬁcance diﬀerences between control and VSL-treated groups were determined using the Student’s t test. A p < 0.05 was considered statistically signiﬁcant. 5. Conclusions Recently, the multi-strain probiotic VSL has been a subject of numerous clinical trials and studies that demonstrated its considerable potential for prevention or therapeutic applications in various gastrointestinal diseases. However, the mechanisms underlying the probiotic mode of action have not been fully elucidated. This research demonstrates for the eﬀects of the probiotic mixture VSL on various cell lineages in the normal gastric mucosa of mice. Our results indicate that VSL enhances mucus, TFF1, and TFF2 production by both types of gastric mucous cells. While TFF2 was induced in chief cells, their production of pepsinogen was inhibited. These were associated with increased production of ghrelin secreting cells. Taken together, these data suggest the beneﬁcial impact of VSL in the mouse stomach and its therapeutic value in diseases involving gastric epithelial cells. Author Contributions: Conceptualization, G.K., F.A.-Y. and S.M.K.; formal analysis, F.A.-Y., P.S., and S.S.; investigation, F.A.-Y., P.S., S.S.; methodology, G.K., F.A., P.S., and S.S.; supervision, G.K., A.A.M. and S.M.K.; project administration, G.K., A.A.M., and S.M.K.; resources, A.A.M., C.T. and S.M.K.; validation, F.A.-Y., G.K. and S.M.K.; writing—original draft, F.A.-Y.; writing—review & editing, all authors; funding acquisition, G.K., A.A.M., C.T. and S.M.K. Funding: This research was funded by UAEU Center-Based research grant number 035108/15 and the University of Sharjah, competitive research grant number 1601110216-P. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Author Contributions: Conceptualization, G.K., F.A.-Y. and S.M.K.; formal analysis, F.A.-Y., P.S., and S.S.; investigation, F.A.-Y., P.S., S.S.; methodology, G.K., F.A., P.S., and S.S.; supervision, G.K., A.A.M. and S.M.K.; project administration, G.K., A.A.M., and S.M.K.; resources, A.A.M., C.T. and S.M.K.; validation, F.A.-Y., G.K. and S.M.K.; writing—original draft, F.A.-Y.; writing—review & editing, all authors; funding acquisition, G.K., A.A.M., C.T. and S.M.K. References 2019, 20, 3901 17 of 20 17 of 20 12. Plaza-Diaz, J.; Gomez-Llorente, C.; Fontana, L.; Gil, A. Modulation of immunity and inﬂammatory gene expression in the gut, in inﬂammatory diseases of the gut and in the liver by probiotics. World J. Gastroenterol. 2014, 20, 15632–15649. [CrossRef] [PubMed] 13. Delia, P.; Sansotta, G.; Donato, V.; Messina, G.; Frosinta, P.; Pergolixxi, S.; De Renzis, C.; Famularo, G. Prevention of radiation-induced diarrhea with the use of VSL#3, a new high potency probiotic preparation. Am. 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https://openalex.org/W1022919448	https://www.reanimatology.com/rmt/article/download/1289/126	Russian	null	Structural Bases of Postresuscitative Heart Failure	Obŝaâ reanimatologiâ	2,005	cc-by	4,240	Structural bases of postresuscitative heart failure V. T. Dolgikh, A. M. Kochetov, S. V. Dolgikh Department of Pathophysiology, Omsk State Medical Academy В эксперименте на 106 белых беспородных крысахсамцах, перенесших 4минутную клиническую смерть от острой кровопотери, установлено, что в первые трое суток после оживления отмечается сочетание сосудистых нарушений и дистрофических изменений кардиомиоцитов, причем ведущую роль играют расстройства микрогемоциркуляции: сладж, стаз, тромбоз, повышение сосудистой проницаемости, периваскулярный отек и кровоизлияния. Деструкция кардиомиоцитов: контрактуры различной степени, глыбчатый распад миофибрилл, миоцитолизис, являются структурной основой постреанимационнои сердечной недостаточности. После трех суток в сердце отмечается соче тание процессов восстановления и повреждения. В зависимости от соотношения этих процессов выделено три варианта морфологических изменений сердца. Рис. 1. Библ. 12. An experiment on 106 noninbred male albino rats undergone 4minute clinical death from acute blood loss has revealed that the first three days after resuscitation are marked by a concomitance of vascular disorders and cardiomyocytic dys trophic changes, the leading role being played by sludge, stasis, thrombosis, increased vascular permeability, perivascu lar edema, and hemorrhages. Cardiomyocytic destruction (various contractures, blocklike myofibrillolysis, myocytoly sis) is a structural basis of postresuscitative heart failure. Three days later the heart displayed concomitant processes of recovery and damage. Three types of cardiac morphological changes have been identified in relation to the ratio of these processes. мг/кг внутрибрюшинно). 4минутную клиническую смерть вы зывали острой кровопотерей из сонной артерии. Для предупреж дения свертывания крови за 15 мин до кровопускания внутривен но вводили гепарин из расчета 500 МЕ/кг массы и интубировали их. Контролем служили 10 интактных наркотизированных и ин тубированных животных. Оживление осуществляли центрипе тальным нагнетанием аутокрови, закрытым массажем сердца и искусственной вентиляцией легких в режиме умеренной гипер вентиляции. Миокард для морфологических исследований заби рали в различные сроки постреанимационного периода. Для све товой микроскопии кусочки миокарда фиксировали в 10% нейтральном формалине и жидкости Карнуа и заливали их в па рафин. На микротоме получали поперечные срезы обоих желу дочков и продольные — через миокард левого желудочка. Окра шенные гематоксилинэозином препараты изучали в обычном и поляризованном свете для выявления контрактур [5]. На попе речных срезах миокарда, окрашенных гематоксилином — основ ным фуксином — пикриновой кислотой [6], оценивали поврежде ния миокарда по количеству фуксинофильных волокон в 10 произвольно взятых полях зрения. Микроскопирование и фото графирование препаратов осуществляли с помощью микроскопа МБИ15 при увеличении в 400—500 раз. О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 Structural bases of postresuscitative heart failure Для электронной микро скопии из верхушки сердца, оставленного на льду, вырезали ку сочки миокарда размером 0,5—1,0 мм 3, фиксировали их в 2,5% растворе глютарового диальдегида на фосфатном буфере при 4 °С с последующей дофиксацией в течение часа при комнатной тем пературе в 1% растворе четырехокиси осмия. После фиксации ку сочки обезвоживали в этаноле возрастающей концентрации, аце тоне и заключали в эпон812 и аралдит [7]. Ультратонкие срезы контрастировали 2% раствором уранилацетата в 70° этаноле 10 мин и цитрате свинца — 15 мин, просматривали и фотографиро вали в электронном микроскопе. Электроннограммы оценива Изучение природы постреанимационных ге модинамических нарушений — фундаментальная проблема реаниматологии и патофизиологии тер минальных состояний [1]. Недостаточность крово обращения, возникающая в раннем периоде после оживления, во многом определяет неблагоприят ный исход реанимации [2]. В развитии постреани мационной недостаточности кровообращения на ряду с дефицитом объема циркулирующей крови, метаболическим ацидозом, вторичной гипоксией, эндотоксемией, нарушениями гемостаза и реоло гических свойств крови важную роль играют по вреждения самого сердца. Функциональномета болические нарушения, возникающие в сердечной мышце во время умирания и после оживления ор ганизма, изучены достаточно полно как в условиях целостного организма, так и изолированного серд ца, и изолированной папиллярной мышцы [3, 4]. Целью настоящего исследования явилось изуче ние структурных основ постреанимационной не достаточности сердца. Материал и методы исследования Материал и методы исследования Результаты исследований и обсуждение Острая смертельная кровопотеря и последую щая реанимация организма вызывают значитель ные повреждения как кардиомиоцитов, так и вне клеточного матрикса. В частности, во время клинической смерти гистологически выявляется неравномерное кровенаполнение и дистония сосу дов сердца, сочетающаяся с набуханием эндотелия, базальной мембраны и периваскулярным отеком. В кардиомиоцитах отмечаются мелкоочаговые дис трофические изменения в виде единичных фокусов глыбчатого распада, субсегментарных (рис. 1а) и сегментарных контрактур I степени (рис. 1б), осо бенно под эпикардом и эндокардом. Спустя 30 мин после оживления выявляется отчетливое нарастание повреждений сердца. В сосу дах, кроме дистонии, отмечается нарушение крово обращения в форме сладжей, стазов и тромбов и усиление деструктивных изменений их стенок: на бухание и десквамация эндотелия, отечность и раз волокненность базальной мембраны, очаги плазма тического пропитывания. В некоторых сосудах с микротромбами выявляются некрозы их стенок. Параллельно с сосудистыми нарушениями увеличивается и число дистрофически изменен ных кардиомиоцитов, среди которых преобладают контрактурные изменения: субсегментарные кон трактуры и контрактуры I и II степени. В поляри зованном свете клетки с субсегментарными кон трактурами частично или полностью теряют поперечную исчерченность, а участки миофиб рилл, находящиеся в контрактурном состоянии, образуют яркие светящиеся поперечные полосы или множественные анизотропные глыбки. Не прерывность миофибрилл сохраняется. Элек тронномикроскопически в поврежденных клетках по ходу отдельных пучков миофибрилл распола гаются группы саркомеров, находящихся в состо янии гиперконтрактуры. Сарколемма сохраняет двухконтурное строе ние на всем протяжении кардиомиоцита. В сарко плазме выявляется умеренное количество рибо сом и гранул гликогена. Гранулы гликогена располагаются под сарколеммой, вокруг митохон дрий и между миофиламентами. Митохондрии выглядят умеренно набухшими с частично гомо генизированными кристами. Матрикс митохонд рий обычной электронной плотности. Однако встречаются единичные митохондрии с расплыв чатой наружной мембраной и частичной гомоге низацией крист, содержащие аморфные, округлой формы электронноплотные включения — фосфа ты кальция. Миофибриллы имеют различную длину, в участках релаксации миофибрилл Iдис ки разрыхлены и принимают извитую форму. К полутора часам в патоморфологической кар тине сердца продолжают преобладать сосудистые нарушения, которые у одних животных нарастают по тяжести, а у других остаются такими же, как и в предыдущие сроки. У животных с более тяже лыми нарушениями микроциркуляции отмечаются и более тяжелые повреждения кардиомиоцитов в виде контрактур II степени, а в отдельных клет ках — III степени (рис. 1в). Очаговые контрактур ные изменения начинают выявляться уже и в толще миокарда. Увеличивается число кардиомиоцитов, находящихся в состоянии глыбчатого распада. р р р у ф р у Через 5—15 мин после оживления явления сосудистой дистонии увеличиваются. Отчетливо выявляется паретическое расширение сосудов всех калибров при выраженном их полнокровии. Материал и методы исследования Эксперименты проведены на 106 белых беспородных кры сахсамцах массой 240±12 г, наркотизированных нембуталом (25 О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 20 П о с т р е а н и м а ц и о н н а я п а т о л о г и я я лись путем анализа общей структуры кардиомиоцитов, сосудис того русла, внутриклеточных органелл: ядра, митохондрий, лизо сом и саркоплазматического ретикулума [8]. местами содержат истонченные фибриллы. Ядра сохраняют четкую нуклеолемму. Повреждение кардиомиоцитов объясняется и активацией апоптоза (высвобождение митохон дриями цитохрома С, который способствует пре вращению прокаспазы9 в каспазу9, расщепление катазами белков клеточных мембран, деградация ДНК в раннем периоде оживления, контролируе мая протеином р53) [9]. ф ц р ф р ц , р е — лимфоидная инфильтрация сосудистой стенки, периваскулярный склероз, окр. гематоксилином и эозином. Ув. 308. О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 Рис. 1. Морфологические изменения в сердце в постреанимационном периоде. а — субсегментарные контрактуры. Ув. 10 300. б — сегментарные контрактуры I степени, окр. гематоксилином и эозином, съемка в поляризованном свете. Ув. 280. в — контрактуры II степени, окр. гематоксилином и эозином, очаги глыбчатого распада и миоцитолизис, съемка в поляризован ном свете. Ув. 308. г — внутриклеточный отек, миоцитолизис, окр. гематоксилином и эозином. Ув. 336. д — лимфогистиоцитарная инфильтрация, окр. гематоксилином и эозином. Ув. 123. е — лимфоидная инфильтрация сосудистой стенки, периваскулярный склероз, окр. гематоксилином и эозином. Ув. 308. Результаты исследований и обсуждение Наряду с утяжелением расстройств микроцирку ляции прогрессируют дистрофические изменения кардиомиоцитов (рис. 1г). Значительно нарастает количество контрактурно измененных сердечных клеток, очагово располагающихся по всей толще миокарда. Выявляются группы кардиомиоцитов в состоянии глыбчатого распада и миоцитолизиса. тивных изменений кардиомиоцитов, можно выде лить три группы повреждений: легкие (число кар диомиоцитов с контрактурным типом повреждения не превышает 20%), средней тяжести (число кардио миоцитов с контрактурными повреждениями со ставляет от 20% до 30%) и тяжелые (свыше 30%). Животные с тяжелыми повреждениями сердца по гибали, как правило, от сердечной недостаточности. тивных изменений кардиомиоцитов, можно выде лить три группы повреждений: легкие (число кар диомиоцитов с контрактурным типом повреждения не превышает 20%), средней тяжести (число кардио миоцитов с контрактурными повреждениями со ставляет от 20% до 30%) и тяжелые (свыше 30%). Животные с тяжелыми повреждениями сердца по гибали, как правило, от сердечной недостаточности. Принимая во внимание факт, что сосудистые нарушения и повреждения кардиомиоцитов тесно взаимосвязаны, нами для количественной оценки тяжести повреждений в каждой группе был ис пользован способ подсчета «фуксиноррагичес ких» кардиомиоцитов [10], окрашенных по Lie [6]. Оказалось, что в сердцах контрольных животных количество «фуксиноррагических» кардиомиоци тов не превышает 2%, а после клинической смерти их число постепенно возрастает, достигая к 6 ч 30%, а затем уменьшается и через 3 недели не от личается от контроля. р Сарколемма на значительном протяжении становится расплывчатой, а местами полностью деструктурированной, формируются сарколем мальные аркады. В перицеллюлярном простран стве встречаются измененные органеллы. Мито хондрии выглядят набухшими, среди них доминируют измененные, а сохранных митохонд рий остается совсем немного. Более сохранные митохондрии на значительном протяжении теря ют наружную мембрану, появляются деструктури рованные митохондрии (аутофагосомы). Неболь шую группу составляют мелкие митохондрии с относительно сохранными кристами. Вокруг ми тохондрий и между миофиламентами выявляется обилие гранул гликогена и капли липидов. Мио фибриллы выглядят частично пересокращенны ми, встречаются мелкие фокусы лизиса миофила ментов и деструкции Iдисков. Электронномикроскопически к исходу пер вых суток сарколемма в основном сохраняет свою двухконтурность, но нередко встречаются «размы тые» участки наружного слоя. Межклеточные ще ли вставочных дисков выглядят свободными или заполненными хлопьевидным материалом уме ренной электронной плотности. Поперечные сег менты вставочных дисков становятся повышенно извитыми, выявляется незначительный диастаз. В саркоплазме почти полностью отсутствуют гра нулы гликогена, а в очагах лизиса миофиламентов скапливаются рибосомы. Спустя 6 час после оживления участки дест рукции или расплывчатости мембран становятся более обширными. В саркоплазме еще в больших количествах накапливаются гранулы гликогена, преимущественно вокруг митохондрий; здесь же встречаются капли липидов. Результаты исследований и обсуждение Нарастает периваскулярный и межуточный отек. Более отчетливым становится набухание эндоте лия и сосудистой стенки. В отдельных капилля рах наблюдаются сладжи. Повреждения кардио миоцитов по характеру и локализации остаются примерно такими же, как и во время клинической смерти; увеличивается лишь число поврежден ных кардиомиоцитов. На электроннограммах сарколемма выглядит умеренно волнистой с ее локальным разрыхлением и гомогенизацией. В саркоплазме отмечается резкое снижение коли чества гранул гликогена, и они локализуются преимущественно вокруг митохондрий. Количе ство компактных митохондрий с четкими мемб ранами и кристами существенно уменьшается. Преобладают митохондрии с частично размыты ми внешней и внутренней мембранами. Релакса ция отдельных саркомеров сопровождается раз рыхлением и размытостью Iдисков. Адиски Таким образом, структурные изменения в сердце, развивающиеся в течение первых полуто ра часов после реанимации, характеризуются, преж де всего, нарушением микроциркуляции, которое можно рассматривать как первичную реакцию. Вслед за микроциркуляторными нарушениями раз виваются умеренно выраженные дистрофические и деструктивные изменения кардиомиоцитов. В последующие 3—6 часов расстройства мик роциркуляции в миокарде нарастают — выявляют ся сладжи и стазы в капиллярах и венулах. Это со провождается дистрофическими изменениями О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 21 21 Рис. 1. Морфологические изменения в сердце в постреанимационном периоде. а — субсегментарные контрактуры. Ув. 10 300. б — сегментарные контрактуры I степени, окр. гематоксилином и эозином, съемка в поляризованном свете. Ув. 280. в — контрактуры II степени, окр. гематоксилином и эозином, очаги глыбчатого распада и миоцитолизис, съемка в поляризован ном свете. Ув. 308. г — внутриклеточный отек, миоцитолизис, окр. гематоксилином и эозином. Ув. 336. д — лимфогистиоцитарная инфильтрация, окр. гематоксилином и эозином. Ув. 123. е — лимфоидная инфильтрация сосудистой стенки, периваскулярный склероз, окр. гематоксилином и эозином. Ув. 308. Рис. 1. Морфологические изменения в сердце в постреанимационном периоде. О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 22 П о с т р е а н и м а ц и о н н а я п а т о л о г и я я эндотелия, внутренней мембраны, мышечного слоя и адвентиции, что повышает порозность сосудов и обусловливает периваскулярные кровоизлияния. Результаты исследований и обсуждение Вопер вых, лимфоцит секретирует на поверхность клетки перфорин, который открывает трансмембранные каналы, через которые проникает белок гранзим В, активирующий внутриклеточные прокаспазы и таким образом запускающий каспазный каскад. Вовторых, на поверхности многих клеток сердца имеются рецепторы Fas, а лимфоциты имеют ли ганд Fas;FasL. При агрегации Fas и FasL активиру ется цитоплазматический протеин FADD, который способствует переходу прокаспазы8 в каспазу8, а последняя запускает весь каскад протеаз [11, 12]. Через неделю после оживления отмечается дальнейшее развитие репаративных процессов на фоне сохраняющихся участков повреждения. По соотношению процессов репарации и повреж дения можно выделить три варианта патоморфо логических изменений сердца. Попрежнему выявляются измененные мито хондрии. Встречаются большие, до двух саркоме ров длиной, митохондрии с гомогенизированными кристами. Сарколемма приобретает четкие грани цы и двухконтурность. В саркоплазме появляется умеренное количество гранул гликогена. Канальцы Тсистемы выглядят значительно расширенными. Миофибриллы, в основном, сохраняют обычную структуру, в отдельных участках лизиса отмечается накопление гликогена и усиление синтеза миофи ламентов. Этому способствует скопление рибосом и полирибосом в зоне лизиса миофибрилл. Для первого варианта характерны следующие особенности. Вопервых, отмечается уменьшение тяжести сосудистых нарушений, сохраняется дис тония сосудов, наблюдается полнокровие отдель ных вен при пустых артериолах и капиллярах. Снижается проницаемость сосудов, более четкой становится структура сосудистой стенки, умень шается отек, появляются признаки регенерации эндотелия. Строма выглядит более компактной, имеются лишь отдельные очаги, где сохраняется разрыхленность. Вовторых, объем деструктивных изменений значительно уменьшается. Количество контрактурно измененных кардиомиоцитов не превышает 20%. Втретьих, на фоне нормализации микроциркуляции и уменьшения деструктивных изменений кардиомиоцитов выявляются лимфо гистиоцитарная инфильтрация и новообразован ные волокнистые структуры, что является наибо лее характерной и отличительной чертой патоморфологии сердца этой группы животных. Лимфогистиоцитарные инфильтраты обнаружи ваются в виде очагов в строме в зоне поврежден ных кардиомиоцитов и вокруг сосудов. Формиру ющийся склероз имеет очаговый характер. Сравнивая признаки трех вариантов морфо логических изменений сердца, выявленных в кон це первой недели после оживления, следует отме тить, что общим для них является развитие склероза на месте погибших кардиомиоцитов и вокруг сосудов, как проявление репаративной регенерации. В первом варианте этот признак со четается с уменьшением деструктивных измене ний кардиомиоцитов и развитием репарации в ос тавшихся клетках, что можно расценивать как благоприятный вариант течения восстановитель ных процессов в сердце к 7м суткам. Второй вариант характеризуется течением двух противоположных процессов: регенерации и повреждения, выраженных примерно в равной степени. Очаги поврежденных кардиомиоцитов отличаются небольшими размерами и реактивной лимфоидной инфильтрацией, которая в последу ющем сменяется пролиферацией гистиоцитов, а затем склерозом. Результаты исследований и обсуждение Нарастает число крупных набухших митохондрий с полностью го могенизированными кристами, а сохранные обна руживаются крайне редко. Матрикс митохондрий обычной плотности и содержит большое количест во гранулярных электронноплотных включений. Встречаются группы пересокращенных саркоме ров, очаги лизиса миофиламентов и чередование лизиса с пересокращением. Митохондрии выглядят либо набухшими с гомогенизированными кристами и расплывчатой наружной мембраной, либо мелкими с хорошо вы раженными кристами. В области вставочных дис ков на месте бывших митохондрий появляются ре зидуальные тельца и миелиноподобные фигуры. Увеличивается доля сохранных митохондрий, ок руженных мелкими липидными включениями. Ка нальцы Тсистемы саркоплазматического ретику лума выглядят дилятированными и содержат хлопьевидный материал. Встречаются участки пе ресокращения саркомеров и крупные очаги лизиса миофиламентов. В зоне деструкции митохондрий появляются аутофагосомы. К концу первых суток соотношение между сосудистыми нарушениями и изменениями кар диомиоцитов становятся примерно равным. Морфологически выявляется наибольшее число признаков, свидетельствующих о тяжелых по вреждениях сердца: определяется большое число сосудов с тромбами, некрозами сосудистых сте нок и массивными периваскулярными и очаго выми кровоизлияниями. Поврежденные кардио миоциты сливаются в крупные очаги, состоящие из контрактур III степени, глыбчатого распада и миоцитолизиса. Через 3 суток в миокарде встречаются в рав ной степени повреждения как легкой, так и средней тяжести, отмечается уменьшение сосудистых нару шений — они проявляются в основном дистонией и неравномерным кровенаполнением. На этом фоне отчетливо выявляется клеточная реакция стромы в виде лимфоидной инфильтрации (рис. 1д) между «фуксиноррагическими» кардиомиоцитами, очаго вой лимфогистиоцитарной инфильтрацией вокруг сосудов и на месте гибели кардиомиоцитов. Лимфоциты, инфильтрирующие миокард, Вместе с тем, в эти же самые сроки встречают ся животные и с легкими структурными поврежде ниями сердца. На основании этого, с учетом тяжести сосудистых нарушений, дистрофических и деструк Лимфоциты, инфильтрирующие миокард, могут и в эти сроки активировать апоптоз различ О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 23 Для третьего варианта повреждения сердца характерны выраженные сосудистые нарушения в виде полнокровия и стазов в сосудах всех типов, значительный межуточный и периваскулярный отек и деструктивные изменения кардиомиоцитов типа контрактур и глыбчатого распада, площадь которых превышает 30%. Эти изменения очень схожи с теми, которые обнаруживаются у живот ных в течение первых трех суток после оживления, но отличаются от них тем, что сосудистые наруше ния и деструкция кардиомиоцитов формируются на фоне очаговой межуточной лимфогистиоцитар ной инфильтрации и развивающегося периваску лярного склероза (рис. 1е). ных клеток сердца несколькими путями. О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 Результаты исследований и обсуждение Таким образом, второй вариант структурных изменений сердца отражает такой тип течения восстановительного периода, для ко торого характерен медленно протекающий про цесс повреждения кардиомиоцитов и замещения их соединительной тканью, что со временем мо жет привести к развитию массивного склероза и послужить основой сердечной недостаточности. Для второго варианта наиболее характерны ми структурными особенностями являются лим фоидная инфильтрация стенок отдельных сосудов и околососудистых пространств (как начальные проявления васкулита) и значительный объем де структивных изменений кардиомиоцитов типа контрактур (от 25% до 30%) и глыбчатого распада, которые выявляются чаще всего в субэндокарди альных зонах. Поврежденные кардиомиоциты ок ружают скопления лимфоцитов. Эти отличитель ные для второго варианта морфологические признаки выявляются на фоне маловыраженных сосудистых нарушений и формирующегося очаго вого периваскулярного склероза. Артериолы и ка пилляры выглядят пустыми, а отек в строме и во круг сосудов носит умеренный характер. Третий вариант морфологии сердца отражает такое течение восстановительного периода, при котором сохраняются расстройства микроге моциркуляции, кардиомиоциты повреждаются большими группами, объем репаративных изме О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2 24 П о с т р е а н и м а ц и о н н а я п а т о л о г и я я нений уступает повреждениям и развивается сер дечная недостаточность. нений уступает повреждениям и развивается сер дечная недостаточность. ление вызывают структурные повреждения серд ца как в ближайшем, так и в отдаленном восста новительном периоде. В первые трое суток отме чается сочетание сосудистых нарушений, дистрофических и деструктивных изменений кардиомиоцитов, причем ведущую роль играют расстройства микроциркуляции: сладж, стаз, тромбоз, повышение сосудистой проницаемости, периваскулярный отек и кровоизлияния. Дест рукция кардиомиоцитов: контрактуры различной степени, глыбчатый распад, миоцитолизис, явля ются основой постреанимационной сердечной не достаточности. ление вызывают структурные повреждения серд ца как в ближайшем, так и в отдаленном восста новительном периоде. В первые трое суток отме чается сочетание сосудистых нарушений, дистрофических и деструктивных изменений кардиомиоцитов, причем ведущую роль играют расстройства микроциркуляции: сладж, стаз, тромбоз, повышение сосудистой проницаемости, периваскулярный отек и кровоизлияния. Дест рукция кардиомиоцитов: контрактуры различной степени, глыбчатый распад, миоцитолизис, явля ются основой постреанимационной сердечной не достаточности. Через две недели сарколемма постепенно ут рачивает складчатость. Базальная мембрана мес тами становится вакуолизированной; на этом же уровне разрыхляется и деструктурируется плаз матическая мембрана. Митохондрии отличаются разнообразием формы и величины: встречаются как мелкие, так и гигантские с частично или пол ностью гомогенизированными кристами. Заключение Таким образом, клиническая смерть, вызван ная острой кровопотерей, и последующее ожив Результаты исследований и обсуждение В очагах лизиса миофиламентов накапливаются рибосомы, увеличивается количество лизосом, особенно в зо не деструкции митохондрий. В некоторых случаях при наличии выражен ных признаков репарации выявляются свежие, дополнительно возникающие повреждения кар диомиоцитов. Структурный анализ позволяет предполагать перманентный характер таких мел коочаговых повреждений миокарда с последую щим развитием склероза, что в конечном итоге должно сказаться неблагоприятно на функции сердца в отдаленные сроки после перенесенной смертельной кровопотери. Морфология таких по врежденных кардиомиоцитов стереотипна и ана логична повреждениям, которые определяются в остром периоде после реанимации. Спустя 3 недели в сердечной мышце выявля ется слабо выраженный очаговый и периваскуляр ный склероз, неравномерное кровенаполнение со судов, незначительный очаговый отек стромы. На этом фоне отчетливо выявляются очаги «фукси норрагических» кардиомиоцитов, часть из которых отличается особенно интенсивной окраской. Рядом с ними имеются кардиомиоциты с явлениями глыб чатого распада, окруженные лимфоцитами. Эти структурные изменения укладываются в морфоло гическую картину выделенного нами второго типа изменений сердца в постреанимационном периоде. К концу месяца у всех животных морфологи ческие изменения в сердце выглядят однотипно и занимают как бы промежуточное положение между первым и вторым вариантом постреанима ционных изменений сердца. В значительной сте пени нормализуется ультраструктура сердечной мышцы. В саркоплазме увеличивается содержа ние гликогена. Митохондрии располагаются в ви де многорядных скоплений (до 2—4 рядов) между миофибриллами; большинство из них сохранны, лишь в некоторых выявляется гомогенизация крист и встречаются остаточные тельца. Происхо дит постепенное восстановление канальцев Тсис темы. Миофибриллы лишь в отдельных участках выглядят слегка релаксированными. Анализ результатов динамического исследо вания структуры миокарда на протяжении одного месяца после острой смертельной кровопотери позволяет утверждать, что сердце не столько по вреждается во время клинической смерти, сколь ко в первые часы после оживления за счет воздей ствия на него целого ряда патогенных факторов, отсутствовавших или слабо выраженных при уми рании и появляющихся или нарастающих только с началом рециркуляции и реоксигенации орга низма. К их числу следует отнести значительное усиление адренергических влияний на сердце, чрезмерную активацию процессов перекисного окисления липидов с развитием признаков мемб ранодеструкции, длительное ингибирование мем бранных ионных насосов, иммунные нарушения и развитие гиперчувствительности замедленного типа на первичные и вторичные кардиальные ау тоантигены [3]. 8. Пауков В. С., Фролов В. А. Элементы теории патологии сердца. М.: Медицина; 1982. 9. Dixon S., Soriano B. J., Lush R. M. et al. Apoptosis: Its role in the devel opment malignancies and its potential as a novel therapeutic target // Ann. Pharmacother. 1997; 31 (1): 76—82. 8. Пауков В. С., Фролов В. А. Элементы теории патологии сердца. М.: Медицина; 1982. 9. Dixon S., Soriano B. J., Lush R. M. et al. Apoptosis: Its role in the devel opment malignancies and its potential as a novel therapeutic target // Ann. Pharmacother. 1997; 31 (1): 76—82. 10. Меерсон Ф. З. Патогенез и предупреждение стрессорных и ишеми ческих повреждений сердца. М.: Медицина; 1984. 11. Rupp H., Maisch B. Control of apoptosis of cardiovascular fibroblast: A novel drug targer. Herz. 1999; 24 (3): 225—231. 12. Schaper B., Elsasser A., Kostin S. The role of cell death in heart failure Circ. Res. 1999; 85: 867—869. 12. Schaper B., Elsasser A., Kostin S. The role of cell death in heart failure Circ. Res. 1999; 85: 867—869. 1. Неговский В. А., Мороз В. В. Теоретические и клинические проблемы реаниматологии. Анестезиология и реаниматология. 2000; № 6: 4—6. 2. Неговский В. А. Очерки по реаниматологии. М.: Медицина; 1986. 3. Долгих В. Т. Повреждение и защита сердца при острой смертельной кровопотере. Омск; 2002. 4. Долгих В. Т., Мордык А. В., Баранец Н. А. Нарушение сократимости сердца после клинической смерти, вызванной острой кровопотерей. Анестезиология и реаниматология. 1996; 5: 42—45. 5. Пирс Э. Гистохимия теоретическая и прикладная / Под ред. В. В. Португалова; Пер с англ. М.: Иниздат; 1962. 6. Lie J. T., Holley K. E, Kampa W. R., Titus J. L. New hystochemical method for morphologic diagnosis of early stages of myocardial ischemia // Mayo. Clin. Proc. 1971; 46 (5): 319—327. 7. Уикли Б. Электронная микроскопия для начинающих / Пер. с англ. М.: Мир; 1975. 5. Пирс Э. Гистохимия теоретическая и прикладная / Под ред. В. В. Португалова; Пер с англ. М.: Иниздат; 1962. 11. Rupp H., Maisch B. Control of apoptosis of cardiovascular fibroblast: A novel drug targer. Herz. 1999; 24 (3): 225—231. 6. Lie J. T., Holley K. E, Kampa W. R., Titus J. L. New hystochemical method for morphologic diagnosis of early stages of myocardial ischemia // Mayo. Clin. Proc. 1971; 46 (5): 319—327. 10. Меерсон Ф. З. Патогенез и предупреждение стрессорных и ишеми ческих повреждений сердца. М.: Медицина; 1984. 8. Пауков В. С., Фролов В. А. Элементы теории патологии сердца. М.: Медицина; 1982. 2. Неговский В. А. Очерки по реаниматологии. М.: Медицина; 1986. 4. Долгих В. Т., Мордык А. В., Баранец Н. А. Нарушение сократимости сердца после клинической смерти, вызванной острой кровопотерей. Анестезиология и реаниматология. 1996; 5: 42—45. 3. Долгих В. Т. Повреждение и защита сердца при острой смертельной кровопотере. Омск; 2002. Литература 1. Неговский В. А., Мороз В. В. Теоретические и клинические проблемы реаниматологии. Анестезиология и реаниматология. 2000; № 6: 4—6. 2. Неговский В. А. Очерки по реаниматологии. М.: Медицина; 1986. 3. Долгих В. Т. Повреждение и защита сердца при острой смертельной кровопотере. Омск; 2002. 3. Долгих В. Т. Повреждение и защита сердца при острой смертельной кровопотере. Омск; 2002. 4. Долгих В. Т., Мордык А. В., Баранец Н. А. Нарушение сократимости сердца после клинической смерти, вызванной острой кровопотерей. Анестезиология и реаниматология. 1996; 5: 42—45. 7. Уикли Б. Электронная микроскопия для начинающих / Пер. с англ. М.: Мир; 1975. Поступила 25.10.04 Поступила 25.10.04 25 25 О Б Щ А Я Р Е А Н И М А Т О Л О Г И Я , 2 0 0 5 , I ; 2
https://openalex.org/W2169118380	https://nottingham-repository.worktribe.com/file/722231/1/Quiet%20Americans%20in%20India%20.pdf	English	null	"Quiet Americans in India": The CIA and the Politics of Intelligence in Cold War South Asia	Diplomatic history	2,014	public-domain	21,951	1 The phrase ‘Quiet Americans in India’ is taken from an article on CIA activity in India published by the Soviet journalist, A. Shalnev, in the Moscow daily Sovetskaya Rossiya [Soviet Russia], 3 November 1972. The term, ‘Quiet American’, became synonymous with CIA officers following the publication of Graham Greene’s novel, The Quiet American (London: William Heinemann Ltd, 1955), a semi-fictional account of the tragically misguided intrusion into the First Indochina War of an American intelligence officer, Alden Pyle. 1 The phrase ‘Quiet Americans in India’ is taken from an article on CIA activity in India published by the Soviet journalist, A. Shalnev, in the Moscow daily Sovetskaya Rossiya [Soviet Russia], 3 November 1972. The term, ‘Quiet American’, became synonymous with CIA officers following the publication of Graham Greene’s novel, The Quiet American (London: William Heinemann Ltd 1955) a semi fictional account of the tragically 1 The phrase ‘Quiet Americans in India’ is taken from an article on CIA activity in India published by th journalist, A. Shalnev, in the Moscow daily Sovetskaya Rossiya [Soviet Russia], 3 November 1972. T ‘Quiet American’, became synonymous with CIA officers following the publication of Graham Greene The Quiet American (London: William Heinemann Ltd, 1955), a semi-fictional account of the t misguided intrusion into the First Indochina War of an American intelligence officer, Alden Pyle. 1 The phrase ‘Quiet Americans in India’ is taken from an article on CIA activity in India published by the Soviet journalist A Shalnev in the Moscow daily Sovetskaya Rossiya [Soviet Russia] 3 November 1972 The term ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia Chester Bowles, American 1 ambassador in New Delhi at the time, lamented that fallout from the Ramparts furore was likely to prove particularly damaging to United States’ standing in India, ‘where we [the US] had developed especially close and extensive relationships with Indian universities and with individual scholars, none of which were in any way connected with intelligence operations.’2 The public relations challenge Bowles faced in India as a result of the CIA’s activities was complicated the following month when The Washington Post published an essay written by his predecessor in New Delhi, the Harvard economist, John Kenneth Galbraith. With no little irony, Galbraith observed that recent events had confirmed the CIA to be, ‘a secret agency...with an excellent instinct for headlines.’ More significantly, Galbraith went on to recount his experiences of working alongside the CIA in the subcontinent, noting in the process, that the Agency’s, ‘activities were generally known to, and involved no conflict with, local [Indian government] authorities.’3 In March 1961, before leaving to take up his ambassadorial posting, Galbraith had taken exception to the scale and scope of the CIA’s interference in India’s internal affairs. During a briefing provided by Richard M. Bissell Jr., the Agency’s Deputy Director of Plans, or clandestine operations, Galbraith was ‘appalled and depressed’ to learn of the CIA’s intention to spend a sum ‘well into the millions [of dollars]’ to bankroll the election campaigns of pro-Western politicians, and subsidize anti- communist Indian newspapers and magazines. Such activity, Galbraith lamented, was unlikely to prove effective in swaying Indian opinion, but was almost certain to leak into the public domain, damaging Indo-US relations and compromising his position as ambassador. Emboldened by the CIA’s public humiliation following its disastrous Bay of Pigs operation against the Castro regime in Cuba that April, Galbraith attempted to rein in the Agency’s activities in India. Although only partially successful, the ambassador’s resolve to limit The public relations challenge Bowles faced in India as a result of the CIA’s activities was complicated the following month when The Washington Post published an essay written by his predecessor in New Delhi, the Harvard economist, John Kenneth Galbraith. 544. CIA Needs a Tug on Its purse Strings’, John Kenneth Galbraith, The Washington Post, 12 March 1967. 2 Chester Bowles, Promises to Keep: My Years in Public Life, 1941-1969 (New York: Harper and Row, 1971), p 544 2 Chester Bowles, Promises to Keep: My Years in Public Life, 1941-1969 (New York: Harper and Row, 1971), p. 544. 3 ‘CIA Needs a Tug on Its purse Strings’, John Kenneth Galbraith, The Washington Post, 12 March 1967. ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia How is it that the poet got no applause when he recited his poem on the stage? How is it that the other poet’s rhyme was not published in any paper? Why is so much tension in husband and wife? Why are the prices rising? Why the pupils gherao [lock-in] their principal? Why the parents are afraid of their adolescent daughters? Why there is so much nudity in films? And why our development plans fail to make any progress? The root cause of all these is just one. This is all due to the secret operations of the C.I.A. How is it that the poet got no applause when he recited his poem on the How is it that the other poet’s rhyme was not published in any paper? Why is so much tension in husband and wife? Why are the prices rising? Why the pupils gherao [lock-in] their principal? Why the parents are afraid of their adolescent daughters? Why there is so much nudity in films? And why our development plans fail to make any progress? The root cause of all these is just one. This is all due to the secret operations of the C.I.A. Dinkar Sonwalkar, ‘The Root Cause’, Dharm Yug [Hindi Literary Magazine], November 1972. In February 1967, senior officials from the Central Intelligence Agency (CIA) were horrified when the American west-coast magazine, Ramparts, exposed the US intelligence organisation’s longstanding financial relationships with a number of international educational and cultural bodies. In a series of damning articles, reproduced in The New York Times and The Washington Post, Ramparts documented the CIA’s provision of covert funding to, amongst others, the National Students Association, Asia Foundation, and Congress for Cultural Freedom (CCF). In India, where the fourth general election campaign to be held since the end of British imperial rule in August 1947 was then in full swing, confirmation that the Indian Committee for Cultural Freedom, a local offshoot of the CCF, had accepted money from the CIA provoked an outpouring of public indignation. p. 544. ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia With no little irony, Galbraith observed that recent events had confirmed the CIA to be, ‘a secret agency...with an excellent instinct for headlines.’ More significantly, Galbraith went on to recount his experiences of working alongside the CIA in the subcontinent, noting in the process, that the Agency’s, ‘activities were generally known to, and involved no conflict with, local [Indian government] authorities.’3 In March 1961, before leaving to take up his ambassadorial posting, Galbraith had taken exception to the scale and scope of the CIA’s interference in India’s internal affairs. During a briefing provided by Richard M. Bissell Jr., the Agency’s Deputy Director of Plans, or clandestine operations, Galbraith was ‘appalled and depressed’ to learn of the CIA’s intention to spend a sum ‘well into the millions [of dollars]’ to bankroll the election campaigns of pro-Western politicians, and subsidize anti- communist Indian newspapers and magazines. Such activity, Galbraith lamented, was unlikely to prove effective in swaying Indian opinion, but was almost certain to leak into the public domain, damaging Indo-US relations and compromising his position as ambassador. Emboldened by the CIA’s public humiliation following its disastrous Bay of Pigs operation against the Castro regime in Cuba that April, Galbraith attempted to rein in the Agency’s activities in India. Although only partially successful, the ambassador’s resolve to limit 2 covert American intelligence operations in the subcontinent earned the disapprobation of the Agency, which dismissed him as, ‘basically anti-CIA.’4 Back in 1967, opposition groups on the left of India’s political spectrum seized upon Galbraith’s comments in The Washington Post as confirmation that the CIA had been actively subverting democracy in South Asia. Exasperated by Galbraith’s indiscretion, the CIA’s Director, Richard Helms, curtly informed the former ambassador that he had, ‘raised unshirted hell in India and [had]...provided the central point of an acrimonious debate in the Lok Sabha [India’s lower parliamentary chamber].’5 Fresh from the campaign hustings, India’s parliamentarians fed off rumour and suspicion surrounding America’s foreign intelligence service, and competed eagerly with each other to exhibit the toughest and most populist anti-CIA line possible. On 23 March, India’s Foreign Minister, M. C. Chagla, bowed to mounting pressure for government action and announced that a ‘thorough’ official inquiry would be conducted to ascertain whether the CIA had interfered in Indian politics. y, , ( ) 6 ‘India to Conduct Inquiry on C.I.A.’, The New York Times, 24 March 1967. 4 John Kenneth Galbraith A Life in our Times: Memoirs (London: Andre Deutsch, 1981), pp. 394-7. Harry Rositzke, the CIA Station chief in New Delhi during Galbraith’s tenure as ambassador, later confirmed that much of the Agency’s political activity in India, and use of the subcontinent as a support base for operations mounted in Chinese controlled Tibet, continued through the 1960s. Richard Parker, John Kenneth Galbraith: His Life, His Politics, His Economics (New York: Farrar, Straus and Giroux, 2005), pp. 354-55. See also, Galbraith to Averell Harriman George Ball and George McGhee 30 November 1961 Box 463 John Kenneth Seasons: My Life in the CIA (New York: Scribner, 1997), p. 84. 5 Richard Helms to Lyndon Johnson, 28 March 1967, Box 9, Folder CIA Vol. 3 [1 of 2], NSF, AF, Lyndon Johnson Library, Austin, Texas (hereafter LBJL). 4 John Kenneth Galbraith A Life in our Times: Memoirs (London: Andre Deutsch, 1981), pp. 394-7. Harry Rositzke, the CIA Station chief in New Delhi during Galbraith’s tenure as ambassador, later confirmed that much of the Agency’s political activity in India, and use of the subcontinent as a support base for operations mounted in Chinese controlled Tibet, continued through the 1960s. Richard Parker, John Kenneth Galbraith: His Life, His Politics, His Economics (New York: Farrar, Straus and Giroux, 2005), pp. 354-55. See also, Galbraith to Averell Harriman, George Ball, and George McGhee, 30 November 1961, Box 463, John Kenneth Galbraith, Folder 2, Averell W. Harriman Papers, Library of Congress, Manuscripts Division, Washington DC.; Diary entry for 29 March 1961, Washington, John Kenneth Galbraith, Ambassador’s Journal: A Personal A f h d ( h iffli 1969) 1 d Cl id A S f All p y g p g Diary entry for 29 March 1961, Washington, John Kenneth Galbraith, Ambassador’s Journal: A Personal Account of the Kennedy Years, (Boston: Houghton Mifflin, 1969), p. 51; and, Duane R. Clarridge, A Spy for All Seasons: My Life in the CIA (New York: Scribner, 1997), p. 84. albraith to Averell Harriman, George Ball, and George McGhee, 30 November 1961, Box 463, John Ke albraith, Folder 2, Averell W. Harriman Papers, Library of Congress, Manuscripts Division, Washington y , , Johnson Library, Austin, Texas (hereafter LBJL). Intelligence Activities, Washington D. C. INTERNET, http://www.aarclibrary.org/publib/church/reports/vol2/pdf/ChurchV2_3_Brennan.pdf 8 8 Russell Jack Smith, The Unknown CIA: My Three Decades with the Agency (New York: Berkley Books, 1992), p. 13. 7 See Frank Church, 25 September 1975, Select Committee to Study Governmental Operations with Respect to Intelligence Activities, Washington D. C. INTERNET, Intelligence Activities, Washington D. C. INTERNET, http://www.aarclibrary.org/publib/church/reports/vol2/pdf/ChurchV2_3_Brennan.pdf 8 y g p p p _ _ p l Jack Smith, The Unknown CIA: My Three Decades with the Agency (New York: Berkley Books, 13. y, g ( ) 11 George W. Bush to R.N. Kao, 19 June 1984, Country File India 1984 [1] [OA-ID 19779], GWBL. 12 L. Natarajan, American Shadow over India (Bombay: People’s Publishing House, 1952). 10 ‘US Congressional Perspectives of India’, Peter Tomsen, Twenty-Sixth Session, 1983-84, United States Department of State, Foreign Service Institute, Country File India 1985 [3] [OA-ID 19797], George W. Bush Library, College Station Texas (hereafter GWBL). 11 ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia ‘We cannot permit foreigners or foreign governments to dictate to us what sort of a government we should have or what sort of people should be elected,’ Chagla asserted. ‘We will unearth any activity that is objectionable, that is against the national interests.’6 The global spotlight that America’s press cast upon some of the CIA’s more questionable activities in early 1967, was to have a profound and enduring impact upon Indian perceptions of America and its intelligence services. In the wake of the Ramparts 3 scandal, the CIA came to occupy a prominent place in mainstream Indo-US cultural and political discourse. Indeed, for the remainder of the twentieth century, and beyond, anti- American elements inside and outside India drew repeatedly upon the spectre of CIA subversion as a means of destabilising New Delhi’s relations with Washington. The blanket exposure given by the world’s press to CIA indiscretions after 1967, exemplified by the international media circus’ that developed around Congressional probes into the US intelligence community in the mid-1970s, made a deep psychological impression in India. Having publicly catalogued the CIA’s involvement in a series of plots to assassinate national leaders and subvert foreign governments, the chairman of one influential investigative committee, the Democrat Senator from Idaho, Frank Church, famously characterised the Agency’s behaviour as akin to, ‘a rogue elephant on a rampage.’7 Moreover, in India’s Prime Minister, Indira Gandhi, whose Congress Party saw its parliamentary majority slashed at the polls in 1967, India was governed for much of the next two decades by a leader with a visceral mistrust of Western intelligence services in general, and the CIA in particular. One former CIA officer, who served in India in the early 1970s, recalled that, ‘CIA agents...were to be found according to Madame Gandhi, beneath every charpoy and behind every neem tree.’8 Remarkably, some reference to the CIA and its purportedly nefarious activities in the subcontinent can be detected in almost every significant exchange that occurred between Indian and American diplomats between the late 1960s and the late 1980s. 9 Richard J. Aldrich, ‘“The Value of Residual Empire”: Anglo-American Intelligence Co-operation in Asia after 1945’ in Intelligence, Defence and Diplomacy: British policy in the Post-war World, (eds.) Richard J. Aldrich and Michael F. Hopkins (London: Frank Cass, 1994), p. 246. 10 11 George W. Bush to R.N. Kao, 19 June 1984, Country File India 1984 [1] [OA-ID 19779], GWB 12 L. Natarajan, American Shadow over India (Bombay: People’s Publishing House, 1952). 1945 in Intelligence, Defence and Diplomacy: British policy in the Post war World, (eds.) Richard J and Michael F. Hopkins (London: Frank Cass, 1994), p. 246. 10 13 John D. Smith, I Was a CIA Agent in India (New Delhi: New Age Printing Press, 1967); Pauly V. Parakal, CIA Dagger against India (New Delhi: New Age Press, 1973); Kunhanandan Nair, Devil and His Dart: How the CIA is Plotting in the Third World (New Delhi: Sterling Publishers, 1986); and, Pandit Sheel Bhadra Yajee, CIA: Manipulating Arm of U.S. Foreign Policy: 40 Years of CIA Manoeuvres against Freedom and Dignity (New Delhi: Criterion Publications, 1987). See also, Satish Kumar, CIA and the Third World: A Study in Crypto-Diplomacy (New Delhi: Vikas Publishing House, 1981); and, Rustem Galiullin, The CIA in Asia: Covert Operations against India and Afghanistan, (Moscow: Progress Publishers, 1988). Leaping on to the CIA bandwagon, L. Natarajan produced a flurry of further books on the Agency following the Ramparts revelations. See, L. Natarajan, Assassination Bureau, Inc. (New Delhi: Perspective Publications, 1967); and, America’s Two Pincers (Lucknow: Pradip Prakashan, 1970). 16 Little light is shed on the CIA’s activities in India, for instance, in works such as, Victor Marchetti and John D. Marks, The CIA and the Cult of Intelligence (London: Jonathan Cape, 1974); John Ranelagh, The Agency: The Rise and Decline of the CIA (London: Weidenfeld & Nicolson, 1986); Rhodri Jeffreys-Jones, The CIA and American Democracy (London: Yale University Press, 1998); John Prados, Safe for Democracy: The Secret Wars of the CIA, (New Amsterdam Books 2006); or, Tim Weiner, Legacy of Ashes: The History of the CIA, (London: Allen Lane, 2007). 15 Naipaul recounts sardonically how, during his travels across India between February 1962 and February 1964, he, ‘had grown tired of meeting young Americans in unlikely places. It was amusing, and charitable, to think that some of them were spies for the CIA or whatever it was.’ V. S. Naipaul, An Area of Darkness: A Discovery of India (London: Andre Deutsch, 1964), p. 172. ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia In fact, during the latter half of the Cold War, what some commentators have referenced as an Indian national ‘paranoia’ toward the CIA and its clandestine activities, came to represent a constant and frustrating impediment whenever US policymakers sought to forge closer and more 4 constructive relations with India.9 In the mid-1980s, senior officials in the Reagan administration continued to bemoan that, ‘Mrs. Gandhi’s periodic swipes at the U.S. for interference in Indian internal affairs infuriate Congressmen and staffers alike.’10 In June 1984, having recently returned from a visit to the subcontinent, US Vice-President, and former CIA Director, George W. Bush, felt compelled to write a letter of complaint to R. N. Kao, the head of India’s external intelligence service, the Research and Analysis Wing (RAW). Bush had taken exception to comments made by Indian government officials that linked the CIA with a secessionist movement in the Punjab, in northern India. In a private letter to Kao, an exasperated Bush emphasised: ...how taken aback I was to hear of recent Indian accusations concerning alleged CIA involvement in the Punjab. Media articles and statements by government officials linking CIA operations with occurrences in Amritsar are completely contrary to the fact and quite distressing. This turn of events is particularly unfortunate coming so closely on the heels of my statement in New Delhi respecting the unity and integrity of India and my long discussions with your Prime Minister.11 Some three decades earlier, one of the very first books to reference the CIA, L. Natarajan’s, American Shadow over India, had been published in Bombay. The account that Natarajan provided of the CIA’s activities in India in 1952, which, if the author is to be believed, encompassed bribing Indian publishers, journalists and politicians to follow a pro- American line, generated little public or political comment in the subcontinent.12 By the end of the following decade, however, Indian authors and journalists had turned the chronicling of CIA misdeeds in South Asia, real and imagined, into something of a cottage industry. In 5 the late 1960s, communist publishing houses in India churned out evocatively entitled works such as, I Was a CIA Agent in India; CIA: Manipulating Arm of U.S. ( p ) 14 ‘Conspiracy Theories’, Hindustan Times, 1 October 2005, ‘India talks of CIA role in Unrest’, The New York Times, 16 June 1984; ‘India's Search for Villain Finds Old Culprit: The CIA’, The Washington Post, 14 June 1991. 15 ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia Foreign Policy; Devil and his Dart: How the CIA is Plotting in the Third World; and, CIA: Dagger against India.13 The ubiquitous hand of the CIA had, such works proclaimed, been behind plots to oust India’s first Prime Minister, Jawaharlal Nehru, from power; assassinate Indira Gandhi, and her son Rajiv; encourage separatist movements in the north, west and south of India; and perhaps most bizarrely, implement a evangelisation plan to covert India’s overwhelming Hindu population to Christianity.14 References to the CIA also began to cross over into conventional Indian literature. Notably, in 1964, the Agency appeared briefly in V. S. Naipaul’s classic work, An Area of Darkness: A Discovery of India.15 In contrast, a comprehensive account of the CIA’s cold war operations in India and, more precisely, the Agency’s wider impact on Indo-US relations remains a notable lacuna within the considerable body of scholarly work addressing America’s intelligence community.16 The memoirs of CIA Directors and former Agency officials largely omit reference to India, or the late 1960s, communist publishing houses in India churned out evocatively entitled works such as, I Was a CIA Agent in India; CIA: Manipulating Arm of U.S. Foreign Policy; Devil and his Dart: How the CIA is Plotting in the Third World; and, CIA: Dagger against India.13 The ubiquitous hand of the CIA had, such works proclaimed, been behind plots to oust India’s first Prime Minister, Jawaharlal Nehru, from power; assassinate Indira Gandhi, and her son Rajiv; encourage separatist movements in the north, west and south of India; and perhaps most bizarrely, implement a evangelisation plan to covert India’s overwhelming Hindu population to Christianity.14 References to the CIA also began to cross over into conventional Indian literature. Notably, in 1964, the Agency appeared briefly in V. S. 17 See, for example, Richard Helms, A Look Over My Shoulder: A Life in the Central Intelligence Agency (New York: Random House, 2003); George Tenet, At the Center of the Storm: My Years at the CIA, (London: Harper Press, 2007); Harry Rositzke, The CIA’s Secret Operations: Espionage, Counterespionage and Covert Action (Boulder, Co.: Westview Press, 1988); J.B. Smith, Portrait of a Cold Warrior (New York: Scribner’s, 1976). A significant exception is Clarridge’s, A Spy for All Seasons, which details aspects of Clarridge’s work as a CIA field officer in New Delhi and Madras in the early 1960s. 19 See, in particular, C. A. Bayly, Empire and Information: Intelligence Gathering and Social communication in India, 1780-1870 (Cambridge: Cambridge University Press, 1996); R. J. Popplewell, Intelligence and Imperial Defence (London: Routledge, 1995); and, Milton Israel, Communications and Power: Propaganda and the Press in the Indian Nationalist Struggle, 1920-47 (Cambridge: Cambridge University Press, 1994). The concept of ‘imagined communities’ was popularised by Benedict Anderson, Imagined Communities: Reflections of the Origins and Spread of Nationalism (London: Verso, 1983). ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia Naipaul’s classic work, An Area of Darkness: A Discovery of India.15 In contrast, a comprehensive account of the CIA’s cold war operations in India and, more precisely, the Agency’s wider impact on Indo-US relations remains a notable lacuna within the considerable body of scholarly work addressing America’s intelligence community.16 The memoirs of CIA Directors and former Agency officials largely omit reference to India, or 6 skim over intelligence operations in the subcontinent.17 Likewise, accounts of ambassadorial tours in the subcontinent, penned by such luminaries as Galbraith, Bowles, and Daniel Patrick Moynihan, offer only tantalisingly brief glimpses of the scale, scope and broader significance of CIA activity inside ‘the world’s largest democracy’.18 India’s historic association with the development of modern intelligence practice is, in fact, particularly strong. Scholars of the subcontinent have documented the extensive networks of indigenous spies, political informants and propagandists that were co-opted by the East India Company from the eighteenth century to safeguard British interests in South Asia from internal revolt and external threats, originally from France, and latterly from imperial Russia. By the time the British retreated from the subcontinent in the aftermath of the Second World War, the manipulation by colonial security agencies of a deep and flexible system of social communication in India had contributed to a proliferation of amorphous ‘imagined’ communities with purportedly sinister and subversive anti-nationalist agendas.19 India’s historic association with the development of modern intelligence practice is, in fact, particularly strong. Scholars of the subcontinent have documented the extensive networks of indigenous spies, political informants and propagandists that were co-opted by the East India Company from the eighteenth century to safeguard British interests in South Asia from internal revolt and external threats, originally from France, and latterly from imperial Russia. By the time the British retreated from the subcontinent in the aftermath of the Second World War, the manipulation by colonial security agencies of a deep and flexible system of social communication in India had contributed to a proliferation of amorphous ‘imagined’ communities with purportedly sinister and subversive anti-nationalist agendas.19 In this context, the deployment of preponderant post-war American power and wealth in Europe and Asia appeared redolent to many Indians of a hidden neo-imperialist US foreign policy. y 18 Galbraith, Ambassador’s Journal, and Life in Our Times; Bowles, Promises to Keep; Daniel Patrick Moynihan, A Dangerous Place (Boston: Little Brown, 1978). 20 For insights into the political dimensions of conspiracy theories, see, Michael Barkun, A Culture of Conspiracy: Apocalyptic Visions in Contemporary America (Berkeley: University of California Press, 2003); Mark Fenster, Conspiracy Theories: Secrecy and Power in American Culture (Minneapolis: University of Minnesota Press, 2008); Daniel Pipes (1997). Conspiracy: How the Paranoid Style Flourishes and Where It Comes from (New York: The Free Press, 1997); and, Kathryn S. Olmsted, Real Enemies: Conspiracy Theories and American Democracy, World War I to 9/11 (New York: Oxford University Press, 2009.) y ( y , ) e, for example, Daniel Pipes, The Hidden Hand: Middle East Fears of Conspiracy (New York: S n's Press, 1998). Comes from (New York: The Free Press, 1997); and, Kathryn S. Olmsted, Real Enemies: Conspiracy Theories and American Democracy, World War I to 9/11 (New York: Oxford University Press, 2009.) 20 For insights into the political dimensions of conspiracy theories, see, Michael Barkun, A C Minnesota Press, 2008); Daniel Pipes (1997). Conspiracy: How the Paranoid Style Flourishes and Wh omes from (New York: The Free Press, 1997); and, Kathryn S. Olmsted, Real Enemies: Conspiracy Th nd American Democracy, World War I to 9/11 (New York: Oxford University Press, 2009.) Conspiracy: Apocalyptic Visions in Contemporary America (Berkeley: University of California Pres Mark Fenster, Conspiracy Theories: Secrecy and Power in American Culture (Minneapolis: Univ p Martin's Press, 1998). ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia More specifically, the CIA, as the ‘covert’ foreign policy tool of choice for US President’s in the early Cold War period, acquired a uniquely invidious status across the In this context, the deployment of preponderant post-war American power and wealth in Europe and Asia appeared redolent to many Indians of a hidden neo-imperialist US foreign policy. More specifically, the CIA, as the ‘covert’ foreign policy tool of choice for US President’s in the early Cold War period, acquired a uniquely invidious status across the developing world as an anti-democratic socio-political malefactor. Indeed, in India, as elsewhere in the global South, from the late 1960s the symbolism attached to the CIA 7 overshadowed American diplomatic initiatives designed to win ‘hearts and minds’. Trust and confidence in US domestic institutions and amongst America’s international partners, and perhaps none more so than India, was corroded by a climate of conspiracy surrounding the CIA, which, in turn, fostered a political culture in the subcontinent that at times verged on paranoia. By drawing upon theoretical frameworks utilized in recent studies of conspiracy theory, important new light can be shed on the political and cultural influences that have conditioned Indian perceptions of the CIA. Specifically, the manner in which the CIA’s notoriety in the subcontinent after 1967 was harnessed by left-wing sections of India’s political class to nurture populist anti-American sentiment, is suggestive of a systematic effort, whether conscious or subconscious, to associate the Agency with a broad conspiracy, centred on a plot to secure control of India, and the wider developing world.20 Here, parallels are evident with work conducted on the political dimension of conspiracy theories in the Middle East. In this context, scholars have demonstrated how notions of ‘conspiracism’ gained currency in mainstream political and media circles, and were subsequently exploited by authoritarian regimes to attribute poverty, economic decline and social stagnation to the imaginary construction of hostile external forces.21 Furthermore, in interpreting Indira Gandhi’s interaction with Western intelligence agencies in general, and the CIA in particular, emphasis is placed on the extent to which Gandhi’s family history, psychological make-up, and familiarity with the Agency’s covert action record in the developing world, amplified traits of insecurity and paranoia buried deep within the Indian premier’s psyche. 22 Richard Hofstadter, ‘The Paranoid Style in American Politics’, Harper's Magazine (1964), pp. 77–86; Robert Robins and Jerrold Post, Political Paranoia: The Psychopolitics of Hatred (New Haven: Yale University Press, 1997). ‘Quiet Americans in India’:1 The Central Intelligence Agency and the Politics of Intelligence in Cold War South Asia Informed by 8 the insights into psychological projection provided by Richard Hofstadter back in the 1960s, and more recent investigations into the malevolent ‘disease’ of political paranoia by Robert Robins and Jerrold Post, it is argued that the emergence of a conspiratorial paradigm in Indian politics around the turn of the 1970s, centred on the CIA, had a significant, enduring and detrimental impact upon Indo-US relations.22 ‘The most satisfactory relationship’: Indian intelligence liaison with the West in the Following the end of British colonial rule in South Asia, both the CIA and the United Kingdom’s Security Service (MI5) moved quickly to establish close liaison relationships with, and provide training and support to, the India’s intelligence service, the Delhi Intelligence Bureau (IB). In June 1949, the IB’s first Indian director, Tirupattur Gangadharam (T. G.) Sanjevi, a 49-year-old former District Superintendent of Police from Madras, travelled to the United States for three weeks of talks with representatives from the CIA, Federal Bureau of Investigation (FBI), and the State Department. The importance that Washington attached to Sanjevi’s visit was underlined by George Kennan, then Director of Policy Planning at the State Department, and one of the driving forces behind the creation of the Office of Policy Coordination, which until the creation of the CIA’s Directorate of Plans in 1952, oversaw covert US psychological and paramilitary operations. In a memorandum distributed to the heads of America’s intelligence agencies, Kennan stressed that Sanjevi, who was known to be ‘very close’ to Jawaharlal Nehru, should ‘not only be given a cordial reception, but that high officials of our Government receive him.’ Reinforcing Kennan’s message, America’s ambassador in New Delhi, Loy Henderson, cautioned Washington that 9 the impressions Sanjevi took away from his trip to the United States were likely to have, ‘wide ramifications in our over-all relations with India.’23 At the time, India’s security and intelligence forces were battling to suppress a violent communist-led insurrection in Telegana, in the south of the country. American diplomats were hopeful that the on-going threat posed by militant communism inside India would encourage Sanjevi to solicit advice and support from his counterparts in the United States. In the event, after meeting with a number of senior CIA figures, including Colonel Richard Stilwell, chief of the Agency’s Far East Division, Kermit ‘Kim’ Roosevelt, shortly to achieve notoriety for his exploits in Iran, and Director of Central Intelligence, Roscoe Hillenkoetter, Sanjevi’s visit was deemed by Washington to have passed off successfully.24 Significantly, and to the CIA’s immense satisfaction, Sanjevi welcomed an American offer to explore, ‘the possibility of establishing an official liaison on Communist matters.’25 Intriguingly, a detailed profile of India’s intelligence director that was distributed to US officials ahead of his visit to the United States, was marked out for ‘most careful handling’ in view of the ‘delicacy’ of its principal source. y 25 Sanjevi was a good deal less enamoured with the reception that he received from the FBI, and in particular, the Bureau’s imperious Director, J. Edgar Hoover. To the distress of Loy Henderson, Sanjevi subsequently confided to the US ambassador that having, ‘been looking forward with particular enthusiasm to meeting and having a heart-to-heart with...J. Edgar Hoover’, he had been left ‘boiling with resentment’ by the off-hand manner in which Hoover had treated him. The ‘deep-seated pique’, which Sanjevi harboured toward Hoover was underscored when the Indian intelligence chief stated unequivocally to a shocked Henderson that, ‘if a liaison was contemplated [between US and Indian intelligence services] even remotely involving the F.B.I....he [Sanjevi] would not only advise against us making such a proposal but would personally oppose it if it were made.’ See, Loy W. Henderson to Joseph S. Sparks, 17 April 1950, and Richard W. Klise to Loy Henderson, 18 April 1950, RG59, Office of South Asian Affairs India Affairs 1944-57, Lot file 57D373, Box 2, Folder Official informal Jan-May 1950, NARA. 26 J. C. Satterthwaite to James E. Webb, 15 June 1949, RG59, Office of South Asian Affairs India Affairs 1944- 57, Lot file 57D373, Box 2, Folder Memorandum to the Secretary 1949, NARA. 23 J. C. Satterthwaite to James E. Webb, 15 June 1949, RG59, Office of South Asian Affairs India Affairs 1944- 57, Lot file 57D373, Box 2, Folder Memorandum to the Secretary 1949, National Archive and Records Administration, College Park, Maryland (hereafter NARA). , g , y ( ) 24 Joseph S. Sparks to Loy W. Henderson, 8 July 1949, RG59, Office of South Asian Affairs India Affairs 1944- 57, Lot file 57D373, Box 2, Folder Official Informal July 1949, NARA. , , , 57, Lot file 57D373, Box 2, Folder Memorandum to the Secretary 1949, National Archive and Records Administration, College Park, Maryland (hereafter NARA). p p y , y , , 57, Lot file 57D373, Box 2, Folder Official Informal July 1949, NARA. 25 23 J. C. Satterthwaite to James E. Webb, 15 June 1949, RG59, Office of South Asian Affairs India Affa 24 Joseph S. Sparks to Loy W. Henderson, 8 July 1949, RG59, Office of South Asia 57, Lot file 57D373, Box 2, Folder Official Informal July 1949, NARA. 25 , g , y ( ) 24 Joseph S. Sparks to Loy W. Henderson, 8 July 1949, RG59, Office of South Asian Affairs India Affairs 1944- 57 L fil 57D373 B 2 F ld Offi i l I f l J l 1949 NARA , g , y ( ) 24 Joseph S. Sparks to Loy W. Henderson, 8 July 1949, RG59, Office of South 28 See, Kenneth Conboy and James Morrison, The CIA’s Secret War in Tibet (Lawrence: University of Kansas Press, 2002); and, John Kenneth Knaus, Orphans of the Cold War: America and the Tibetan Struggle for Survival (New York: Public Affairs, 1999). Knaus, who headed the CIA’s Tibetan Task force around the turn of the 1960s, has asserted that, between 1957 and 1961, over 500,000 pounds of arms, ammunition, communications equipment and medical supplies were delivered to Tibetan resistance groups by the CIA and its proprietary airline, Civil Air Transport. See Knaus, Orphans of the Cold War, p. 155. 27 MI5 stationed SLO’s across Asia and Africa after 1945, where under the ‘Attlee Directive’, the Empire and Commonwealth remained primarily the preserve of the Security Service, and insulated from the clandestine activities of the British Secret Intelligence Service (MI6). The SLO role was to provide advice and support to local security agencies, whilst at the same time acting as conduit for the exchange of security related information between London and Britain’s former imperial outposts. It was not to engage in acts of subterfuge or espionage. The first SLO in India, Lieutenant Colonel Kenneth Bourne, arrived in New Delhi in 1947, while 27 MI5 stationed SLO’s across Asia and Africa after 1945, where under the ‘Attlee Directive’, the Empire and Commonwealth remained primarily the preserve of the Security Service, and insulated from the clandestine activities of the British Secret Intelligence Service (MI6). The SLO role was to provide advice and support to local security agencies, whilst at the same time acting as conduit for the exchange of security related information between London and Britain’s former imperial outposts. It was not to engage in acts of subterfuge or espionage. The first SLO in India, Lieutenant Colonel Kenneth Bourne, arrived in New Delhi in 1947, while the last, a victim of Whitehall economies, was withdrawn, against the wishes of the then head of IB, S. P. Varma, in the late 1960s. See, Visit of Captain Liddell (Security Service) to the Middles East’, Confidential Annex to J.I.C. (47) 33rd Meeting (0), 9 June 1947, CAB 159/1; and Roger Hollis to Sir Burke Trend, 13 November 1965, CO 1035/171, British National Archives, Kew London (hereafter, TNA). See also, Christopher Andrew, The Defence of the Realm: The Authorized History of MI5 (London: Allen Lane, 2009) p. 137 and p. 481. 28 the last, a victim of Whitehall economies, was withdrawn, against the wishes of the then head of IB, S. P. Varma, in the late 1960s. See, Visit of Captain Liddell (Security Service) to the Middles East’, Confidential Annex to J.I.C. (47) 33rd Meeting (0), 9 June 1947, CAB 159/1; and Roger Hollis to Sir Burke Trend, 13 November 1965, CO 1035/171, British National Archives, Kew London (hereafter, TNA). See also, Christopher Andrew, The Defence of the Realm: The Authorized History of MI5 (London: Allen Lane, 2009) p. 137 and p. 481. 28 ‘The most satisfactory relationship’: Indian intelligence liaison with the West in the Sanjevi, it seems, may not have been the first senior Indian government official to forge a working relationship with the CIA.26 10 Under Sanjevi, and his successor, B. N. Mullik, who assumed control of IB in July 1950, the CIA and MI5, the latter of which maintained a declared, or overt, Security Liaison Officer (SLO) in New Delhi, enjoyed close collaborative relations with India’s intelligence services into the 1970s.27 Notably, the IB chose to ‘look the other way’ as CIA aircraft transited through Indian airspace in support of Agency sponsored resistance operations in Chinese controlled Tibet, and CIA operatives spirited the Dalai Lama out of Lhasa and into northern India following an abortive Tibetan uprising in 1959.28 Indeed, by the early 1960s, the CIA had a sizable, growing and active in-country presence in India. Having initially operated from a single ‘station’, or base of operations, in New Delhi, the Agency progressively extended the geographical scope of its activities, establishing a network of smaller out-stations in Bombay, Calcutta, and Madras. Inevitably, as the scale of CIA activity in India increased, the Agency found it increasingly difficult to preserve its anonymity. Abraham Michael Rosenthal, the New York Times’ correspondent in New Delhi for much of the 1950s, confided to his editor back in the United States that local CIA staff working under the guise of Treasury experts, Air Force contractors or members of specialised bodies, such as the Asia Foundation, were easily identified by Indian government officials and journalists. Agency personnel in India, Rosenthal noted, were widely referred to by Americans and 11 Indians alike, as ‘the Halicrafter boys’, ‘because whatever their Embassy cover they all had offices in the same part of the Embassy basement and all had identical Halicrafter radios.’ The CIA’s involvement in Indian political circles, Rosenthal observed, appeared to be directed, ‘more than anything else in getting inside the Congress Party for purposes of information or influence.’29 In 1959, the Agency demonstrated its ability and willingness to work with India’s ruling political party, when it helped to pave the way for the removal of a democratically elected Communist Party of India (CPI) government in the southern state of Kerala. Affairs Oral History Project (hereafter FAOHP), INTERNET, http://memory.loc.gov/ammem/collections/diplomacy/. Daniel Patrick Moynihan, US ambassador to India in the early 1970s, has confirmed that the CIA was used to fund Congress Party campaigns against the Communist Party of India in Kerala and West Bengal. See, Moynihan, A Dangerous Place, p. 41. y p , y, y ( ) 30 Ellsworth Bunker, US ambassador to India between 1956 and 1961, confirmed in 1979 that the CIA had funnelled money to the Congress Party in the late 1950s, through S.K. Patil, a Congress leader from Maharashtra, to sustain its campaign of opposition to the communist administration in Kerala. See, Ellsworth Bunker, Oral History, 18 Jun. & 17 Jul. 1979, New York, Butler Library, Columbia University, pp. 67-8. More junior US officials, such as David S. Burgess, Labour attaché at the US Embassy in New Delhi between 1955 and 1960, have corroborated bunker’s testimony. See, David. S. Burgess, Oral History, 7 April 1991, Foreign Affairs Oral Histor Project (hereafter FAOHP) INTERNET Abraham Michael Rosenthal to Mr. Salisbury, undated, Box 159, CIA Series 1965-66, Raw Data, Salisbury Papers, Butler Library, Columbia University (hereafter HSP). 29 Abraham Michael Rosenthal to Mr. Salisbury, undated, Box 159, CIA Series 1965-66, Raw Data, Harrison Salisbury Papers, Butler Library, Columbia University (hereafter HSP). u e , O s o y, 8 Ju . & 7 Ju . 979, ew o , u e b y, Co u b U ve s y, pp. 67 junior US officials, such as David S. Burgess, Labour attaché at the US Embassy in New Delhi betw and 1960, have corroborated bunker’s testimony. See, David. S. Burgess, Oral History, 7 April 1991 Aff i O l Hi t P j t (h ft FAOHP) INT 33 See, Robert Komer to McGeorge Bundy, 14 October 1965, NSF, Robert W. Komer Papers, Box 13, Folder 6 Bundy, McG - Decisions 1965-66; and, Walt Rostow to President Johnson, 30 April 1966, NSF, Intelligence File, Box 2, Folder India's Unconventional Warfare Force, LBJL. Also, M.S. Kohli and Kenneth Conboy, Spies 31 The evolution of Indo-US relations under Eisenhower and Kennedy has received significant scholarly attention. See, for example, Robert J. McMahon, The Cold War on the Periphery: The United States, India and Pakistan (New York: Columbia University Press, 1994); Dennis Merrill, Bread and the Ballot: The United States and India’s Economic Development (Chapel Hill: The University of North Carolina Press, 1990); Andrew Rotter, Comrades at Odds: The United States and India, 1947-1964 (Ithaca: Cornell University Press, 2000); and, Richard P. Dauer, A North-South Mind in an East-West World: Chester Bowles and the Making of United States Cold War Foreign Policy, 1951-1969 (Westport, CT: Praeger Publishers, 2005). ‘The most satisfactory relationship’: Indian intelligence liaison with the West in the By secretly channelling funds to Congress Party officials and local anti-communist labour leaders, the CIA helped to destabilise, and ultimately bring down, the incumbent CPI administration.30 In a wider sense, in the late 1950s, toward the end of Dwight D. Eisenhower’s second presidential term, Washington had become increasingly concerned both at the growth of indigenous communism in the subcontinent, and the Soviet Union’s burgeoning ties with New Delhi. Eisenhower’s enthusiasm for bringing India and the United States closer together, primarily through the provision of generous American economic aid to Nehru’s administration, was subsequently embraced by his successor, John F. Kennedy, who looked upon democratic India as a strategic counterweight to the expansion of Communist Chinese influence in Asia. Indeed, at the beginning of the 1960s, and in particular for a brief period following the Sino-Indian border war of 1962, the locus of Washington’s effort to contain 12 Asian communism was located not in South Vietnam, but in India.31 A series of very public CIA ‘failures’ around this time, beginning with the loss of a U-2 spy-plane over the Soviet Union in May 1960, and culminating in the Bay of Pigs fiasco the following April, had little immediate impact on Indo-US relations, or more pertinently, co-operation in the intelligence field. In July 1961, whilst on a tour of the southern India, John Kenneth Galbraith was surprised, and faintly amused, when in the course of being introduced to a group of local dignitaries, a man stepped forward and exclaimed exuberantly, “'Mr. Ambassador, I am the superintendent of police here in Madras. I would like to tell you that I have the most satisfactory relationship with your spies.”32 Asian communism was located not in South Vietnam, but in India.31 A series of very public CIA ‘failures’ around this time, beginning with the loss of a U-2 spy-plane over the Soviet Union in May 1960, and culminating in the Bay of Pigs fiasco the following April, had little immediate impact on Indo-US relations, or more pertinently, co-operation in the intelligence field. In July 1961, whilst on a tour of the southern India, John Kenneth Galbraith was surprised, and faintly amused, when in the course of being introduced to a group of local dignitaries, a man stepped forward and exclaimed exuberantly, “'Mr. Ambassador, I am the superintendent of police here in Madras. 32 ‘The Year of the Spy (in a Manner of Speaking)’, John Kenneth Galbraith, The New York Times, 5 January 1986. 33 nd, Richard P. Dauer, A North South Mind in an East West World: Chester Bowles and the Making of ates Cold War Foreign Policy, 1951-1969 (Westport, CT: Praeger Publishers, 2005). r, Comrades at Odds: The United States and India, 1947-1964 (Ithaca: Cornell University Press, 2000 Richard P. Dauer, A North-South Mind in an East-West World: Chester Bowles and the Making of Unite s Cold War Foreign Policy, 1951-1969 (Westport, CT: Praeger Publishers, 2005). ‘The most satisfactory relationship’: Indian intelligence liaison with the West in the I would like to tell you that I have the most satisfactory relationship with your spies.”32 In the aftermath of the following year’s Sino-Indian border war, Indian and American intelligence agencies further strengthened their working relationships. Notably, the CIA assisted the IB in equipping and training a clandestine warfare unit tasked with monitoring Chinese military supply routes into Tibet. Under an agreement reached between James Critchfield, chief of the CIA’s Near East operations, and B. N. Mullik, Langley furnished support to the Indo-Tibetan Special Frontier Force (SFF), a unit modelled on the US Army Green Berets, or special forces. From the winter of 1964, SFF operations along the Sino- Indian border were co-ordinated through a joint Indo-US command centre in New Delhi. The Agency also oversaw the insertion of nuclear-powered surveillance equipment on two of India’s Himalayan peaks, with a view to collecting data on Chinese atomic tests.33 One US 13 diplomat, who served in the US Embassy in New Delhi throughout the first half of the 1960s, subsequently verified that the Agency’s presence in India at the time was, ‘very large, and very invasive...the CIA was deeply involved in the Indian Government.’34 In fact, although many Indian government officials were aware of the existence of liaison relationships between the IB and their British and American counterparts, India’s intelligence chiefs worked hard to disguise the true extent and significance of their collaboration with Western intelligence agencies. In large part, such caution reflected a realisation on the part of senior IB officers that, as B. N. Mullik reflected, India’s premier, Jawaharlal Nehru, held a ‘natural’ and ‘strong prejudice’ against much of the work performed by intelligence organisations.35 In April 1950, T. G. Sanjevi had reassured American officials that, ‘he frequently had to take independent action without the knowledge of his government’, and, ‘regardless of the official attitude of his government, he would welcome the continuance of...unofficial contacts.’36 Once he had succeeded Sanjevi, Mullik deemed it equally prudent to keep IB’s links with British and American intelligence agencies as quiet as possible. chard W. Klise to Loy Henderson, 18 April 1950, RG59, Office of South Asian Affairs India Affairs 1944 ot file 57D373, Box 2, Folder Official informal Jan-May 1950, NARA. p f pp 34 Ambassador Mary Seymour Olmsted, Oral History, 8 April 1992, FAOHP, INTERNET, http://memory.loc.gov/ammem/collections/diplomacy/. in the Himalayas: Secret Missions and Perilous Climbs (Lawrence: University of Kansas Press, 2003); and, Knaus, Orphans of the Cold War, pp. 265-276. 34 , y , ( , ), p chard W. Klise to Loy Henderson, 18 April 1950, RG59, Office of South Asian Affairs India Affairs 1944 , , , 37 Andrew, Defence of the Realm, p. 445-6. , y , ( , ), p 36 Richard W. Klise to Loy Henderson, 18 April 1950, RG59, Office of South Asian Affairs India Affairs 1944- 57 Lot file 57D373 Box 2 Folder Official informal Jan-May 1950 NARA Ambassador Mary Seymour Olmsted, Oral History, 8 April 1992, FAOHP, INTERNET /memory.loc.gov/ammem/collections/diplomacy/. N Mullik My Years with Nehru 1948-1964 (New Delhi: Allied Publishers 1972) p 57 y drew, Defence of the Realm, p. 445-6. y y , y, p , , , mory.loc.gov/ammem/collections/diplomacy/. Mullik, My Years with Nehru, 1948-1964 (New Delhi: Allied Publishers, 1972), p. 57. y , p , , 57, Lot file 57D373, Box 2, Folder Official informal Jan-May 1950, NARA. 37 39 Indira Gandhi, ‘Women on the March’, September 1963, reproduced in, India: The Speeches and Reminiscences of Indira Gandhi, Prime Minister of India (London: Hodder and Stoughton, 1975), p. 17. 40 Nehru’s speech to conference of the heads of Indian Missions in Europe, Salzburg, Austria, 28-30 June1955, Selected Works of Jawaharlal Nehru, Second Series, Volume Twenty-Nine, 1 June-31 August 1955, eds. H.Y Sharad Prasad, A.K. Damodaran (New Delhi: Oxford University Press, 2001), p. 257. Nehru received indications from Indian government sources that the United States was subsidising the production of pro- Western Indian newspapers, such as Thought, and the providing financial payments to Indian journalists in return for ‘propaganda work.’ See, for example, Nehru’s note to Secretary General and Foreign Secretary, 5 Sep. 1956, Selected Works of Jawaharlal Nehru, Second Series, Volume Thirty-Five, 1 September 1956-30 November 1956, ed. Mushirul Hasan, H.Y Sharad Prasad, A.K. Damodaran (New Delhi: Oxford University Press, 2005), pp. 527-8. See also, Subimal Dutt, With Nehru in the Foreign Office (Calcutta: Minerva Associates 1977) p. 241. 38 An observation made in September 1945, by Joseph P. McCarthy, the head of American counter-intelligence in the American India-Burma theatre during the Second World War. Cited in Richard J. Aldrich, ‘Imperial Rivalry: British and American Intelligence in Asia, 1942-1946’, Intelligence and National Security, 3 (1), (January 1988), p. 38. Gandhi, ‘Women on the March’, September 1963, reproduced in, India: The Speeches and ences of Indira Gandhi, Prime Minister of India (London: Hodder and Stoughton, 1975), p. 17. 38 An observation made in September 1945, by Joseph P. McCarthy, the head of American counter-in ‘The most satisfactory relationship’: Indian intelligence liaison with the West in the Were Nehru to get wind of the scale of India’s partnership with the CIA and MI5, Mullik explained to one British intelligence officer, much of the liaison activity would have to be curtailed.37 When viewed through the prism of Nehru’s family history, the suspicion and distrust that the Indian premier, and more significantly, his daughter, Indira Gandhi, harboured toward foreign intelligence agencies is unsurprising. Before India’s independence in 1947, when not languishing in British prisons, both Nehru and Gandhi had been subjected to 14 oppressive surveillance by a British imperial security service that one contemporary American observer lauded as, ‘the finest political intelligence organisation in the world.’38 In later life, Gandhi emphasised the deep psychological impression left by witnessing ‘a large number of relatives, on both my father’s and mother’s side’ hounded and imprisoned for expressing political opinions inimical to the British Raj. ‘I do not know of any other family,’ Gandhi observed, ‘which was so involved in the freedom struggle and its hardships.’39 In post-independence South Asia, however, it was anxiety over CIA activity undertaken without the knowledge and approval of the Indian government, that most exercised the Nehru clan. In June 1955, in a speech delivered to a gathering of Indian diplomats in Salzburg, Austria, Jawaharlal Nehru made it clear that he regarded the CIA as an especially invidious threat to Indian democracy. ‘The United States are carrying on their espionage and secret service activities [inside India],’ Nehru assured his audience. ‘They have also been buying up newspapers and spreading a network of publicity organisations... We are more concerned with what the Americans are trying to do than the others.’40 The unease that India’s political leadership felt towards the CIA were reflected in the Agency’s endeavours to maintain a low public profile. In 1965, shortly after being posted to New Delhi, the New York Times’ correspondent, Anthony Lukas, noted that in India the CIA did its best to operate, ‘very much on the hush-hush.’ In contrast to the more overt presence 15 that it adopted in other parts of the developing world, such as the Congo, in India, Lukas found that the Agency went to, ‘great efforts to pretend that it doesn’t exist.’ The Agency’s challenge in disguising the ever-greater numbers of its officers seconded to the American embassy faintly amused Lukas, who much like Rosenthal before him, found little difficulty in identifying US intelligence personnel. y ‘Ducks Reprieved at a U.S. Embassy’, The New York Times, 6 June 1965. Anthony Lukas to Tom Wicker, 18 October 1965, Box 159 CIA Series 1965-66 Raw Data, HSP. See ‘The most satisfactory relationship’: Indian intelligence liaison with the West in the One of the Indian Prime Minister’s foremost biographer’s has gone as far as to state that throughout Mathai’s period of government service between 1946 and 1959, ‘the CIA had access to every paper passing through Nehru’s Secretariat.’43 in the 1960s, India’s increasing reliance on American surplus wheat and rice to stave off famine, generated more resentment than goodwill. ‘The American role here,’ one Western journalist in India opined, ‘has been an object lesson in how to give aid and win enemies.’42 ‘The most satisfactory relationship’: Indian intelligence liaison with the West in the The CIA’s resolve to keep its presence in India out of the public spotlight was made abundantly clear to the American journalist after he published a ‘light yarn’ in the Times. Lukas’ report referenced the emergence of a ‘protest movement’ amongst American diplomats, led by an unnamed CIA official, against plans to cull some ducks that had taken up residence in a pool within the US Embassy compound. Within days of the stories publication, Lukas was summoned to the Embassy by the resident press attaché, and tersely informed that he had been decreed persona non grata by the local CIA station chief. ‘I was told,’ Lukas advised his superiors back in New York, ‘that I had gravely compromised the agency’s security here.’ ‘What, I asked incredulously, had I done? The answer: I had informed the Indians that the C.I.A. was operating out of the Embassy.’41 The CIA’s reaction to Lukas’ article was undoubtedly influenced by the fact that India’s broader relationship with the United States was under considerable strain at that time. Once Lyndon Johnson had entered the White House in November 1963, America’s focus shifted inexorably away from the Indian subcontinent, and toward South East Asia. Consequently, diplomatic tensions between Washington and New Delhi on issues ranging from the provision of military assistance, the supply of food aid, and the escalating conflict in Vietnam, were left to fester. In particular, having received the lion’s share of American overseas developmental assistance and food aid from the late 1950s, many Indians felt humiliated by the cycle of dependency that had come to characterise their country’s relationship with the United States. As the subcontinent experienced a succession of droughts 16 in the 1960s, India’s increasing reliance on American surplus wheat and rice to stave off famine, generated more resentment than goodwill. ‘The American role here,’ one Western journalist in India opined, ‘has been an object lesson in how to give aid and win enemies.’42 Efforts by American officials in India, led by Chester Bowles, to maintain constructive diplomatic relations with New Delhi were not helped by a series of revelations that suggested the CIA had recruited assets, or informers, at the very top levels of the Indian government. Among those alleged to have worked for US intelligence, was Mac Mathai, Nehru’s private secretary. Gopal based his assertion on records of discussions that had taken place between his father, and India’s President, Sarvepalli Radhakrishnan, and V. Sahay, India’s Cabinet Secretary, in 1966. Nehru had asked the Cabinet Secretary to investigate allegations linking Mathai to the CIA back in 1959. In contrast, a subsequent biographer of Nehru’s, who was afforded privileged access to his private papers, was unable to unearth any evidence linking Mathai to the CIA. See, Judith M. Brown, Nehru: A Political Life (London: Yale University Press, 2003), pp. 382-3. ‘Not so quiet Americans’, The Guardian, 28 October 1972. q , , alli Gopal, Jawaharlal Nehru: A Biography, Vol.3, 1956-1964 (London: Jonathan Cape, 1984), p. 122. 43 Sarvepalli Gopal, Jawaharlal Nehru: A Biography, Vol.3, 1956-1964 (London: Jonathan Cape, 1984 Gopal based his assertion on records of discussions that had taken place between his father, and President, Sarvepalli Radhakrishnan, and V. Sahay, India’s Cabinet Secretary, in 1966. Nehru had a vepalli Gopal, Jawaharlal Nehru: A Biography, Vol.3, 1956 1964 (London: Jonathan Cape, 1984), p. 12 l based his assertion on records of discussions that had taken place between his father, and India dent, Sarvepalli Radhakrishnan, and V. Sahay, India’s Cabinet Secretary, in 1966. Nehru had asked th 44 Christopher Andrew and Vasili Mitrokhin, The Mitrokhin Archive II: The KGB and the Wider World (London: Penguin, 2005), pp. 9-10; pp. 312-4. 45 Rostow to LBJ, 2 May 1966, NSF Country File, India, Box 131 [1 of 2], Folder 2 India memos & Misc [2 of 3] Vol. VII 1-66 to 8-66, LBJL. 46 Oleg Kalugin, Spymaster: My Thirty-Two Years in Intelligence and Espionage against the West (New York: Basic Books, 2009), p. 141. ‘Spies and Saboteurs’: The intelligence Cold War comes to India At the same time that the United States’ association with India was coming under strain, the Soviet Union’s relationship with New Delhi began to blossom. Until Joseph Stalin’s death in 1953, the Soviet government had disparaged India as an imperialist puppet. Both Nehru, and India’s spiritual leader, Mohandas Karamchand Gandhi, had routinely been dismissed by Moscow as bourgeois reactionaries. Under Nikita Khrushchev’s leadership, however, with ideological fervour having been displaced with a more pragmatic approach to international affairs, the Soviet Union embarked upon a concerted effort to woo nascent post-colonial states. India’s huge population, untapped economic potential, shared anxiety at China’s 17 international assertiveness, and in the form of Nehru, influence within the Non-Aligned Movement, all attracted Soviet interest. Moreover, where Soviet politicians led, the nation’s foreign intelligence bodies, in the form of the KGB and Soviet military intelligence, the GRU, soon followed. In 1961, with Khrushchev’s blessing, the young and dynamic KGB chairman, Aleksandr Shelepin, launched an aggressive covert campaign designed to undermine Western influence across the developing world. By 1967, Leonid Brezhnev had replaced Khrushchev, and the future Soviet premier, Yuri Andropov, had taken control of the KGB. The strategy of fomenting revolution across Asia, Africa and Latin America, however, remained unchanged. Indeed, during the Brezhnev era, the conservative Soviet Foreign Minister, Andrei Gromyko, was happy to cede the initiative for much of the Soviet forward policy in the developing world to Andropov and the KGB. In turn, Soviet foreign intelligence agencies concentrated a large proportion of their operational effort, outside of Europe and North America, on India.44 In May 1966, Chester Bowles warned Lyndon Johnson’s National Security Advisor, Walt Rostow, that the United States faced, ‘a period of acute competition with the USSR for leverage in India.’45 Oleg Kalugin, then a rising star in the KGB’s First Chief (Foreign Intelligence) Directorate, has stated that around the time of the Ramparts disclosures: ...we [the KGB] had scores of sources throughout the Indian government – in intelligence, counterintelligence, the defense and foreign ministries, and the police. The entire country was seemingly for sale, and the KGB and the CIA had deeply penetrated the Indian government. ] , 46 Oleg Kalugin, Spymaster: My Thirty-Two Years in Intelligence and Espionage against the West (New York: Basic Books, 2009), p. 141. ‘Spies and Saboteurs’: The intelligence Cold War comes to India After a while, neither side entrusted sensitive information to the Indians, realizing their enemy would know all about it the next day.46 ...we [the KGB] had scores of sources throughout the Indian government – in intelligence, counterintelligence, the defense and foreign ministries, and the police. The entire country was seemingly for sale, and the KGB and the CIA had deeply penetrated the Indian government. After a while, neither side entrusted sensitive information to the Indians, realizing their enemy would know all about it the next day.46 18 The following year, Bowles again urged an increasingly disinterested Washington to take heed of, ‘the massive effort the Communist press and the Communist party [of India] are now making to disrupt Indo-American relationships.’47 In April 1967, in an article published in the American Reporter, a newspaper distributed by the US government in the subcontinent, Bowles complained bitterly of the ‘international character assassination’ being undertaken by sections of the Indian media. Communist propaganda in India, the ambassador fulminated, had maliciously charged American ‘“spies and saboteurs”’ with ‘plotting turn this country over to “Wall Street imperialists”, “Neo-Colonialists”, and “Neo-Cultural penetrationists.”’48 Following that year’s Indian general election, for the first time since independence the ruling Congress party was faced with a credible parliamentary opposition. At the same time, Indira Gandhi’s government was assailed by sluggish economic growth, a food crisis, and a slump in export orders. In unfamiliar political territory, senior Congress party figures began to question the appetite of the Johnson administration to come to India’s rescue. ‘‘Unfortunately,’ a dispirited Bowles informed Lyndon Johnson, ‘the atmosphere in which the estimate of [the] future U.S. stance [on India] is being made, is heavily fogged by widespread public and Parliamentary uneasiness regarding admitted and alleged CIA activities... and general concern about the ability of the U.S. Government to maintain the required level of assistance here in view of demands in Vietnam.’49 The following year, Bowles again urged an increasingly disinterested Washington to take heed of, ‘the massive effort the Communist press and the Communist party [of India] are now making to disrupt Indo-American relationships.’47 In April 1967, in an article published in the American Reporter, a newspaper distributed by the US government in the subcontinent, Bowles complained bitterly of the ‘international character assassination’ being undertaken by sections of the Indian media. , p p , p , p 49 Bowles to Johnson, No. 13991, 29 March 1967, NSF Country File, India, Box 131 [2 of 2], Folde Cables Vol. IX 3-67 to 7-67, LBJL. wles to Rusk, 27 April 1967, RG59, Bureau of Near Eastern and South Asian Affairs Records Relating India 1966-75, Lot file 71D385, Box 4, Folder Political Affairs & Relations India 1967 Chester Bowles, NARA. 48 Chester Bowles, ‘Ambassador’s Report: Keep the Cold War out of India’, American Reporter, 26 April 1967. , , Cables Vol. IX 3-67 to 7-67, LBJL. India 1966 75, Lot file 71D385, Box 4, Folder Political Affairs & Relations India 1967 Chester Bowles, NARA. 48 Chester Bowles, ‘Ambassador’s Report: Keep the Cold War out of India’, American Reporter, 26 April 1967. ‘Spies and Saboteurs’: The intelligence Cold War comes to India Communist propaganda in India, the ambassador fulminated, had maliciously charged American ‘“spies and saboteurs”’ with ‘plotting turn this country over to “Wall Street imperialists”, “Neo-Colonialists”, and “Neo-Cultural penetrationists.”’48 Following that year’s Indian general election, for the first time since independence the ruling Congress party was faced with a credible parliamentary opposition. At the same time, Indira Gandhi’s government was assailed by sluggish economic growth, a food crisis, and a slump in export orders. In unfamiliar political territory, senior Congress party figures began to question the appetite of the Johnson administration to come to India’s rescue. ‘‘Unfortunately,’ a dispirited Bowles informed Lyndon Johnson, ‘the atmosphere in which the estimate of [the] future U.S. stance [on India] is being made, is heavily fogged by widespread public and Parliamentary uneasiness regarding admitted and alleged CIA activities... and general concern about the ability of the U.S. Government to maintain the required level of assistance here in view of demands in Vietnam.’49 In actuality, the psychological writing had been on the wall for Indo-US relations from the moment that Indira Gandhi became India’s Prime Minister, in January 1966. As one CIA report from September 1973 observed, ‘left-of-centre Indian officials, including Mrs. Gandhi, have long held a conspiratorial view of U.S. activities in India which has been a 19 smouldering source of resentment against the United States.’50 To many Western diplomats Gandhi appeared ‘vain’, ‘emotional’, ‘authoritarian’, and prone to ‘irrational’ fits of pique when events turned against her.51 Officials in Washington were particularly disconcerted by an anti-American undercurrent that appeared to influence many of Gandhi’s actions and utterances, a character trait that the Indian premier was commonly perceived to have inherited from her father.52 Following one bruising encounter with Gandhi in October 1970, US Secretary of State, William Rogers, complained that although the Nixon administration had, ‘been in office only 20 months’, the Indian premier was, ‘holding against us a paranoia going back to John Foster Dulles.’53 An avid consumer of literature with a strong CIA theme, in 1974 alone Gandhi devoured Victor Marchetti and John Marks’, The CIA and the Cult of Intelligence, Antony Sampson’s, The Sovereign State: The Secret History of ITT, and David Halberstam’s, The Best and the Brightest. y g 53 Telecon between Secretary of State Rogers and the President's Assistant for National Security Affairs (Kissinger), Washington, 24 October 1970, Foreign Relations of the United, 1969–1976 Volume E–7, , , y , 52 Moynihan to Kissinger, No. 3458, ‘Mrs Gandhi on the Hustings’, 27 March 1973, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1973-21/31/1973, NARA. p y g 54 Saxbe to Secretary of State, No. 03530, ‘Prime Minister Gandhi Comments on CIA Activity’, 13 March 1975, RG59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. Documents on South Asia, 1969–1972, Document 89, INTERNET, http://history.state.gov/historicaldocuments/frus1969-76ve07/d89 50 ‘National Intelligence Survey: India September 1973’, NARA, CIA-RDP01-00707000200070032-3, CREST. 51 ‘Mrs. Indira Gandhi’, 15 January 1964, RG59, Records Relating to Indian Political Affairs, 1964-1966, Lot 68D207, Box 5, Prime Minister Nehru Jan-May 1964, NARA. g y p 51 ‘Mrs. Indira Gandhi’, 15 January 1964, RG59, Records Relating to Indian Political Affairs, 1964-1966, Lot 68D207, Box 5, Prime Minister Nehru Jan-May 1964, NARA. 50 ‘National Intelligence Survey: India September 1973’, NARA, CIA-RDP01-00707000200070032-3, C 51 y g p 56 One of Gandhi’s political opponents, S. K. Patil, observed sardonically that after taking delivery of KGB ‘donation’s’, Gandhi did not even have the decency to return the suitcases in which they had arrived to their Soviet owners. Inder Malhotra, Indira Gandhi: A Personal and Political Biography (London: Hodder & Stoughton, 1989), p. 143, f.; Although the KGB had taken a considerable interest in Gandhi from the early 1950s, assigning her the codename VANO, no evidence has emerged to link the Indian premier directly with Soviet intelligence. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 322. g p 57 Saxbe to Secretary of State, No. 03530, ‘Prime Minister Gandhi Comments on CIA Activity’, 13 March 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975; Saxbe to Secretary of State, No. 03606, ‘Indo-US Intelligence Cooperation Reported in Press’, 14 March 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. ‘Spies and Saboteurs’: The intelligence Cold War comes to India ‘The picture she would have [drawn of the CIA] from this selection,’ the American Embassy in New Delhi bemoaned, ‘...would hardly be objective.’54 Equally, as one US ambassador to India, Daniel Patrick Moynihan, has pointed out, Gandhi had few qualms about co-operating with foreign intelligence agencies, including those of the United States, when it suited her interests to do so. In his 1978 memoir, A Dangerous Place, Moynihan confirmed that to his knowledge the CIA had twice intervened in Indian politics. On both occasions the Agency had funnelled money to the ruling Congress Party in a bid to head off the election of communist governments in Kerala and West Bengal. 20 In one instance, the ambassador charged, CIA money was passed directly to Gandhi in her capacity as Congress Party President.55 Others have pointed out, that whether she realised it or not, Gandhi’s principal political fundraiser during the early 1970s, Lalit Narayan Mishra, actively solicited money from the KGB. Rumours abounded in New Delhi at the time of suitcases stuffed full of banknotes arriving at the Prime Minister’s residence in the dead of night. Leonid Shebarshin, a KGB officer stationed in India in the 1970s, has documented an occasion on which he personally handed over a gift of two million Indian rupees to Mishra on behalf of the Soviet Politburo.56 Moreover, as Nehru’s closest political confidante and, after 1964, a Cabinet Minister in her own right, it is hard to conceive that Gandhi was not at least aware of, if not complicit in, joint initiatives with the CIA which the Indian government sanctioned in the wake of the Sino-Indian border war. In 1975, Gandhi’s links to the CIA would come back to haunt her when, in the midst of a crusade against the Agency’s subversive practices, the Hindustan Times began to publish details of the history of the Indian government’s relationship with American intelligence. In response, Indira Gandhi affected an awkward, and none to convincing, case of prime ministerial amnesia.57 55 Moynihan, A Dangerous Place, p. 41. 56 55 Moynihan, A Dangerous Place, p. 41. 56 han, A Dangerous Place, p. 41. f G dhi’ li i l S K P il b d d i ll h f ki d li f KGB , , , 61 India’, Visit of Nigel Clive (IRD) to India and Pakistan, 5 December 1967, FCO 95/290, TNA. g p p 59 The IRD’s role in the Cold War has received considerable scholarly attention. The majority of studies to date, however, have concentrated on IRD activity during the 1950s. Moreover, little focus has been placed on the organisations work on the Indian subcontinent. See for example, Andrew Defty, Britain, America and Anti- Communist Propaganda, 1945-53: The Information Research Department (Abingdon: Routledge, 2004); James R. Vaughan, The Failure of American and British Propaganda in the Middle East, 1945–1957: Unconquerable Minds (London: Palgrave Macmillan, 2005), and ‘“Cloak Without Dagger”: How the Information Research Department Fought Britain's Cold War in the Middle East, 1948-1956’, Cold War History (2004) 4(3): 56-84; Hugh Wilford, ‘The Information Research Department: Britain's Secret Cold War Weapon Revealed’, Review of International Studies (1998), 24: 353-369; Christopher Mayhew, A War of Words: A Cold War Witness (London: IB Tauris, 1998); Paul Lashmar and James Oliver, Britain's Secret Propaganda War 1948-1977 (Stroud: Sutton Publishing, 1998); and Tony Shaw, The Information Research Department of the British Foreign office and the Korean War, 1950-53, Journal of Contemporary History, 34, 2: 263-281. 58 The British led a Commonwealth of Nations that included India, and its neighbour, Pakistan; supplied India’s armed forces with much of its training and equipment into the late 1960s; and remained South Asia’s principal trading partner. John Freeman to CRO, 15 April 1966, PREM 13/970, TNA. 59 g , , f p y y, , 60 By 1953, Mullik was receiving regular deliveries of several IRD publications. See, V.C. Martin to G.S. Bozman, 7 October 1953, FO 1110/603, TNA 61 The CIA and Cold War propaganda in India The success enjoyed by Indian left-wing groups in exploiting the Ramparts disclosures to fan concern over CIA interference in India’s internal affairs alarmed the United States’ British 21 ally. After two hundred years of colonial rule in South Asia, the British retained a significant political, economic, and cultural stake in India after 1947.58 London also maintained an important intelligence presence in India, both overt, in the form of MI5’s SLO, and covert, through the work of the Information Research Department (IRD). The IRD had been established in 1948 to counter the spread of Soviet propaganda in Britain and throughout the developing world.59 During the 1950s, its personnel were seconded to British diplomatic missions abroad, and tasked with feeding anti-communist literature to networks of local journalists and politicians. IRD cultivated a wide range of contacts in India, and counted B. N. Mullik, and other senior Intelligence Bureau officers, amongst its customers.60 It was not until January 1962, however, that the first permanent IRD representative in India, Peter Joy, was posted to the British High Commission in New Delhi under cover provided by the British Information Service.61 Over the next five years, IRD expanded its work in the subcontinent, establishing relationships with independent research centres and Indian government departments, including the Ministry of External Affairs, Armed Forces Information Office, Press Information Bureau, and All India Radio. By early 1967, Joy was working with over 400 ‘well-placed and influential individuals throughout India’, some of who received secret 22 payments for their services. In addition, two publishing houses in the Indian capital, and a regional newspaper article redistribution scheme, were paid to disseminate IRD material on a non-attributable basis through a British cover organisation, the International Forum.62 Under Joy’s guidance, the IRD was particularly successful in cultivating ‘assets’ in the Indian press. One IRD survey estimated that its material had appeared in over 500 Indian newspaper articles during the course of 1964 alone. ‘We are able,’ the IRD crowed, ‘to get the right article into the right paper at the right time.’63 In 1967, having diversified its operations from New Delhi to incorporate work in Calcutta and Madras, IRD began lobbying Whitehall for permission to take on a more interventionist role in the subcontinent. Ibid. 63 One former Director General of MI5, Stella Rimington, worked for IRD in India for a brief period in the mid- 1960s, distributing covert propaganda to Indian journalists, politicians and academics who, ‘had been recruited to use the material unattributably.’ Rimington confirmed the IRD’s success in placing material, some of which ‘was quite personal stuff about [Indian] politicians’, in Indian newspapers and magazines. Stella Rimington. Open Secret: The Autobiography of the Former Director-General of MI5, (London: Arrow Books, 2002), 75. Secret: The Autobiography of the Former Director General of MI5, (London: Arrow Books, 2002), 75. er Joy to C.F.R. Barclay, 8 May 1964, FO 1110/182; ‘India’, Visit of Nigel Clive to India and Pakistan, mber 1967 FCO 95/290 TNA 64 Peter Joy to C.F.R. Barclay, 8 May 1964, FO 1110/182; ‘India’, Visit of Nigel Clive to India and Pa December 1967, FCO 95/290, TNA. 63 One former Director General of MI5, Stella Rimington, worked for IRD in India for a brief period in the mid- 1960s, distributing covert propaganda to Indian journalists, politicians and academics who, ‘had been recruited to use the material unattributably.’ Rimington confirmed the IRD’s success in placing material, some of which ‘was quite personal stuff about [Indian] politicians’, in Indian newspapers and magazines. Stella Rimington. y y, y December 1967, FCO 95/290, TNA. Open Secret: The Autobiography of the Former Director-General of MI5, (London: Arrow Books, 2002 64 Peter Joy to C.F.R. Barclay, 8 May 1964, FO 1110/182; ‘India’, Visit of Nigel Clive to India and Pa 1960s, distributing covert propaganda to Indian journalists, politicians and academics who, ‘had been recruited to use the material unattributably.’ Rimington confirmed the IRD’s success in placing material, some of which ‘was quite personal stuff about [Indian] politicians’, in Indian newspapers and magazines. Stella Rimington. Open Secret: The Autobiography of the Former Director-General of MI5, (London: Arrow Books, 2002), 75. y y, y , ; , g December 1967, FCO 95/290, TNA. p g p y f f , ( , ), 64 Peter Joy to C.F.R. Barclay, 8 May 1964, FO 1110/182; ‘India’, Visit of Nigel Clive to India and Pakistan, 5 December 1967, FCO 95/290, TNA. The CIA and Cold War propaganda in India ‘IRD should,’ its management argued, ‘concentrate more than hitherto on the cultivation of influential Congress Ministers, M.P.’s and senior civil servants.’64 Fearful that ‘“blowback”’ from the anti-CIA campaign in India would compromise Britain’s covert propaganda operation, the United Kingdom’s High Commissioner in New Delhi, John Freeman, instructed the IRD to proceed with ‘particular caution’ and temporarily curtail some of its riskier activities. IRD officers were temporarily prohibited for seeking new Indian contacts, suspended meetings with existing ‘assets’, and implemented tighter security measures around the distribution of financial ‘incentives’. In rationalising his decision to order a ‘pause’ in IRD activity, Freeman argued that the spotlight which had been thrown on the CIA in India threatened to, ‘unearth the activities of other Western Missions and perhaps link these with C.I.A. Here we should be an obvious target.’ Furthermore, with communist dominated coalition governments having assumed power in Kerala and West Bengal, 23 Freeman was conscious that, ‘the spread of communist influence is now likely to enter the field of Indian domestic politics, and... in the process, the ability of the State Governments to uncover – or fabricate – “foreign influences” is of course increased.’65 American diplomats in India evinced frustration that Indira Gandhi’s government, if not actively directing India’s leading communist leaning newspapers and magazines, such as Patriot, Blitz and Link, to carry salacious and defamatory anti-CIA content, was certainly doing very little to discourage the practice. The US Embassy in New Delhi noted that in one edition of Patriot, 11½ of its 28 pages were taking up with Government advertising. Likewise, an American analysis conducted on Link’s advertising income revealed that it was derived, in large part, from Gandhi’s Congress Party, at both national and state level. Moreover, All India Radio, a potent source of information in a country where two out of every three adults were illiterate, and the Press Trust of India, both of which were, as the British noted, ‘open to official pressure’, infuriated US officials by giving blanket coverage to spurious allegations implicating the CIA in subversive activity.66 Throughout India’s general election in 1967, the KGB had employed a variety of ‘active measures’ in a bid to smear the CIA. Many of these utilised fabricated American documents drafted by Service A of the KGB’s First Chief Directorate, which specialised in disinformation. 66 Memorandum from the Deputy Chief of Mission to Moynihan, undated, c. August 1973, I-376 Folder India Press General 4 1972-7; and, Robert A. Collinge (First Secretary, Press) to Moynihan, ‘Wretched Excess!’, 14 August 1973, I-376 Folder India Press General 4 1972-75, Daniel P. Moynihan Papers, Manuscript Division, Library of Congress, Washington, D.C.]; P. H. Roberts to Mr Hayday, 2 November 1972, ‘Influence of KGB and CIA Organisations on Press in India’, FCO 95/1388, TNA. 65 John Freeman to Sir Saville Garner (CRO), PL. 34/51D, 11 April 1967, FCO 95/200; ‘Brief Progres of IRD work in India, 1 January-30 June 1967’, FCO95/290, TNA. p y y g Press General 4 1972-7; and, Robert A. Collinge (First Secretary, Press) to Moynihan, ‘Wretched Excess!’, 14 August 1973, I-376 Folder India Press General 4 1972-75, Daniel P. Moynihan Papers, Manuscript Division, Library of Congress, Washington, D.C.]; P. H. Roberts to Mr Hayday, 2 November 1972, ‘Influence of KGB d CIA O i ti P i I di ’ FCO 95/1388 TNA 65 John Freeman to Sir Saville Garner (CRO), PL. 34/51D, 11 April 1967, FCO 95/200; ‘Brief Progress Report of IRD work in India, 1 January-30 June 1967’, FCO95/290, TNA. g , , y p , p Library of Congress, Washington, D.C.]; P. H. Roberts to Mr Hayday, 2 November 1972, ‘Influence and CIA Organisations on Press in India’, FCO 95/1388, TNA. , y , , 66 Memorandum from the Deputy Chief of Mission to Moynihan, undated, c. August 1973, I-376 Fol , pp 70 Peter Joy to Mr. Clive, Mr. Tucker and Mr. Welser, IR 1/209/29, 20 April 1967, FCO 95/200, TNA. , , , , 69 Leonid Shebarshin, Ruka Moskvy (Moscow: Tsentr-100, 1992), cited in Andrew and Mitrokhin, The Mitrokhin Archive II, pp. 317-8. 70 Peter Joy to Mr. Clive, Mr. Tucker and Mr. Welser, IR 1/209/29, 20 April 1967, FCO 95/200, TNA. , , , , 69 Leonid Shebarshin, Ruka Moskvy (Moscow: Tsentr-100, 1992), cited in Andrew and Mitrokhin, The Mitrokhin Archive II, pp. 317-8. 67 Andrew and Mitrokhin, The Mitrokhin Archive II, pp. 317-8. See also, John Barron, KGB Today: The Hidden Hand (London: Hodder and Stoughton, 1984). 68 Andrew and Mitrokhin, The Mitrokhin Archive II, pp. 317-8. See also, John Barron, KGB Today: The Hidden Hand (London: Hodder and Stoughton, 1984). ( g ) 68 ‘India’, IRI/545/5, 15 November 1967, FCO95/290, TNA. 69 Leonid Shebarshin Ruka Moskvy (Moscow: Tsentr-100 1992) cited in Andrew and Mitrokhin The M , , pp , , y Hand (London: Hodder and Stoughton, 1984). 68 ( g , ) dia’, IRI/545/5, 15 November 1967, FCO95/290, TNA. id Sh b hi R k M k (M T 100 1992) i d i A d d Mi khi Th Mi kh ( g , ) ia’, IRI/545/5, 15 November 1967, FCO95/290, TNA The CIA and Cold War propaganda in India In one instance, a Soviet agent inside the American Embassy in New Delhi was able to pass templates of official US documents and sample signatures to the KGB. These were then used by Service A to forge a letter nominally from the US Consul-General in Bombay, in which it was suggested that the CIA had been channelling large sums of money 24 to S.K. Patil, a right-wing Congress Party MP.67 By leaking a number of similar counterfeit letters to the Indian press, the KGB was able to keep the CIA firmly in the public spotlight. The Soviet disinformation campaign in India was assisted by the fact that, the CPI journal New Age aside, an estimated 16 English language Indian broadsheets, and a far greater number of vernacular newspapers, were regarded as, ‘fundamentally in sympathy with the Communist line’.68 One Soviet intelligence officer, who served in the KGB residency, or station, in New Delhi, subsequently confirmed that in seeking to blacken the CIA’s reputation in the subcontinent, the KGB had made full use of, ‘extensive contacts within political parties, among journalists and public organizations. All were enthusiastically brought into play.’69 The scale of the Soviet ‘black’ propaganda initiative was such that, in the IRD’s estimation, by April 1967 the anti-CIA campaign run in the Indian press had begun, ‘over- reaching itself and thus blurring the target.’ Even the New Delhi law practice run by the rabidly anti-American former Indian Defence Minister, Krishna Menon, one IRD official noted with wry amusement, ‘has been accused of receiving C.I.A. funds!’70 The Soviets problems were compounded when the results of the enquiry into CIA ‘interference’ in India’s general election, which M. C. Chagla had promised the country’s exercised parliamentarians, became available in June. In the report delivered to Chagla by the IB, both the CIA and the KGB were implicated in funding the election campaigns of their preferred candidates. 72 Douglas Heck to Carleton S. Coon Jr. ‘Soviet Policy towards India’, 28 February 1968, RG59, Bureau of Near Eastern and South Asian Affairs Records Relating to India 1966-75, Lot file 72D5, Box 5 India-USSR 1968, NARA. The CIA and Cold War propaganda in India So much evidence was accumulated by India’s intelligence service relating to KGB interference in the election, however, that the Soviet Embassy successfully lobbied Chagla to suppress the 25 reports findings.71 Although never quite recapturing the same level of intensity reached in the first half of 1967, the KGB and CIA continued to fight a ‘bitter’ and protracted propaganda war in the subcontinent during the final years of the decade.72 In New Delhi, Chester Bowles repeatedly voiced concern that the distracted and politically weakened Johnson administration was steadily ceding American influence to Soviet Union in South Asia. ‘The U.S. Government, diverted and confused by Vietnam and social tensions, seems to be pulling back [in India],’ a disconsolate Bowles wrote to Harrison Salisbury, the New York Times journalist, in May 1968. ‘In the meantime the Soviets are successfully stepping up their efforts here with a great amount of skills and resources.’73 Bowles warning proved to be prescient. Under the Nixon administration in the early 1970s, the Soviet Union was to redouble its efforts to use the CIA as a political wedge with which to unhinge the United States’ relationship with Indira Gandhi’s government. 71 ‘Brief Progress Report on IRD work in India, 1 January-30 June 1967’, and ‘India’, IRI/545/5, 15 November 1967, FCO 95/290, TNA. , 73 Chester Bowles to Harrison Salisbury, 2 May 1968, Box 1, Folder Chester Bowles, HSP. ARA. r Bowles to Harrison Salisbury, 2 May 1968, Box 1, Folder Chester Bowles, HSP. p y y ( ) ( p ) pp 76 See, for example, Conversation between President Nixon and Kissinger, Washington, 26 May 1971, and Conversation Nixon, Kissinger, and Haldeman, Washington, 5 November 1971, Foreign Relations of the United States, 1969–1976, Volume E–7, Documents on South Asia, 1969–1972, Documents 135 and 150, INTERNET http://history.state.gov/historicaldocuments/frus1969-76ve07. See also, Kissinger, White House Years, p. 848. Kissinger, The White House Years (London: Weidenfeld & Nicolson, 1979), p. 849. 75 Nixon, RN, pp. 526-31. For an assessment of Indo-US relations under Nixon, see, in particular, R. J. McMahon, “The Danger of Geopolitical Fantasies: Nixon, Kissinger, and the South Asia Crisis of 1971," in Nixon and the World: American Foreign Relations, 1969-197, eds. Fredrik Logevall and Andrew Preston (Oxford, 2008), Sanjit Gandhi, ‘The Tilt: The U.S. and the South Asian Crisis of 1971’, National Security Archive Electronic Briefing Book No. 79, 16 December 2002, INTERNET, http://www.gwu.edu/~nsarchiv/NSAEBB/NSAEBB79/; and, Dennis Kux, India and the United States: Estranged Democracies (Washington: National Defense University Press, 1993). For a British take on the South Asia Crisis of 1971, see, Simon C. Smith, ‘Coming Down on the Winning Side: Britain and the South Asia Crisis, 1971’, Contemporary British History, 24 (4), (September 2010), pp. 451-70. 76 74 Richard Nixon, RN: The Memoirs of Richard Nixon (New York: Simon & Schuster, 1978), p. 131; Henry Kissinger, The White House Years (London: Weidenfeld & Nicolson, 1979), p. 849. Richard Nixon, RN: The Memoirs of Richard Nixon (New York: Simon & Schuster, 1978), p. 13 Kissinger, The White House Years (London: Weidenfeld & Nicolson, 1979), p. 849. Richard Nixon, RN: The Memoirs of Richard Nixon (New York: Simon & Schuster, 1978), p. 131; H Indira Gandhi, the Nixon administration and the politics of intelligence On entering the White House in January 1969, Richard Nixon quickly concluded that peace and stability in South Asia could best be maintained by furnishing India’s rival, Pakistan, with sufficient American economic and military assistance to counter-balance New Delhi’s preponderant regional power. Back in the 1950s, Nixon had earned India’s disfavour by enthusiastically supporting the Eisenhower administration’s decision to enter into an alliance with Pakistan. At the time, the then Vice-President had failed to warm to Nehru, whom he considered cold, aloof and bent on consolidating India’s dominant influence not only in South 26 Asia, but across the Middle East and Africa. Nixon’s National Security Advisor, and latterly Secretary of State, Henry Kissinger, subsequently reflected that the President always felt more comfortable dealing with the, ‘bluff, direct military chiefs of Pakistan…than the complex and apparently haughty Brahmin leaders of India.’74 In December 1971, to Indira Gandhi’s fury, Nixon ‘tilted’ decisively towards Pakistan following the outbreak of Indo-Pakistan hostilities. In turn, having frustrated his effort to prevent East Pakistan’s transformation into the independent nation state of Bangladesh, Nixon returned Gandhi’s animus.75 After 1971, an atmosphere of deep mutual distrust pervaded Washington’s relations with New Delhi. Strained Indo-US tensions were subsequently aggravated by differences over issues as diverse as the Gandhi government’s ties to the Soviet Union; the on going war in Vietnam; an outstanding rupee debt which New Delhi had accumulated purchasing US grain shipments in 1960s; and, India’s fledgling nuclear weapons programme. Within the confines of Nixon’s Oval Office, Indians came to be characterised as ‘bastards’, while Gandhi herself was derided as an ‘old witch’ and a ‘bitch’.76 It was with an understandable degree of trepidation therefore, that on 24 July 1972, Kenneth Keating, Nixon’s ambassador to India, made a final call on Indira Gandhi before returning to the United States to oversee the President’s re-election campaign. Keating had 27 anticipated that his encounter with Gandhi might prove awkward. During a thirty-minute audience with the Indian premier, however, the ambassador was left stunned by the force of the ‘emotional and distorted’ attack that Gandhi launched against the Nixon administration. Significantly, Gandhi chose to vent her spleen by presenting Keating with a long and well- worn series of allegations linking the CIA with subversive activities in India. 77 Keating to the Department of State, 24 July 1972, RG 59, Central Files 1970-1973, POL INDIA-US, NARA. Indira Gandhi, the Nixon administration and the politics of intelligence Forces inside American government, Gandhi assured an incredulous Keating, were ‘working against us in India’; ‘cooperating with communist extremists’ to destabilise her administration; and encouraging ‘a lot of American professors...to engage in improper activities injurious to India.’ ‘Incredible!’, the dumbstruck Keating subsequently cabled to Washington, ‘My successor has an even tougher task ahead than I anticipated.’77 The rhetorical barbs that Indira Gandhi threw Keating’s way were, in part, driven by domestic political expediency. Whenever Gandhi’s domestic fortunes flagged, the Indian economy tanked, or the Congress Party found itself in political difficulty, it was all too easy to blame the malign influence of a ‘foreign hand’. During 1969, Gandhi had fallen afoul of the Congress Party’s elder statesman, known as the Syndicate, by shifting decisively to the political left. In July that year, Gandhi nationalised fourteen of India’s commercial banks, and sacked her conservative finance minister, and bitter political rival, Morarji Desai. The syndicate reacted by dismissing Gandhi from the Congress Party. In response, Gandhi formed a new breakaway party, Congress (R). In February 1971, India held its fifth general election, and Gandhi’s reconstituted party, which drew on support from the Moscow sponsored wing of the CPI, was returned to power on the back of the slogan, Garibi Hatao, or abolish poverty. In the months that followed, the CPI exerted pressure on its Congress (R) partner to adopt a radical socialist agenda by organising periodic demonstrations of civil disobedience, or ‘mass satyagraha’, in support of land reform, full employment, and wealth redistribution. 28 In the autumn of 1972, confronted with mass CPI orchestrated protests against rampant inflation, food shortages, and rising unemployment, Gandhi and senior Congress (R) officials elected not to admonish their communist allies, but instead to implicate he CIA, and the Agency’s domestic “accomplices”, with fomenting unrest inside India. Beginning in late September, Congress (R) president, S. D. 78 ‘The Poodle Not House-Trained’, P. H. Roberts to Mr Martin, Mr Chittenden and Mr Everett, 19 October 1972, FCO 95/1347, TNA. 79 Garvey to FCO, No. 2595, 23 October 1972, FCO 95/1388, TNA. 78 ‘The Poodle Not House-Trained’, P. H. Roberts to Mr Martin, Mr Chittenden and Mr Everett, 19 October 1972 FCO 95/1347 TNA 1972, FCO 95/1347, TNA. 79 Garvey to FCO, No. 2595, 23 October 1972, FCO 95/1388, TNA. , , 1972, FCO 95/1347, TNA. Indira Gandhi, the Nixon administration and the politics of intelligence Sharma, delivered a string of public speeches in which he claimed to have firm evidence, which despite repeated demands from the Indian press he declined to produce, proving that the CIA was colluding with right wing opponents of the government to, ‘throttle the Indian economy.’ On 9 October, on the eve of a national Congress (R) convention in Gujarat, Gandhi added her voice to the chorus of anti-CIA rhetoric by openly asserting that: ...elements in India, who had always been voicing opposition to the Government’s political economic and foreign policies, were receiving encouragement from foreign sources. The opposition was identical with criticism from abroad not only in regard to ideas, but also in the choice of words used. This made it clear that there were contacts between India’s critics at home and abroad.78 British officials in India, and much of the country’s English language press, dismissed the allegations levelled by Sharma and Gandhi at he CIA, as a ‘barefaced political stunt.’ The British High Commission in New Delhi informed London that the, ‘CPI have plainly been up to their necks in recent agitation’, whilst, ‘Indian security professionals do not put down [the] agitation to [a] CIA conspiracy.’79 The right-wing Indian periodical, Thought, observed sardonically that Gandhi had chosen to deliver her anti-CIA polemic in the very location where Mahatma Gandhi’s autobiography, Experiments with Truth, had played out. In an editorial entitled, ‘Hitting Out Wildly’, Calcutta’s respected daily, The Statesman, disparaged Gandhi’s implication that India’s economic difficulties were ‘due to CIA “machinations”’, as 29 simply ‘too infantile to be considered seriously.’80 Casting a satirical, if equally damning eye on the CIA rumpus, the Indian Express printed a cartoon on its front page which depicted Sharma advising the Indian premier that, ‘“This week’s CIA activities include four price-rise demonstrations, seven buses hijacked by students, plus one cyclone in Orissa.”’81 In lashing out at the CIA, Indira Gandhi undoubtedly calculated that the Agency would serve as a convenient external scapegoat for internal troubles, and at the same time provide a useful means of placating her CPI ally with a show of anti-Western bluster. g , , , IS Media Reaction Report, New Delhi, 26 September 1972, FCO 95/1388, TNA. p , , p , , 82 Garvey to FCO, No. 2595, 23 October 1972, FCO 95/1388, TNA. itting Out Wildly , The Statesman, 12 October 1972. See also, Queering the Pitch , Hindustan Times, 2 mber 1972. Thought, Vol. XXIV, No. 42, 14 October 1972. ‘Hitting Out Wildly’, The Statesman, 12 October 1972. See also, ‘Queering the Pitch’, Hindustan Time p g , , , 81 USIS Media Reaction Report, New Delhi, 26 September 1972, FCO 95/1388, TNA. 82 g y , , , Q g September 1972. Thought, Vol. XXIV, No. 42, 14 October 1972. Hitting Out Wildly , The Statesman, 12 October 1972. See also, Queering the Pitch , Hindustan Times, 29 September 1972. Thought, Vol. XXIV, No. 42, 14 October 1972. 81 USIS Media Reaction Report New Delhi 26 September 1972 FCO 95/1388 TNA Indira Gandhi, the Nixon administration and the politics of intelligence Moreover, the British suspected that Gandhi would derive no little satisfaction from the knowledge that, ‘these allegations would stick in President Nixon’s gizzard.’ Not least, Indian government officials were fully aware that the CIA, which Nixon denigrated as a haven for Ivy League liberals at odds with his political philosophy, represented a particularly sensitive pressure point where the President was concerned. While on the surface India’s wider economic and strategic interests suggested that choosing to pick a quarrel with the United States made little sense, Gandhi was deemed shrewd enough to reason that, with Nixon seemingly assured of a second presidential term, ‘the US would be in no hurry anyway to resume aid, participate in debt rescheduling or make any other gesture towards India. Hence further deterioration in Indo/US relations was no skin off her nose while bringing internal benefits.’82 Having beaten so hard on the anti-CIA drum, senior US officials began to worry that Gandhi risked creating a domestic political monster over which she would be able to wield increasingly less control. The Congress left wing, it seemed, aided and abetted by the CPI, had ‘gradually undercut’ Indian officials and politicians seeking better Indo-US relations and more pragmatic economic policies. ‘Ironically this latter group probably includes Mrs. 30 Gandhi…’, one US official diplomat, ‘But the [anti-CIA] campaign is tailored to cater to her prejudices and sensitivities.’83 Gandhi’s tendency to conflate political opportunism and the CIA appeared to be equally in evidence in June 1975, after the Allahabad High Court controversially found the Indian Prime Minister guilty of minor electoral mal-practice during the 1971 general election. If upheld by India’s Supreme Court, the ruling threatened to invalidate Gandhi’s status as an MP and bring down her government. Later the same month, with her political opponents scenting blood and protestors having taken to India’s streets in almost equal numbers to both support and denounce her, Gandhi declared a State of Emergency, suspended civil liberties, and jailed her political opponents. Socialist Weekly, 15 August 1975. 86 Schneider to Secretary of State, No. 08067, ‘Indo-US Relations’, 19 June 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. See also, ‘Conspiracy Theories’, Hindustan Times, 1 October 2005; and, Moynihan, A Dangerous Place, p. 150. , 84 Schneider (Embassy New Delhi) to Secretary of State, No. 07903, ‘Allahabad Court Case’, 16 June 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. 85 83 Moynihan to Secretary of State, No. 03617, ‘Thunder on the Left: “Left” Attacks on the US and “Pragmatic” Domestic Policies’, 16 March 1974, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1974- 21/31/1974, NARA. y y , , Domestic Policies’, 16 March 1974, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1 21/31/1974, NARA. 83 Moynihan to Secretary of State, No. 03617, ‘Thunder on the Left: “Left” Attacks on the US and “Pr hneider (Embassy New Delhi) to Secretary of State, No. 07903, ‘Allahabad Court Case’, 16 June 1975, R entral Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. cialist Weekly, 15 August 1975. , g y , 85 Socialist Weekly, 15 August 1975. Indira Gandhi, the Nixon administration and the politics of intelligence Almost immediately, senior Congress Party figures loyal to Gandhi began attributing the unrest that had preceded the imposition of martial law to a ‘foreign hand’, and declared defiantly that the government, ‘would not allow Delhi to be turned into Chile.’84 On 15 August, as India commemorated its independence from British imperial rule, Gandhi gave an interview to a Congress Party newssheet, Socialist Weekly, in which she observed pointedly, ‘Have these several Western countries not given full moral and material support to the most authoritarian regimes of Africa and Asia? Have we so soon forgotten what happened to Chile?’85 Those members of India’s political opposition that had avoided imprisonment openly mocked the idea that external forces had been plotting to subvert the government. One Indian MP took to wearing a badge that proclaimed, ‘I am a CIA agent’, and made a tidy profit into the bargain by selling on copies to his parliamentary colleagues.86 Within the American 31 Embassy, however, a file of Indian press clippings linking the United States government, and more particularly the CIA, with a conspiracy to overthrow Gandhi, grew larger by the day.87 The KGB residency in New Delhi, which in a reflection of the importance that the Soviet intelligence service attached to India, had been upgraded to the status of a main residency in the early 1970s, played an especially active part in feeding Gandhi’s suspicion that the CIA was actively plotting her demise.88 In doing so, the KGB was able to call upon the services of an Indian press agency, two daily and eight weekly Indian newspapers, and four popular magazines, all which received covert Soviet funding. During the course of 1972 alone, the Soviet residency in New Delhi claimed credit for placing almost 4,000 pro-Soviet and anti- US articles in the Indian press.89 Whilst it remains likely that KGB officers on the subcontinent exaggerated the extent to which they were able to manipulate India’s media, one of the world’s leading authorities on the KGB has gone as far as to state that, ‘India under Indira Gandhi was...probably the area for more KGB active measures than anywhere in the world.’90 One of the KGB’s notable propaganda successes in India occurred in December 1972. That month, three of India’s leading newspapers, The Times of India, Hindustan Times, and Indian Express, reproduced a story which had first appeared in the Kuwaiti daily, Al Siyassa. p , , , p 90 KGB operations in India had become so extensive by 1974, that a new department, the Seventeenth department, was established within its First Chief (Foreign Intelligence) Directorate, which dealt exclusively with South Asia. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 324. 87 Saxbe to Secretary of State, No. 08751, ‘Press Allegations of US Involvement in India Domestic Situation’, 1 July 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. 88 The KGB’s main resident, or station chief, in India between 1970 and 1975, Yakov Profofyevih Medyanik, was responsible for four KGB outposts in the subcontinent, in New Delhi, Bombay, Calcutta and Madras. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 321. , , p 89 Figures from the KGB’s archives list 3,789 articles as being planted in the Indian press in 1972; 2,760 in 1973; 4,486 in 1975; and 5,510 in 1975. By way of comparison, in Italy, the KGB took credit for placing only 48 articles in the local press in 1975, and 63 in 1976. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 324. July 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. 88 The KGB’s main resident, or station chief, in India between 1970 and 1975, Yakov Profofyevih Medyanik, was responsible for four KGB outposts in the subcontinent, in New Delhi, Bombay, Calcutta and Madras. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 321. 4,486 in 1975; and 5,510 in 1975. By way of comparison, in Italy, the KGB took credit for placing only 48 articles in the local press in 1975, and 63 in 1976. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 324. 90 KGB operations in India had become so extensive by 1974, that a new department, the Seventeenth department, was established within its First Chief (Foreign Intelligence) Directorate, which dealt exclusively with South Asia. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 324. , , p 89 Figures from the KGB’s archives list 3,789 articles as being planted in the Indian press in 1972; 2,760 in 1973; 4,486 in 1975; and 5,510 in 1975. By way of comparison, in Italy, the KGB took credit for placing only 48 articles in the local press in 1975, and 63 in 1976. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 324. 90 KGB operations in India had become so extensive by 1974, that a new department, the Seventeenth department, was established within its First Chief (Foreign Intelligence) Directorate, which dealt exclusively xbe to Secretary of State, No. 08751, ‘Press Allegations of US Involvement in India Domestic Situation’, 87 Saxbe to Secretary of State, No. 08751, ‘Press Allegations of US Involvement in India Domestic Situation’, 1 July 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. 88 The KGB’s main resident, or station chief, in India between 1970 and 1975, Yakov Profofyevih Medyanik, was responsible for four KGB outposts in the subcontinent in New Delhi Bombay Calcutta and Madras 87 Saxbe to Secretary of State, No. 08751, ‘Press Allegations of US Involvement in India Domestic Situ J l 1975 RG 59 C t l F i P li Fil El t i T l 1/1/1975 21/31/1975 NARA y , , g 975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. 87 Saxbe to Secretary of State, No. 08751, ‘Press Allegations of US Involvement in India Domestic Situation’, 1 July 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. 88 The KGB’s main resident, or station chief, in India between 1970 and 1975, Yakov Profofyevih Medyanik, was responsible for four KGB outposts in the subcontinent, in New Delhi, Bombay, Calcutta and Madras. Andrew and Mitrokhin, The Mitrokhin Archive II, p. 321. 89 Figures from the KGB’s archives list 3,789 articles as being planted in the Indian press in 1972; 2,760 in 1973; 4,486 in 1975; and 5,510 in 1975. By way of comparison, in Italy, the KGB took credit for placing only 48 articles in the local press in 1975 and 63 in 1976 Andrew and Mitrokhin The Mitrokhin Archive II p 324 Indira Gandhi, the Nixon administration and the politics of intelligence The report alleged that the CIA had been complicit in a number of plots to assassinate Indira Gandhi. In the Indian parliament, communist MPs seized on the news, and demanded an 32 official investigation to establish its veracity. D. D. Puri, the CPI Member of Parliament for Haryana, declared theatrically that the allegation represented: official investigation to establish its veracity. D. D. Puri, the CPI Member of Parliament for Haryana, declared theatrically that the allegation represented: ...the most earth-shaking news...I would like to get an unequivocal assurance from the Government that if these is an iota of truth in this, if one-hundredth or even one-thousandth of this is true, there is no question of opening trade relations or any other type of relations with the United States of America. The Government must give us an assurance to this effect here and now.91 To the British, the Al Siyassa story displayed all the hallmarks of a Soviet ‘smear’, and constituted a clear, ‘step up in the CIA/KGB slanging match in India.’92 Above all, the growing sophistication of the Soviet disinformation campaign in India impressed the British. Al Siyassa, it was noted, had established a reputation as an independent, politically moderate, and well-informed Arab newspaper, with no apparent connection to Soviet intelligence. Its editor, Ahmed Jarallah, who was known to senior IRD officers, was regarded as a generally pro-Western and conservative figure. In the past, Jarallah’s criticism of the Soviet presence in the Middle East had even seen him labelled as a CIA agent. By filtering anti-CIA propaganda through Jarallah, or more plausibly a more junior member of the Al Siyassa staff, the British conceded that the Soviets had ensured that any suggestion that the newspaper was a vehicle for KGB propaganda, ‘would probably ring false.’93 To the British, the Al Siyassa story displayed all the hallmarks of a Soviet ‘smear’, and constituted a clear, ‘step up in the CIA/KGB slanging match in India.’92 Above all, the growing sophistication of the Soviet disinformation campaign in India impressed the British. Moreover, to the intense frustration of American and British officials, the expansion of KGB operations in India continued to go largely unreported in the Indian press. 92 ‘Alleged CIA Plot’, P H Roberts to H J Spence, 7 December 1972; Background note on Al Siyassa, 6 December 1972, FCO 95/1388, TNA. 93 91 Transcript of Indian Parliamentary debate, Rajya Sabha, RB: GDS T-1, 4 December 1972, FCO 95/1388, TNA. 92 93 B. R. Berry background note on Al Siyassa, 6 December 1972; Peter Joy to P.H. Roberts, 20 December 1972, FCO 95/1388, TNA. , 95 Draft copy of Henderson address to Conference of US Foreign Service Officers, February 1951, Box 8, India Misc folder, Loy W. Henderson Papers, Library of Congress, Manuscripts Division, Washington DC. 94 ‘Alleged CIA and KGB Activity in India’, H J Spence to P H Roberts, Ref. PRG2/302/5, 27 October 1972, FCO 95/1388, TNA. 95 94 ‘Alleged CIA and KGB Activity in India’, H J Spence to P H Roberts, Ref. PRG2/302/5, 27 October 1972, FCO 95/1388, TNA. 95 Draft copy of Henderson address to Conference of US Foreign Service Officers, February 1951, Box 8, India Indira Gandhi, the Nixon administration and the politics of intelligence Efforts made by the CIA, local United States Information Service officials, and the IRD to counter Soviet inspired attacks on the CIA by placing articles critical of the KGB in the mainstream Indian press, generated relatively modest results. At the height of the 1972 anti-CIA 33 campaign in India, the British could, ‘only find a handful of recent references to suspected KGB activity’ in the subcontinent’s newspapers.94 In one sense, the KGB’s comparative anonymity was undoubtedly reflective of the generally positive tenor of Indo-Soviet relations under Indira Gandhi. As previously indicated, from the mid-1960s onward, the strength of India’s economic and strategic links with the West steadily dissipated. Increasingly, New Delhi looked to Moscow to meet its economic and security needs, rather than Washington or London, a shift underlined by the signing of the Indo-Soviet Treaty of Peace, Friendship and Co-operation in August 1971. Deeper cultural factors, however, may also go some way toward explaining the varying levels of Indian interest shown in the CIA and the KGB. At the turn of the 1940s, Loy Henderson detected an almost unconscious tendency on the part of Indians to, ‘exercise less restraint in finding fault with the United States than with the Soviet Union.’ Henderson attributed this phenomenon to the ‘deeper emotional responses’ evoked in Indians by American attitudes to questions of race, colonialism, and wealth redistribution, than by Soviet constraints on civil liberties and freedom of expression. Or put another way, the ambassador observed, ‘a general feeling [in the subcontinent] that the United States, as the most prosperous and technically the most advanced nation, is just a little too smug and that a bit of criticism now and then is good for the soul.’95 United States Information Agency (USIA) surveys broadly supported Henderson’s impressions. One poll conducted by the USIA on ‘Indian Attitudes towards the U.S and U.S.S.R’ in 1956, revealed that respondents in India typically perceived America as ‘aggressive’ and ‘exploitative’, while the Soviet Union was 34 generally deemed to be, ‘friendly to Asian countries’, and possess an, ‘economic system [that] leads to a rise in the standard of living of all in the country.’96 Specific actions undertaken by the United States government and the CIA in the early 1970s can also be seen to have played a part in reinforcing the negative perception of America’s foreign intelligence service held many by Indians, and by Indira Gandhi in particular. g y p 98 Gandhi’s political confidantes and biographers have made much of the ‘Chilean’ effect on the Indian premier. See, P. N. Dhar, Indira Gandhi, the ‘Emergency’ and Indian Democracy, (New Delhi: Oxford University Press, 2000), p. 254; Katherine Frank. Indira: The Life of Indira Nehru Gandhi (London: Harper Collins, 2002), pp. 373-4; and, Inder Malhotra, Indira Gandhi: A Personal and Political Biography (London: Hodder & Stoughton, 1989), pp. 291-2. 99 Moynihan to Secretary of State, No. 12063, ‘The United States as a Counter-Revolutionary Power: The Case of India and Chile’, 10 September 1974, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1974- 21/31/1974, NARA. p p , , , 97 Kissinger to Moynihan, No. 242175, ‘Press Allegations’10 December 1973, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1973-21/31/1973, NARA. See, also Henry, White House Years, p. 901. 96 ‘Indian Attitudes Towards U.S. and U.S.S.R’, 25 May 1956, RG306, Records of the US Information Agency, Office of Research Special 'S' Reports 1953-1983, Box 11, Folder S-25 56, NARA. y p p y g g 102 Moynihan to Secretary of State, No. 12063, ‘The United States as a Counter-Revolutionary Power: The Case of India and Chile’, 10 September 1974, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1974- 21/31/1974, NARA. 101 Moynihan to Kissinger, 11 March 1973, I-377 Folder India Subject File White House 1973, Daniel P. Moynihan Papers, Manuscript Division, Library of Congress, Washington, D.C. 100 For details of the impact Colby’s testimony had in India, see The Patriot and The Times of India, 9 September 1974. Indira Gandhi, the Nixon administration and the politics of intelligence Circumstantial evidence linking the United States with those responsible for the assassination of Bangladesh’s premier, Sheikh Mujibur Rahman, in August 1975, and exposes in The New York Times alleging that the CIA had run an agent inside Gandhi’s cabinet during the 1971 Indo-Pakistan War, unsettled the Indian premier.97 Above all, however, the CIA’s complicity in the bloody right-wing coup that removed the government of Salvador Allende in Chile, in September 1973, deeply affected Gandhi. After Allende’s demise, Indira Gandhi was genuinely concerned that she might be the next target on the Nixon administration list for regime change.98 In a bid to reassure Gandhi that Washington wished her no harm, the American Deputy Chief of Mission in New Delhi paid a call on the Indian premier and stated unequivocally, ‘that of course, the US had not’ meddled in Chilean domestic politics.99 Weeks later, Gandhi heard the CIA’s Director, William E. Colby, confirm to a US Congressional committee that, between 1970 and 1973, the Agency had spent more than $8 Congressional committee that, between 1970 and 1973, the Agency had spent more than $8 35 million in an effort to destabilize the Allende government.100 Earlier in 1973, shortly after he arrived in India to replace Kenneth Keating as US ambassador, Daniel Patrick Moynihan had cabled to Henry Kissinger that, ‘The paranoia out here is thicker than the dust.’101 Following Colby’s testimony, a dispirited Moynihan lamented that by handling the Chile question in such an inept manner, the United States government had done a first rate job of shooting itself in the foot in India. On 10 September 1974, Moynihan complained to Kissinger that Gandhi was now convinced: ...that we would be content to see her overthrown, as we have, to her mind, been content to see others like her overthrown. She knows full well that we have done our share and more of bloody and dishonourable deeds. This as such is not her concern. She knows all too much of such matters. It is precisely because she is not innocent, not squeamish, and not a moralizer that her concern about American intentions is real and immediate. , 103 ‘Concern by India on C.I.A. Related’, The New York Times, 13 September 1974. Indira Gandhi, the Nixon administration and the politics of intelligence And of course the news from the United States, as printed in the Indian press, repeatedly confirms her worst suspicions and genuine fears.102 Moynihan’s gloom deepened further, when the investigative journalist, Seymour Hersh, obtained a leaked copy of his cable to Kissinger, and splashed the ambassador’s observations on Gandhi across the front page of the New York Times.103 Moreover, to Moynihan’s fury, Colby went on to compound Gandhi’s anxiety that the CIA was out to get her, by mounting a robust public defence of the United States’ need to exercise a covert action capability abroad. On 13 September, in an address before the Fund for Peace Conference, having first acknowledged the Agency’s track record of ‘assist[ing] 36 America’s friends against her adversaries in their contest for control of a foreign nation’s political direction’, Colby proceeded to argue that: America’s friends against her adversaries in their contest for control of a foreign nation’s political direction’, Colby proceeded to argue that: ...a sovereign nation must look ahead to changing circumstances. I can envisage situations in which the United States might well need to conduct covert action in the face of some new threat that developed in the world...I thus would think it mistaken to deprive our nation of the possibility of some moderate covert action response to a foreign problem and leave us with nothing between a diplomatic protest and sending the Marines.104 On 2 December, Colby reiterated his support for covert action in an interview published in US News & World Report. After being replayed in the Indian press, this had the effect of whipping up a ‘wholly predictable storm’ in the subcontinent. In response, a perplexed Moynihan was left ‘groping’ for an explanation as to why Colby considered it necessary, or advisable, to openly debate the merits and morality of CIA clandestine operations. The Director of the KGB, the US ambassador noted ruefully, felt no similar compulsion to air the Soviet intelligence services dirty laundry in public. On 3 December, in a stinging cable sent to Henry Kissinger, Moynihan asked pointedly, ‘It is out of the question that some thought might be given in Washington to the effect in India of statements such as the Director has made? It is that nobody knows? p , p , y g , g , ( ) 105 Moynihan to Kissinger, No. 16066, ‘CIA’, 3 December 1974, I: 371, India: Central Intelligence Agency Folder, DPMP. 104 William E. Colby, ‘The View from Langley’, Address to the Fund for Peace Conference on ‘The CIA and Covert Actions’, 13 September 1974, I: 371, India: Central Intelligence Agency Folder, Daniel P. Moynihan Papers, Manuscript Division, Library of Congress, Washington, D.C. (hereafter DPMP). Covert Actions’, 13 September 1974, I: 371, India: Central Intelligence Agency Folder, Daniel P. Moynihan Papers, Manuscript Division, Library of Congress, Washington, D.C. (hereafter DPMP). 104 William E. Colby, ‘The View from Langley’, Address to the Fund for Peace Conference on ‘The CIA and Covert Actions’ 13 September 1974 I: 371 India: Central Intelligence Agency Folder Daniel P Moynihan Covert Actions , 13 September 1974, I: 371, India: Central Intelligence Agency Folder, Dani Papers, Manuscript Division, Library of Congress, Washington, D.C. (hereafter DPMP). 107 Moynihan to Secretary of State, No. 15610, ‘New York Time Query on CIA in India’, 20 November 1974, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1974-21/31/1974, NARA. See also, ‘Kissinger Assures India That C.I.A. Won’t Interfere’, The New York Times, 31 October 1974. , , 108 Schneider to Secretary of State, No. 12626, ‘Press Treatment of Kissinger, Atherton Congressional Testimony’, 29 October 1974, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1974-21/31/1974, NARA; Andrew and Mitrokhin, The Mitrokhin Archive II, p. 327. 106 Moynihan to Kissinger, No. 16066, ‘CIA’, 3 December 1974, I: 371, India: Central Intelligence Agency Folder, DPMP. 107 , , 108 Schneider to Secretary of State, No. 12626, ‘Press Treatment of Kissinger, Atherton Congressional Testimony’, 29 October 1974, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1974-21/31/1974, y , , , g y , g , NARA; Andrew and Mitrokhin, The Mitrokhin Archive II, p. 327. Indira Gandhi, the Nixon administration and the politics of intelligence Or is it that nobody cares?’105 Kissinger had, Moynihan reminded the Secretary of State, personally affirmed to Gandhi during a visit to New Delhi back in October, that the United States was not ‘directly or indirectly’ engaged in activities designed to destabilise India. ‘In what but an insane situation could a Director of Central Intelligence’, the ambassador speculated, ‘...find himself giving interviews to the press asserting a right to do what the Secretary of State had said was not being done in India?’ Half the Indian government, Moynihan emphasised, already 37 suspected that the CIA was up to no good in India, while the other half were astute enough to recognise that by demonising the Agency and its purportedly nefarious activities in the subcontinent, they could outflank the Congress Party’s domestic critics. Colby’s outburst of candour, Moynihan underlined, had once again increased the likelihood that the CIA would be dragged into India’s internal politics. ‘Is there no way’, the ambassador urged Kissinger, ‘that [Colby’s] statement can be retracted?’106 Indira Gandhi’s repeated assertion that the malevolent hand of the CIA lay behind most of India’s problems, whether genuinely held or not, consistently undermined Washington’s efforts to normalise Indo-US relations for much of the latter Cold War period. As Moynihan had underlined, when visiting India in October 1974, before getting down to any substantive business with his hosts, Henry Kissinger felt compelled to publicly affirm the ‘Kissinger Rule’, which committed Washington to repatriate any American official that the Indian government suspected of interference in its internal affairs.107 With the Ford administration having timed its first major policy statement on Indo-US relations to coincide with Kissinger’s arrival in India, the US Embassy in New Delhi optimistically hoped to draw some much needed positive publicity in the following days press. Instead, to the satisfaction of the KGB, which had passed a familiar batch of fabricated stories of CIA misdeeds on to friendly journalists, India’s newspapers ran with headlines proclaiming, ‘India asked to name CIA agents’, and ‘Suspicious U.S. y, , g ( ) 111 Daniel Patrick Moynihan, ‘We would Like India to be What India Is’, Newsweek, 20 January 1975. g y g 110 Saxbe to the Department of State, No. 1767 February 5, 1976, National Security Adviser, Presidential Country Files for Middle East and South Asia, Box 12, India, State Telegrams to Secretary of State NODIS (3), Gerald Ford Presidential Library, Ann Arbor, Michigan (hereafter GFPL). ou y es o dd e as a d Sou s a, o , d a, S a e e eg a s o Sec e a y o S a e NO erald Ford Presidential Library, Ann Arbor, Michigan (hereafter GFPL). 109 Saxbe to Secretary of State, No. 09951, ‘Ambassador Saxbe’s Meeting with Mrs Gandhi’, 24 July 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. Saxbe to Secretary of State, No. 09951, Ambassador Saxbe s Meeting with Mrs Gandhi , 24 July 1975, RG 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA. y , , g 59, Central Foreign Policy Files, Electronic Telegrams, 1/1/1975-21/31/1975, NARA 110 109 Saxbe to Secretary of State, No. 09951, ‘Ambassador Saxbe’s Meeting with Mrs Gandhi’, 24 July 1 59 C t l F i P li Fil El t i T l 1/1/1975 21/31/1975 NARA y , , gn Policy Files, Electronic Telegrams, 1/1/1975-21/3 Indira Gandhi, the Nixon administration and the politics of intelligence Men to be removed from India.’108 Despite the assurances provided to Gandhi by Moynihan, and his successor, Bill Saxbe, that they would resign if evidence emerged of CIA interference in Indian domestic affairs, Indian politicians and 38 sections of the country’s media continued to accuse the CIA of acts of subversion throughout the 1970s.109 Indeed, an indignant Saxbe spent much of his time as ambassador in India between February 1975 and late 1976, making it clear to every senior Indian official that he encountered, that an improvement in Indo-US relations, ‘just cannot take place while the Prime Minister and other high Indian leaders continue to poke away at the US.’110 By early 1976, however, with Gandhi’s government having disregarded numerous private warnings from the State Department to desist from publicly criticising the CIA’s purported activities in the subcontinent, the Ford administration resolved to punish New Delhi. Over the previous five years, the upsurge in anti-American sentiment in India had prompted both the United States government and US business to disengage from subcontinent. Between 1972 and 1975, the number of staff serving in the US Embassy in New Delhi halved. During the same period American companies turned their backs on India, with fewer than 40 US businessmen living and working amongst 600 million Indians.111 In Washington, India’s ambassador, T. N. Kaul, was frozen out by the White House and denied access to both President Ford and Kissinger. In January 1976, the Ford administration ratcheted up pressure on Gandhi by announcing the curtailment of a range of joint Indo-US scientific and educational programmes, and the postponement plans to resume developmental assistance to India, which had been halted back in 1971 during the Indo-Pakistan War. Given India’s continuing financial problems, the latter measure was expected to hit Gandhi’s government particularly hard. Between 1965 and 1971, India had received $4.2 billion in American economic aid, $1.5 billion of which had been appropriated during the early years of the Nixon administration. In rationalising the United States’ punitive policy to India’s 39 Foreign Minister, Y.B. Chavan, Saxbe confirmed that the Ford administration had simply run out of patience with Indira Gandhi and her government. ‘[We have] reach[ed] a point,’ Saxbe informed Chavan, ‘at which we don't feel can continue to cooperate if [these] attacks [on the CIA] continue. Indira Gandhi, the Nixon administration and the politics of intelligence We have said repeatedly that if the G[overnment] O[f] I[ndia] has any evidence of US interference we will act to eliminate it. I would resign.’112 112 Saxbe to the Department of State, No. 787, 15 January 1976, ‘Mrs. Gandhi’s Attacks on US’, National 112 Saxbe to the Department of State, No. 787, 15 January 1976, ‘Mrs. Gandhi’s Attacks on US’, National Security Adviser, Presidential Country Files Middle East and South Asia, Box 12, India, State to Sec State p , , y , , Security Adviser, Presidential Country Files Middle East and South Asia, Box 12, India, State to Sec State NODIS (3), GPFL. 112 Saxbe to the Department of State, No. 787, 15 January 1976, ‘Mrs. Gandhi’s Attacks on US’, National Security Adviser, Presidential Country Files Middle East and South Asia, Box 12, India, State to Sec State NODIS (3), GPFL. Banishing the rogue elephant: Recasting contemporary Indo-US intelligence relations In the context of the early 1970s, the US government’s contention that the CIA was not involved in subversive activity in India, undoubtedly contained more than an element of truth. There is no doubt, however, that the Central Intelligence Agency had actively sought to influence India’s internal political landscape in the early Cold War period. During the first two decades following India’s independence, the CIA had a complex, and often conflicted relationship with the country’s political elite and the nation’s media. Despite the criticisms levelled at the Agency by India’s first premier, Jawaharlal Nehru, a number of CIA covert operations in the subcontinent were undertaken with the full knowledge and support of India’s intelligence service, and senior figures inside the ruling Congress Party. In the late 1950s, in Kerala, the CIA worked with the Congress Party to destabilise a democratically elected communist administration. For much of the 1960s, following the Sino-Indian border war, the Agency assisted the Indian government in strengthening its defences against an external threat from the People’s Republic of China. From 1967 onwards, the appearance of an embarrassing sequence of very public revelations implicating the CIA in illegal and morally questionable activities, both at home and abroad, propelled the Agency into the 40 centre of a political storm in the subcontinent which, while varying in its intensity, was to remain a dominant feature of wider Indo-US relations for the remainder of the Cold War. It proved an enduring source of frustration to US government officials in the latter Cold War period that, at a time when the American intelligence presence in India was in retreat, and that of the Soviet Union expanding exponentially, the CIA remained firmly in the cross hairs of India’s politicians and press. In part, manifestations of an Indian ‘national paranoia’ when it came to CIA can be explained in terms of the deterioration in wider Indo- US relations that occurred toward the end of the Johnson administration and, more especially, after the Nixon/Kissinger tilt towards Pakistan in 1971. Nonetheless, other political and cultural factors undoubtedly played a significant role in shaping Indian perceptions of the CIA. Specifically, the CIA’s heightened public profile after 1967, both globally and in the subcontinent, transformed it into a convenient vehicle through which Indian politicians were able to vent populist anti-American sentiment. 113 Bayly, Empire and Information, p. 365. 113 Bayly, Empire and Information, p. 365. Banishing the rogue elephant: Recasting contemporary Indo-US intelligence relations Furthermore, Indira Gandhi’s appreciation that the Agency could serve as political lightening-rod, deflecting attention away from her Party’s mismanagement of India’s formidable domestic problems, propelled the CIA to the forefront of official Indo-US discourse. In one sense, the conspiratorial paradigm that gained such traction amongst sections of India’s political elite and national media can be explained in terms of the nation’s historic familiarity with the shadowy work of ‘foreign’ intelligence organisations in the subcontinent, which stretched back to the eighteenth century.113 Equally, the ‘conspiracism’ that gripped hold of India from the late 1960s, is suggestive of a more contemporary post-colonial expression of nationalism, not dissimilar to that evident in the Middle East. In India during the premiership of Indira Gandhi, or for that matter, Gamal Abdul Nasser’s Egypt, or Ayatollah Ruhollah Khomeini’s Iran, an inability to effectively tackle endemic social and 41 41 economic problems invited the temptation to court popular legitimacy by explaining away the national ills in terms of the machinations of a ‘foreign hand.’114 Moreover, the fact that leaders in the developing world were able to point to genuine attempts undertaken by Western intelligence services to subvert the sovereignty of independent nation states, be they in Chile or elsewhere, made it all the easier to divert attention away from uncomfortable daily realties, and towards invariably illusory constructions of external subversion. Likewise, when interpreted through the lens of the Nehru/Gandhi dynasty’s personal interaction with Western intelligence agencies, the CIA’s demonization in India appears less surprising. Having witnessed first hand the full force of a repressive British security system while participating in India’s fight for independence, it is unsurprising that Indira Gandhi acquired a highly developed sense of suspiciousness and distrust which, at times, appeared to border on the paranoid. It appears perfectly plausible, if difficult to establish beyond doubt, that the Nixon’s administration’s tilt toward India’s regional rival Pakistan, coupled with public revelations surrounding CIAs misdeeds, and not least the Agency’s complicity in bloody coups in Bangladesh and Chile, triggered a strain of persecutory paranoia buried within Indira Gandhi’s psyche. p , , pp 115 Further insights into the political manifestations of persecutory paranoia can be found in Robins and Post, Political Paranoia, pp. 5-6. 114 Pipes, The Hidden Hand, pp. 26-7 115 114 Pipes, The Hidden Hand, pp. 26-7 115 g Political Paranoia, pp. 5-6. 116 See for example, ‘C.I.A. Study Details Failures; Scouring of System Is Urged’, The New York Times, 3 June 1998; ‘CIA Faces Heavy Fallout over India Nuclear Tests’ The Wall Street Journal, 13 May 1998. 117 ‘CIA director meets with Indian security officials’, Associated Press, 19 March 2009. 118 ‘One Dead as Two Wild Elephants go Berserk in Mysore’, The Times of India, 8 June 2011. Banishing the rogue elephant: Recasting contemporary Indo-US intelligence relations Certainly behavioural traits associated with persecutory paranoia, principally strong feelings of mistrust and a powerful inclination to deny ones own insecurities and hostilities and to project them onto others, appear all to evident in Gandhi’s words and deeds in relation to the CIA.115 It was not until after the Cold War had ended, the Soviet Union’s relationship with India had faltered, and the strangle hold exercised by the Nehru/Gandhi family on Indian politics had loosened, albeit fleetingly, that the CIA ceased to be a defining factor in Indo-US relations. Surprisingly little attention has been given by contemporary scholars to the wider political dimension of the Indo-American intelligence relationship. This represents a 42 remarkable historical omission given the extent to which Indian rhetorical attacks on the CIA, both official and unofficial, impacted upon Washington’s wider relationship with New Delhi from the late 1960s onwards. Moreover, from a more parochial standpoint, it appears likely that the CIA’s inability to remain out of the Indian political spotlight for long compromised its operational effectiveness in the subcontinent. Here, it is noteworthy that the CIA failed to anticipate nuclear tests conducted by India in 1974 and 1998.116 Since, 1991, the end of one war, and the beginning of another, has transformed the CIA’s relationship with India. Significantly, in 2009, the first overseas trip Leon Panetta made after becoming Director of the Central Intelligence Agency was to New Delhi, to hold talks on intelligence co-operation in the ‘war on terror’ with his Indian counterpart.117 Yet, perhaps the most telling confirmation that the CIA has finally turned a corner in its relationship with India came in June 2011. At the time, the subcontinents newspapers were busy reporting on the destruction that rogue elephants had wrought in the southern Indian city of Mysore, and, not once, was the Agency’s name mentioned.118 remarkable historical omission given the extent to which Indian rhetorical attacks on the CIA, both official and unofficial, impacted upon Washington’s wider relationship with New Delhi from the late 1960s onwards. Moreover, from a more parochial standpoint, it appears likely that the CIA’s inability to remain out of the Indian political spotlight for long compromised its operational effectiveness in the subcontinent. Banishing the rogue elephant: Recasting contemporary Indo-US intelligence relations Here, it is noteworthy that the CIA failed to anticipate nuclear tests conducted by India in 1974 and 1998.116 Since, 1991, the end of one war, and the beginning of another, has transformed the CIA’s relationship with India. Significantly, in 2009, the first overseas trip Leon Panetta made after becoming Director of the Central Intelligence Agency was to New Delhi, to hold talks on intelligence co-operation in the ‘war on terror’ with his Indian counterpart.117 Yet, perhaps the most telling confirmation that the CIA has finally turned a corner in its relationship with India came in June 2011. At the time, the subcontinents newspapers were busy reporting on the destruction that rogue elephants had wrought in the southern Indian city of Mysore, and, not once, was the Agency’s name mentioned.118 43
https://openalex.org/W4234987027	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0213396&type=printable	English	null	Correction: Association of birth weight with corneal power in early adolescence: Results from the National Health and Nutrition Examination Survey (NHANES) 1999-2008	PloS one	2,019	cc-by	409	S1 File. Originally published, uncorrected article. (PDF) S1 File. Originally published, uncorrected article. (PDF) Reference 1. Fieß A, Schuster AK, Pfeiffer N, Nickels S (2017) Association of birth weight with corneal power in early adolescence: Results from the National Health and Nutrition Examination Survey (NHANES) 1999– 2008. PLoS ONE 12(10): e0186723. https://doi.org/10.1371/journal.pone.0186723 PMID: 29073249 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Correction: Association of birth weight with corneal power in early adolescence: Results from the National Health and Nutrition Examination Survey (NHANES) 1999-2008 The PLOS ONE Staff Notice of republication This article was republished on February 21, 2019, to correct for an error in the Data Availabil- ity statement introduced during the production process. The publisher apologizes for the error. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference. OPEN ACCESS Citation: The PLOS ONE Staff (2019) Correction: Association of birth weight with corneal power in early adolescence: Results from the National Health and Nutrition Examination Survey (NHANES) 1999- 2008. PLoS ONE 14(2): e0213396. https://doi.org/ 10.1371/journal.pone.0213396 Published: February 28, 2019 Copyright: © 2019 The PLOS ONE Staff This is an Citation: The PLOS ONE Staff (2019) Correction: Association of birth weight with corneal power in early adolescence: Results from the National Health and Nutrition Examination Survey (NHANES) 1999- 2008. PLoS ONE 14(2): e0213396. https://doi.org/ 10 1371/journal pone 0213396 C t t o OS O S ( ) C Association of birth weight with corneal power in early adolescence: Results from the National Health and Nutrition Examination Survey (NHANES) 1999- 2008. PLoS ONE 14(2): e0213396. https://doi.org/ 10.1371/journal.pone.0213396 Published: February 28, 2019 Copyright: © 2019 The PLOS ONE Staff. 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. 1. Fieß A, Schuster AK, Pfeiffer N, Nickels S (2017) Association of birth weight with corneal power in early adolescence: Results from the National Health and Nutrition Examination Survey (NHANES) 1999– 2008. PLoS ONE 12(10): e0186723. https://doi.org/10.1371/journal.pone.0186723 PMID: 29073249 Published: February 28, 2019 Copyright: © 2019 The PLOS ONE Staff. 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. 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0213396 February 28, 2019
https://openalex.org/W3031537526	https://figshare.com/articles/journal_contribution/Synthetic_Lethality_between_DNA_Polymerase_Epsilon_and_RTEL1_in_Metazoan_DNA_Replication_/12387893/1/files/22826027.pdf	English	null	Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication	Cell reports	2,020	cc-by	13,540	Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication Correspondence simon.boulton@crick.ac.uk Correspondence simon.boulton@crick.ac.uk In Brief Bellelli et al. report that RTEL1 deﬁciency is synthetic lethal with the loss of pole-4 in C. elegans/hypomorphy of Pol epsilon. An analysis of replication dynamics in Rtel1/ Pole4/ mouse cells revealed a combination of dysfunctional fork progression and defective origin activation, which cooperatively drive incomplete genomic replication and genetic instability. Highlights Article Article Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication Graphical Abstract Authors Roberto Bellelli, Jillian Youds, Valerie Borel, Jennifer Svendsen, Visnja Pavicic-Kaltenbrunner, Simon J. Boulton Correspondence simon.boulton@crick.ac.uk INTRODUCTION size and viability, sensitivity to DNA damaging agents, and elevated meiotic recombination. Biochemical studies show that RTEL1 can efﬁciently disassemble D-loop recombination in- termediates, suggesting that RTEL-1 might disassemble these intermediates to promote non-crossover repair, likely through synthesis-dependent strand annealing. Genetic analysis also re- vealed that rtel-1 is synthetic lethal when combined with muta- tions in dog-1/FANCJ, mus-81, him-6/BLM, and rcq-5, all of which are homologs of genes involved in human genetic dis- eases and the maintenance of genome stability at replication forks (Barber et al., 2008). Indeed, mutants of these genes when combined with rtel-1 displayed persistent RAD-51 foci in the germline and embryonic lethality, indicating that RTEL-1 is essential in their absence. However, where and when RTEL1 ac- tivity is essential in nematodes remained to be established. Sub- sequent studies showed that RTEL1 facilitates efﬁcient telomere and genome-wide replication in vertebrates (Vannier et al., 2013), but its precise function during DNA replication remains unclear. DNA replication origins are established at thousands of sites throughout the genome through a combination of structural and functional chromatin determinants that promote loading of inactive MCM2-7 double hexamers around DNA replication ori- gins (Fragkos et al., 2015). At the G1-S transition, DDK- and CDK-dependent phosphorylation events drive the formation and activation of the CMG (CDC45-MCM2-7-GINS), the eukary- otic replicative helicase, followed by establishment of two sym- metric replication forks that initiate DNA synthesis (Bell and La- bib, 2016). The bulk of DNA replication at active replication forks is performed by the conserved DNA polymerase com- plexes Pol delta and Pol epsilon, which act on the lagging and leading strand, respectively (Burgers and Kunkel, 2017). The latter is also considered to be a stable component of the repli- some and is required for efﬁcient CMG formation in budding yeast (Bell and Labib, 2016). DNA replication in metazoans also requires the function of several helicases and replisome- associated factors to prevent inappropriate transactions at the replication fork and to avert persistent fork stalling events (Leo´ n-Ortiz et al., 2014; Dungrawala et al., 2015). To gain an improved understanding of the role of RTEL-1 in maintaining genome stability, we conducted a genome-wide RNAi screen to identify genes that, when knocked down with RNAi in the rtel-1 mutant background, cause synthetic lethality, but not in the wild-type. SUMMARY Genome stability requires coordination of DNA replication origin activation and replication fork progression. RTEL1 is a regulator of homologous recombination (HR) implicated in meiotic cross-over control and DNA repair in C. elegans. Through a genome-wide synthetic lethal screen, we uncovered an essential genetic inter- action between RTEL1 and DNA polymerase (Pol) epsilon. Loss of POLE4, an accessory subunit of Pol epsilon, has no overt phenotype in worms. In contrast, the combined loss of POLE-4 and RTEL-1 results in embryonic lethality, accumulation of HR intermediates, genome instability, and cessation of DNA replica- tion. Similarly, loss of Rtel1 in Pole4/ mouse cells inhibits cellular proliferation, which is associated with persistent HR intermediates and incomplete DNA replication. We propose that RTEL1 facilitates genome- wide fork progression through its ability to metabolize DNA secondary structures that form during DNA repli- cation. Loss of this function becomes incompatible with cell survival under conditions of reduced origin acti- vation, such as Pol epsilon hypomorphy. Article Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication Bellelli,1,2,3 Jillian Youds,1,2 Valerie Borel,1 Jennifer Svendsen,1 Visnja Pavicic-Kaltenbrunner,1 Roberto Bellelli,1,2,3 Jillian Youds,1,2 Valerie Borel,1 Jennifer Svendsen,1 Visnja Pavicic-Kaltenbrunner,1 and Simon J. Boulton1,4,* 1The Francis Crick Institute, 1 Midland Road, NW1 1AT London, UK 2These authors contributed equally 3Present address: Center for Cancer Cell and Molecular Biology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, Barbican, EC1M 6BE London, UK 4Lead Contact Roberto Bellelli,1,2,3 Jillian Youds,1,2 Valerie Borel,1 Jennifer Svendsen,1 Visnja Pavicic-Kaltenbrunner,1 and Simon J. Boulton1,4,* 1The Francis Crick Institute, 1 Midland Road, NW1 1AT London, UK 2These authors contributed equally 3Present address: Center for Cancer Cell and Molecular Biology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, Barbican, EC1M 6BE London, UK 4Lead Contact Correspondence: simon.boulton@crick.ac.uk https://doi.org/10.1016/j.celrep.2020.107675 Correspondence: simon.boulton@crick.ac.uk https://doi.org/10.1016/j.celrep.2020.107675 Highlights d rtel-1 is synthetic lethal with the loss of DNA polymerase epsilon in C. elegans d rtel-1; pole-4 double mutants accumulate Rad51 and RPA foci and fail to replicate d Impaired DNA replication and genome instability in Rtel1 Pole4 knockout mouse cells d Rtel1 Pole4 double knockout mouse cells exhibit fork asymmetry and defective origin activation ll ll ll OPEN ACCESS Cell Reports 31, 107675, May 26, 2020 ª 2020 The Author(s). 1 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). rtel-1 Is Synthetic Lethal with Members of the DNA Polymerase Epsilon Complex in C. elegans RTEL1 is dispensable for viability in the nematode C. elegans, an organism amenable to genetic manipulation and a potent system to identify synthetic interactions in vivo. To further interrogate the functions of RTEL1 in genome stability in C. elegans, we per- formed a genome-wide RNAi screening in N2(wild type) and rtel-1 mutant worms (Kamath et al., 2003) by using a library of 16,256 genes (Figure S1A). Following secondary screens to conﬁrm our initial hits, we identiﬁed a number of genes with es- tablished roles in DNA replication whose RNAi caused lethality in the rtel-1 mutant but not in an N2(wild-type) strain (Figure S1B). These genes included rfc-1/RFC1, mus-101/TOPBP1, crn-1/ FEN1, F31C3.5/PSF2, Y65B4BR.8/PSF3, and cdt-1/CDT1. Interestingly, we also identiﬁed three components of the DNA Pol epsilon complex: F33H2.5 (pole-1), F08B4.5 (pole-2), and T26A5.8 (pole-3) (Figure 1A). A fourth component of the com- plex, Y53F4B.3 (pole-4), was not present in the RNAi library. In secondary screens, all three DNA Pol epsilon components knocked down by RNAi showed dramatic synthetic lethal pheno- types in the rtel-1 mutant (Figure S1B), which we decided to explore further (Figure 1A). Because DNA Pol epsilon has been implicated in activation of the intra-S-phase checkpoint, we questioned whether the absence of pole-4 might also inactivate the replication check- point in worms (Navas et al., 1995). However, in contrast to this hypothesis, DAPI staining of the germlines of rtel-1; pole-4 double mutants showed enlarged mitotic nuclei and fewer nuclei in the mitotic zone than wild-type animals or either single mutant, a phenotype associated with mitotic replication arrest due to checkpoint activation. To quantify the mitotic arrest, we counted the number of nuclei in a single plane of focus within 45 mm of the distal tip cell of the mitotic zone. rtel-1; pole-4 double mutants had fewer mitotic nuclei than N2(wild-type) or either single mutant alone (Figure 2C). Thus, the number of nuclei in rtel-1; pole-4 animals was more similar to wild-type animals treated with 40 mM hydroxyurea (HU) than either single mutant, indi- cating that replication stress and activation of the DNA replica- tion checkpoint were indeed present in rtel-1; pole-4 double mu- tants. We conclude that the absence of pole-4 does not compromise the replication checkpoint in worms. This is in accordance with recent ﬁndings in both Pole4/ mouse embryo ﬁbroblasts and CRISPR knockout human cells (Bellelli et al., 2018; Hustedt et al., 2019). rtel-1 Is Synthetic Lethal with pole-4 in C. elegans To conﬁrm our observations with RNAi, we obtained a genetic deletion of pole-4, tm4613, which removed the majority of the coding region of the gene, apart from the ﬁrst 35 nucleotides of exon 1; this likely represents a bona ﬁde null allele (Figure S2A). pole-4(tm4613) mutants appeared superﬁcially wild type, and the loss of pole-4 did not result in any signiﬁcant loss of viability. rtel-1 mutants show greater than 90% viability as previously described (Barber et al., 2008). In contrast, rtel-1; pole-4 double mutant animals displayed a dramatic synthetic embryonic lethal phenotype (Figure 2A), wherein none of the progeny were viable. This was associated with a dramatic reduction in DNA replica- tion, as observed by attenuated incorporation of Cy3-dUTP in double mutant worms (Figure 2B). Our data, although pointing to conserved functions in meta- zoans for RTEL1 in replication fork progression and POLE4 in maintaining Pol epsilon complex stability, reveal an un-appreci- ated interplay between replication origin activation and fork pro- gression required for genome-wide DNA replication and the maintenance of genome stability. 2 Cell Reports 31, 107675, May 26, 2020 INTRODUCTION This genetic screen identiﬁed multiple genes involved in DNA replication, such as TOPBP1, GINS com- plex subunits PSF2 and PSF3, RFC-1, FEN-1, and CDT1, as well The helicase RTEL1 was identiﬁed as the ﬁrst metazoan anti- recombinase, which facilitates DNA repair and regulates cross over formation in meiosis in C. elegans (Barber et al., 2008; Youds et al., 2010). rtel-1 mutant worms display reduced brood Article A ti l ll OPEN ACCESS as three components of DNA Pol epsilon, which we chose to further investigate. Strains lacking the non-essential subunit of Pol epsilon, pole-4, exhibit no overt phenotype under normal or DNA damaging conditions. Strikingly, however, the pole-4; rtel-1 double mutant is 100% synthetic embryonic lethal and pre- sents with persistent homologous recombination (HR) intermedi- ates, extensive genome instability, and cessation of DNA replication. We proceed to show that this synthetic lethal interac- tion is conserved in mammalian cells. A combined loss of RTEL1 and POLE4 in primary mouse cells also inhibits cellular prolifera- tion and results in extensive genetic instability. Molecular anal- ysis of DNA replication dynamics in Rtel1-Pole4 double knockout cells revealed a combination of dysfunctional fork pro- gression and origin activation, which leads to fork stalling and genome under-replication. extend to DNA Pol delta (Figure S1C). pole-3 is predicted to be a non-essential component of the complex, and therefore, RNAi of this gene had a less dramatic effect on rtel-1 mutants than pole-1 or pole-2 RNAi (Figure 1B). We proceeded to examine the germlines of these animals for defects that could explain the source of the observed synthetic lethality. After RNAi for pole-1 or pole-2, rtel-1 mutants exhibited increased levels of RAD-51 foci in the mitotic zone of the germline, which is the only region of active DNA replication in the C. elegans germline. In contrast, RAD-51 foci were not observed in N2(wild-type) animals fed with the same RNAi, suggesting that replication defects might underlie the lethality in rtel-1; pole-1(RNAi) and rtel-1; pole-2(RNAi) animals (Figure 1C). rtel-1 Is Synthetic Lethal with Members of the DNA Polymerase Epsilon Complex in C. elegans We observed that RNAi knockdown of pole-1, pole-2, or pole-3 caused a signiﬁcant reduction in brood size and viability in rtel-1 mutants compared with N2(wild-type) worms (Fig- ure 1B). Orthologs of pole-1 and pole-2 are essential for viability in budding yeast; thus, RNAi in worms likely produced a partial knockdown of these genes. Furthermore, variability in knockdown efﬁciency could account for the different levels of synthetic lethality between rtel-1, pole-1, and pole-2. Impor- tantly, we did not observe any overt synthetic lethality when we knocked down the major subunit of DNA Pol delta, F12F6.7, in the rtel-1 background, suggesting that the genetic interaction between rtel-1 and DNA Pol epsilon does not Finally, we examined the mitotic regions to determine the types of DNA damage occurring spontaneously in rtel-1; pole- 4 double mutants. Staining with an anti-RPA antibody showed an accumulation of RPA foci in rtel-1; pole-4 animals, but not in single mutants, which is indicative of replication stress and DNA single-strand accumulation in the double mutant, poten- tially due to uncoupling between DNA polymerases and the CMG helicase (Figures 2D and S2B). 2 Cell Reports 31, 107675, May 26, 2020 Mus81- and Rfs1-Dependent Processing of Replication To analyze the consequences of replicative damage accumu- C. elegans gene (name given) Human gene pol epsilon subunit 1 pol epsilon subunit 2 pol epsilon subunit 3 pol epsilon subunit 4 F33H2.5 (pole-1) Y53F4B.3 (pole-4) T26A5.8 (pole-3) pole-2 C rtel-1 rtel-1; pole-2 (RNAi) wild type pole-2 (RNAi) pole-1 (RNAi) rtel-1; pole-1 (RNAi) α-RAD-51 (green); DAPI (blue) α-RAD-51 α-RAD-51 (green); DAPI (blue) α-RAD-51 A no RNAi pole-1 pole-2 pole-3 0 50 100 RNAi feeding of pole complex components % viability (percent of control) p=0.09 ** * no RNAi pole-1 pole-2 pole-3 0 50 100 150 Brood size (percent of control) N2/wild-type rtel-1 n.s. ** * * B RNAi feeding of pole complex components Figure 1. Knockdown of Polymerase Epsilon Components by RNAi Causes Synthetic Lethality in rtel-1 Mutant Background (A) C. elegans gene names of the four polymerase epsilon components and their corresponding human homologs. (B) Total brood size and percent viability after feeding either no RNAi or RNAi for pole-1, pole-2, or pole-3 in the N2(wild-type) or rtel-1 mutant. Brood size and percent viability are both normalized based on untreated N2(wild-type) or rtel-1 control animals. (p < 0.05, p < 0.01, *p < 0.001, *p < 0.0001; n.s., not signiﬁcant). rtel-1 Is Synthetic Lethal with Members of the DNA Polymerase Epsilon Complex in C. elegans Brood size and percent viability are both normalized based on untreated N2(wild-type) or rtel-1 control animals. (p < 0.05, p < 0.01, p < 0.001, *p < 0.0001; n.s., not signiﬁcant). (A) C. elegans gene names of the four polymerase epsilon components and their corresponding human homologs. (B) Total brood size and percent viability after feeding either no RNAi or RNAi for pole-1, pole-2, or pole-3 in the N2(wild-type percent viability are both normalized based on untreated N2(wild-type) or rtel-1 control animals. (p < 0.05, p < 0.01, p signiﬁcant). (C) RAD-51 staining of mitotic zones of N2(wild-type) or rtel-1 animals fed either with no RNAi, pole-1, or pole-2 RNAi. Images are composites of several images stitched together. Error bars in all graphs represent standard deviation (SD) o (C) RAD-51 staining of mitotic zones of N2(wild-type) or rtel-1 animals fed either with no RNAi, pole-1, or pole-2 RNAi. Images are composites of several images stitched together. Error bars in all graphs represent standard deviation (SD) of the mean. rtel-1 Is Synthetic Lethal with Members of the DNA Polymerase Epsilon Complex in C. elegans (C) RAD-51 staining of mitotic zones of N2(wild-type) or rtel-1 animals fed either with no RNAi, pole-1, or pole-2 RNAi. Images are composites of several images stitched together. Error bars in all graphs represent standard deviation (SD) of the mean. Article ll OPEN ACCESS e unit 1 unit 2 unit 3 unit 4 no RNAi pole-1 pole-2 pole-3 0 50 100 RNAi feeding of pole complex components % viability (percent of control) p=0.09 * no RNAi pole-1 pole-2 pole-3 0 50 100 150 Brood size (percent of control) N2/wild-type rtel-1 n.s. ** * * B RNAi feeding of pole complex components C. elegans gene (name given) Human gene pol epsilon subunit 1 pol epsilon subunit 2 pol epsilon subunit 3 pol epsilon subunit 4 F33H2.5 (pole-1) Y53F4B.3 (pole-4) T26A5.8 (pole-3) pole-2 A no RNAi pole-1 pole-2 pole-3 0 50 100 RNAi feeding of pole complex components % viability (percent of control) p=0.09 ** * no RNAi pole-1 pole-2 pole-3 0 50 100 150 Brood size (percent of control) N2/wild-type rtel-1 n.s. ** * * B RNAi feeding of pole complex components B A C rtel-1 rtel-1; pole-2 (RNAi) wild type pole-2 (RNAi) pole-1 (RNAi) rtel-1; pole-1 (RNAi) α-RAD-51 (green); DAPI (blue) α-RAD-51 α-RAD-51 (green); DAPI (blue) α-RAD-51 RNAi feeding of pole complex components RNAi feeding of pole complex components C rtel-1 rtel-1; pole-2 (RNAi) rtel-1; pole-1 (RNAi) α-RAD-51 (green); DAPI (blue) α-RAD-51 Figure 1. Knockdown of Polymerase Epsilon Components by RNAi Causes Synthetic Lethality in rtel-1 Mutant Background (A) C. elegans gene names of the four polymerase epsilon components and their corresponding human homologs. (B) Total brood size and percent viability after feeding either no RNAi or RNAi for pole-1, pole-2, or pole-3 in the N2(wild-type) or rtel-1 mutant. Brood size and percent viability are both normalized based on untreated N2(wild-type) or rtel-1 control animals. (p < 0.05, p < 0.01, *p < 0.001, p < 0.0001; n.s., not signiﬁcant) Figure 1. Knockdown of Polymerase Epsilon Components by RNAi Causes Synthetic Lethality in rtel-1 M Figure 1. Knockdown of Polymerase Epsilon Components by RNAi Causes Synthetic Lethality in rtel-1 Mutant Background (A) C. elegans gene names of the four polymerase epsilon components and their corresponding human homologs. (B) Total brood size and percent viability after feeding either no RNAi or RNAi for pole-1, pole-2, or pole-3 in the N2(wild-type) or rtel-1 mutant. Cell Reports 31, 107675, May 26, 2020 3 Mus81- and Rfs1-Dependent Processing of Replication Intermediates in rtel-1; pole4 Worms N2(wild-type) animals treated for 24 h with 40 mM hydroxyure (D) RPA staining of mitotic zones in the indicated genotypes (see also Figure S2B I i f l i i h d h E b i ll B D wild type wild type rtel-1 (g ); ( ) pole-4 rtel-1; pole-4 α RPA type pole-4 rtel-1; pole-4 rtel-1 rtel-1; pole-4 N2/wild-type pole-4 Figure 2. rtel-1 Is Synthetic Lethal in Combination with pole-4 Deletion gure 2. rtel-1 Is Synthetic Lethal in Combination with po Figure 2. rtel 1 Is Synthetic Lethal in Combination with pole 4 Deletion (A) Progeny viability in N2(wild-type), pole-4, and rtel-1 single mutants and rtel-1; pole-4 double mutants (p < 0.0001; n.s., not signiﬁcant). (B) Top: representative images of Cy3-dUTP incorporation in mitotic nuclei in N2(wild-type), pole-4, and rtel-1 single mutants and rtel-1; pole-4 double mutant worms. Bottom: bar graphs showing the percentage of Cy3-dUTP-positive cells in the described genetic backgrounds (p < 0.0001; n.s., not signiﬁcant). (C) Average number of mitotic nuclei counted in a single plane of focus within 45 mm of the distal tip cell as a measure of mitotic arrest and checkpoint activity in the genotypes shown. N2(wild-type) animals treated for 24 h with 40 mM hydroxyurea (HU) were used as a control (p < 0.0001). (D) RPA staining of mitotic zones in the indicated genotypes (see also Figure S2B for quantiﬁcation). Images are composites of several images stitched together. Error bars in all graphs represent standard deviation (SD) of the mean. meiotic chromosomal defects and the subsequent lethality of rtel-1; pole-4 embryos. strains. With this in mind, we tested whether or not pole-4 might also be synthetic lethal with dog-1. However, dog-1; pole-4 double mutants showed greater than 90% viability (Ta- ble S1). Furthermore, no G-tract deletions were observed in pole-4 or rtel-1 single mutants or in rtel-1; pole-4 double mu- tants. The frequency of G-tract deletions in the dog-1 back- ground was also unchanged after feeding with pole-2(RNAi) (Table S2), indicating that the lethality in rtel-1; pole-4 is unre- lated to poly G-tract instability. In our previous work, we identiﬁed a synthetic lethal genetic interaction between rtel-1 and dog-1, the FANCJ homolog in C. elegans (Barber et al., 2008; Youds et al., 2008). DOG-1 is involved in inter-strand cross-link repair and is also responsible for maintaining the stability of tracts of poly-guanine DNA in metazoans (Cheung et al., 2002). Mus81- and Rfs1-Dependent Processing of Replication Intermediates in rtel-1; pole4 Worms To analyze the consequences of replicative damage accumu- lation in rtel-1; pole-4 double mutants, we analyzed diakinesis nuclei. In N2(wild-type) animals, six DAPI-stained bodies are pre- sent at diakinesis, which correspond to paired homologous chromosomes held together by a single chiasmata. As expected, N2(wild-type) animals, as well as rtel-1 and pole-4 single mu- tants, presented with six intact DAPI-stained bodies. In contrast, rtel-1; pole-4 animals displayed a wide range of chromosomal defects at diakinesis, ranging from chromosome fragments to unpaired sister chromatids, chromosome fusions, and constric- tions on the chromosome arms (Figures 4B and S3B). Quantiﬁ- cation of the number of DAPI-stained bodies at diakinesis showed that 41% of rtel-1; pole-4 diakinesis nuclei had greater than six bodies. However, many of those that showed a correct number of DAPI-stained bodies displayed constrictions on the arms of chromosomes, suggesting the presence of signiﬁcant chromosome damage. Thus, we conclude that DNA damage arising during DNA replication in the absence of rte-l1 and pole-4 persists as nuclei progress through meiosis, resulting in In eukaryotes, persistent fork stalling events are associated with recruitment of the RAD-51 recombinase to protect newly synthe- sized DNA from nucleolytic degradation and promote recombi- nation-dependent fork restart and/or processing into double- strand breaks (DSBs) for canonical DNA repair (Bhat and Cortez, 2018). Thus, we stained mutant strains with RAD-51 antibodies to monitor the presence and resolution of HR intermediates. Strikingly, although mitotic RAD-51 foci were rarely observed in N2(wild-type) or single mutant animals, rtel-1; pole-4 double mutants showed a strong accumulation of RAD51 foci in the mitotic zone (Figure 3A). rtel-1; pole-4 animals also displayed greater numbers of RAD-51 foci throughout the meiotic regions of the germline, and these foci persisted through late pachytene when meiotic DSB repair is normally complete (Figure S3A). This data suggest that DNA damage occurring spontaneously during DNA replication in rtel-1; pole-4 double mutants persists into meiosis. Cell Reports 31, 107675, May 26, 2020 3 Article wild type wild type rtel-1 α-RPA (green); DAPI (blue) pole-4 rtel-1; pole-4 α-RPA A B C D wild type pole-4 rtel-1 rtel-1; pole-4 Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP N2/Wild type pole-4 rtel-1 rtel-1; pole-4 0 50 100 150 % viability n.s. n.s. N2/wild-type pole-4 rtel-1 rtel-1; pole-4 0 20 40 60 80 100 % Cy3 postitive nuclei n.s. n.s. 4 Cell Reports 31, 107675, May 26, 2020 Mus81- and Rfs1-Dependent Processing of Replication Intermediates in rtel-1; pole4 Worms N2/Wild type pole-4 rtel-1 rtel-1; pole-4 wt + 40mM HU 0 10 20 30 40 50 Number of Mitotic Nuclei in single plane within 45mm of distal tip Figure 2. rtel-1 Is Synthetic Lethal in Combination with pole-4 Deletion (A) Progeny viability in N2(wild-type), pole-4, and rtel-1 single mutants and rtel-1; pole-4 double mutants (p < 0.0001; n.s., not signiﬁcant). (B) Top: representative images of Cy3-dUTP incorporation in mitotic nuclei in N2(wild-type), pole-4, and rtel-1 single mutants and rtel-1; pole-4 double mutant worms. Bottom: bar graphs showing the percentage of Cy3-dUTP-positive cells in the described genetic backgrounds (p < 0.0001; n.s., not signiﬁcant). (C) Average number of mitotic nuclei counted in a single plane of focus within 45 mm of the distal tip cell as a measure of mitotic arrest and checkpoint activity in the genotypes shown. N2(wild-type) animals treated for 24 h with 40 mM hydroxyurea (HU) were used as a control (p < 0.0001). (D) RPA staining of mitotic zones in the indicated genotypes (see also Figure S2B for quantiﬁcation). Images are composites of several images stitched together. Error bars in all graphs represent standard deviation (SD) of the mean. Article OPEN ACCESS RPA ( ) DAPI (bl ) C D N2/Wild type pole-4 rtel-1 rtel-1; pole-4 wt + 40mM HU 0 10 20 30 40 50 Number of Mitotic Nuclei in single plane within 45mm of distal tip A N2/Wild type pole-4 rtel-1 rtel-1; pole-4 0 50 100 150 % viability n.s. n.s. A A B wild type pole-4 rtel-1 rtel-1; pole-4 Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP Cy3-dUTP + DAPI Cy3-dUTP N2/Wild type pole-4 rtel-1 rtel-1; pole-4 0 50 100 % viability n.s. n.s. N2/wild-type pole-4 rtel-1 rtel-1; pole-4 0 20 40 60 80 100 % Cy3 postitive nuclei n.s. n.s. Figure 2. rtel-1 Is Synthetic Lethal in Combination with pole-4 Deletion (A) Progeny viability in N2(wild-type), pole-4, and rtel-1 single mutants and rtel-1; (B) Top: representative images of Cy3-dUTP incorporation in mitotic nuclei in N2( worms. Bottom: bar graphs showing the percentage of Cy3-dUTP-positive cells (C) Average number of mitotic nuclei counted in a single plane of focus within 45 mm genotypes shown. Mus81- and Rfs1-Dependent Processing of Replication Intermediates in rtel-1; pole4 Worms DOG-1 is believed to unwind secondary structures that may form in these G-rich sequences during replication. Thus, in its absence, deletions occur in tracts of poly-guanine greater than 18 nucleotides in length. To understand thenature of the DNA damage that accumulates in rtel-1; pole-4 mutant animals, we analyzed the dependence of mitotic RAD-51 foci formation on MUS-81 and RFS-1. MUS-81 is a structure-speciﬁc endonuclease that has been implicated in processing DNA damage intermediates at the replication forks We noted that similar to rtel-1; pole-4 animals, rtel-1; dog-1 double mutants display mitotic RAD-51 foci, suggesting a possible link between the defects observed in these two 4 Cell Reports 31, 107675, May 26, 2020 ll OPEN ACCESS mitotic zone early pachytene late pachytene N2 (wild type) rtel-1; pole-4 pole-4 rtel-1 RAD51 DAPI + RAD51 A B N2 (wild type) rtel-1; pole-4 pole-4 rtel-1 RAD51 DAPI + RAD51 RAD51 DAPI + RAD51 5 6 7 8 9 10 11 12 >12 0 50 100 Number of DAPI-stained bodies at diakinesis N2 (wild type) pole-4 rtel-1 rtel-1; pole-4 % Diakinesis nuclei ure 3. RAD-51 Foci and Chromosome Aberrations Are Elevated in rtel-1; pole-4 Double Mutants Representative images of RAD-51 foci in mitotic zones and early pachytene and late pachytene in the indicated genotypes. Greyscale images show RAD . Left: top panels show representative images of N2(wild-type), pole-4, and rtel-1 single mutant diakinesis nuclei showing six DAPI-stained bivalents pres om panels show examples of the chromosome defects observed in rtel-1; pole-4, including chromosome fragments, constrictions on chromosome a mosome fusions, and unpaired sister chromatids. Right: quantiﬁcation of the number of DAPI-stained bodies present at diakinesis in each of the indic ns (see also Figure S3B). mitotic zone early pachytene late pachytene N2 (wild type) rtel-1; pole-4 pole-4 rtel-1 RAD51 DAPI + RAD51 A B RAD51 DAPI + RAD51 RAD51 DAPI + RAD51 A RAD51 DAPI + RAD51 B N2 (wild type) rtel-1; pole-4 pole-4 rtel-1 RAD51 DAPI + RAD51 RAD51 DAPI + RAD51 5 6 7 8 9 10 11 12 >12 0 50 100 Number of DAPI-stained bodies at diakinesis N2 (wild type) pole-4 rtel-1 rtel-1; pole-4 % Diakinesis nuclei B B % Diakinesis nuclei Figure 3. RAD-51 Foci and Chromosome Aberrations Are Elevated in rtel-1; pole-4 Double Mutants (A) Representative images of RAD-51 foci in mitotic zones and early pachytene and late pachytene in the indicated g only. Figure 3. Article Despite the overall reduction in the number of RAD-51 foci, we did not observe a rescue of viability in pole-4; rtel-1; rfs-1 and rtel-1; mus-81; pole-4 tri- ple mutant animals, thus suggesting that RAD-51-dependent HR events might pro- mote cell survival upon persistent replica- tion fork stalling in pole-4; rtel-1 mutant worms (Table S1). mitotic RAD-51 foci (Figures 4A and 4B), indicating that in rtel-1; pole-4 mutants, RAD-51 foci formation is partially dependent on both MUS-81 and RFS-1. Overall, RAD-51 foci were more signif- icantly reduced in rtel-1; pole-4; rfs-1 than in rtel-1; mus-81; pole- 4, suggesting a greater dependence on RFS-1 than on MUS-81. Given that MUS-81 and HIM-9/XPF-1 exhibit redundancy in generating recombination substrates at inter-strand cross-links (Ward et al., 2007), we speculate that both MUS-81 and HIM-9/ XPF-1 might independently process replication intermediates present in rtel-1; pole-4 animals, explaining the partial depen- B RAD-51 N2(wild-type ) rtel-1 mus-81 rfs-1 pole-4 rtel-1 mus-81 rtel-1; rfs-1 rtel-1; pole-4 rtel-1 mus-81; pole4 rtel-1; pole-4; rfs-1 0 1 2 3 4 5 number of mitotic RAD-51 foci ble knockout cells, as blings analysis (Figur cells showed a variab which complicate the action. In addition to Rtel1 was associated tion, similarly to that suggesting a coopera promoting genome-w lian cells in culture (F Article Despite the overall reductio number of RAD-51 foci, we observe a rescue of viability in rtel-1; rfs-1 and rtel-1; mus-81; p ple mutant animals, thus sugges RAD-51-dependent HR events m mote cell survival upon persisten tion fork stalling in pole-4; rtel-1 worms (Table S1). Proliferative Failure and Impa DNA Replication in Rtel1-Pole Double Knockout Cells We recently reported the genera Pole4 knockout mouse, which with intra- and extra-uterine gr striction, developmental abnor and lymphopenia. In vitro, mouse embryonic ﬁbroblasts exhibit Pol epsilon complex i and spontaneous DNA damage lation, which we attributed to origin activation and replicatio (Bellelli et al., 2018). To investi consequences of the loss of R POLE4+/+ and POLE4/ MEFs fected conditional Rtel1F/F Pole Rtel1F/F Pole4/ primary ME adenovirus expressing GFP-C empty GFP. Transduction of MEFs resulted in the expected the ﬂoxed RTEL1 alleles and eli of endogenous RTEL1 protei 72 h (Figure S4). Strikingly, the both Rtel1 and Pole4 resulted in plete block of cellular proliferatio ble knockout cells, as assessed by cumulative populat blings analysis (Figure 5A). Of note, both Pole4/ and cells showed a variable degree of reduced cellular prol which complicate the identiﬁcation of a speciﬁc synthe action. In addition to this, the concomitant loss of Po Rtel1 was associated with an overall reduction in EdU in tion, similarly to that observed in rtel-1; pole4 mutant suggesting a cooperative function for Pol epsilon and R promoting genome wide replication in both worms and m A N2 (wild type) rtel-1 mus-81 rfs-1 pole-4 rtel-1 mus-81 rtel-1; rfs-1 rtel-1; pole-4 rtel-1 mus-81; pole-4 rtel-1; pole-4; rfs-1 RAD-51 + DAPI RAD-51 RAD-51 + DAPI RAD-51 Mitotic zone nuclei A Figure 4. Mitotic RAD-51 Foci in rtel-1; pole- 4 Double Mutants Are Dependent on Both MUS-81 and RFS-1 (A) Representative images of mitotic nuclei stained with anti-RAD-51 in the indicated genotypes. (B) Quantiﬁcation of the average number of RAD- 51 foci per mitotic nucleus in the described ge- notypes is shown (p < 0.0001). Error bars represent standard deviation (SD) of the mean. (A) Representative images of mitotic nuclei stained with anti-RAD-51 in the indicated genotypes. (B) Quantiﬁcation of the average number of RAD- 51 foci per mitotic nucleus in the described ge- notypes is shown (p < 0.0001). Error bars represent standard deviation (SD) of the mean. dency on MUS-81 for RAD-51 foci forma- tion. Proliferative Failure and Impaired DNA Replication in Rtel1-Pole4 Double Knockout Cells B N2(wild-type ) rtel-1 mus-81 rfs-1 pole-4 rtel-1 mus-81 rtel-1; rfs-1 rtel-1; pole-4 rtel-1 mus-81; pole4 rtel-1; pole-4; rfs-1 0 1 2 3 4 5 number of mitotic RAD-51 foci B We recently reported the generation of a Pole4 knockout mouse, which presents with intra- and extra-uterine growth re- striction, developmental abnormalities, and lymphopenia. In vitro, Pole4/ mouse embryonic ﬁbroblasts (MEFs) exhibit Pol epsilon complex instability and spontaneous DNA damage accumu- lation, which we attributed to reduced origin activation and replication stress (Bellelli et al., 2018). To investigate the consequences of the loss of RTEL1 in POLE4+/+ and POLE4/ MEFs, we in- fected conditional Rtel1F/F Pole4+/+ and Rtel1F/F Pole4/ primary MEFs with adenovirus expressing GFP-CRE or empty GFP. Transduction of Rtel1F/F MEFs resulted in the expected loss of the ﬂoxed RTEL1 alleles and elimination of endogenous RTEL1 protein within 72 h (Figure S4). Strikingly, the loss of both Rtel1 and Pole4 resulted in a com- plete block of cellular proliferation in dou- as assessed by cumulative population dou- re 5A). Of note, both Pole4/ and Rtel1F/F ble degree of reduced cellular proliferation, e identiﬁcation of a speciﬁc synthetic inter- o this, the concomitant loss of Pole4 and d with an overall reduction in EdU incorpora- t observed in rtel-1; pole4 mutant worms, rative function for Pol epsilon and RTEL1 in wide replication in both worms and mamma- Figures 5B and 5C). mus-81; pole4 rtel-1; pole-4; rfs-1 We recently reported the generation of a Pole4 knockout mouse, which presents with intra- and extra-uterine growth re- striction, developmental abnormalities, and lymphopenia. In vitro, Pole4/ mouse embryonic ﬁbroblasts (MEFs) exhibit Pol epsilon complex instability and spontaneous DNA damage accumu- lation, which we attributed to reduced origin activation and replication stress (Bellelli et al., 2018). To investigate the consequences of the loss of RTEL1 in POLE4+/+ and POLE4/ MEFs, we in- fected conditional Rtel1F/F Pole4+/+ and Rtel1F/F Pole4/ primary MEFs with adenovirus expressing GFP-CRE or empty GFP. Transduction of Rtel1F/F MEFs resulted in the expected loss of the ﬂoxed RTEL1 alleles and elimination of endogenous RTEL1 protein within 72 h (Figure S4). Strikingly, the loss of both Rtel1 and Pole4 resulted in a com- plete block of cellular proliferation in dou- ble knockout cells, as assessed by cumulative population dou- blings analysis (Figure 5A). Article ll OPEN ACCESS mitotic RAD-51 foci (Figures 4A and 4B), indicating that in rtel-1; pole-4 mutants, RAD-51 foci formation is partially dependent on both MUS-81 and RFS-1. Overall, RAD-51 foci were more signif- icantly reduced in rtel-1; pole-4; rfs-1 than in rtel-1; mus-81; pole- 4, suggesting a greater dependence on RFS-1 than on MUS-81. Given that MUS-81 and HIM-9/XPF-1 exhibit redundancy in generating recombination substrates at inter-strand cross-links A B N2 (wild type) rtel-1 mus-81 rfs-1 pole-4 rtel-1 mus-81 rtel-1; rfs-1 rtel-1; pole-4 rtel-1 mus-81; pole-4 rtel-1; pole-4; rfs-1 RAD-51 + DAPI RAD-51 RAD-51 + DAPI RAD-51 Mitotic zone nuclei N2(wild-type ) rtel-1 mus-81 rfs-1 pole-4 rtel-1 mus-81 rtel-1; rfs-1 rtel-1; pole-4 rtel-1 mus-81; pole4 rtel-1; pole-4; rfs-1 0 1 2 3 4 5 number of mitotic RAD-51 foci ble knockout cells, blings analysis (Fig cells showed a vari which complicate t action. In addition Rtel1 was associate tion, similarly to th mitotic RAD-51 foci (Figures 4A and 4B), indicating that in rtel-1; pole-4 mutants, RAD-51 foci formation is partially dependent on both MUS-81 and RFS-1. Overall, RAD-51 foci were more signif- icantly reduced in rtel-1; pole-4; rfs-1 than in rtel-1; mus-81; pole- 4, suggesting a greater dependence on RFS-1 than on MUS-81. Given that MUS-81 and HIM-9/XPF-1 exhibit redundancy in generating recombination substrates at inter-strand cross-links (Ward et al., 2007), we speculate that both MUS-81 and HIM-9/ XPF 1 might independently process replication intermediates A B N2 (wild type) rtel-1 mus-81 rfs-1 pole-4 rtel-1 mus-81 rtel-1; rfs-1 rtel-1; pole-4 rtel-1 mus-81; pole-4 rtel-1; pole-4; rfs-1 RAD-51 + DAPI RAD-51 RAD-51 + DAPI RAD-51 Mitotic zone nuclei N2(wild-type ) rtel-1 mus-81 rfs-1 pole-4 rtel-1 mus-81 rtel-1; rfs-1 rtel-1; pole-4 rtel-1 mus-81; pole4 rtel-1; pole-4; rfs-1 0 1 2 3 4 5 number of mitotic RAD-51 foci Figure 4. Mitotic RAD-51 Foci in rte 4 Double Mutants Are Dependent MUS-81 and RFS-1 (A) Representative images of mitotic nuc with anti-RAD-51 in the indicated genot (B) Quantiﬁcation of the average numb 51 foci per mitotic nucleus in the des notypes is shown (p < 0.0001). represent standard deviation (SD) of the dency on MUS-81 for RAD-51 fo tion. Mus81- and Rfs1-Dependent Processing of Replication Intermediates in rtel-1; pole4 Worms RAD-51 Foci and Chromosome Aberrations Are Elevated in rtel-1; pole-4 Double Mutants (A) Representative images of RAD-51 foci in mitotic zones and early pachytene and late pachytene in the indicated g only. (B) Left: top panels show representative images of N2(wild-type), pole-4, and rtel-1 single mutant diakinesis nuclei showing six DAPI-stained bivalents present; bottom panels show examples of the chromosome defects observed in rtel-1; pole-4, including chromosome fragments, constrictions on chromosome arms, chromosome fusions, and unpaired sister chromatids. Right: quantiﬁcation of the number of DAPI-stained bodies present at diakinesis in each of the indicated strains (see also Figure S3B). breaks. To this end, we constructed two balanced strains: rtel- 1 mus-81/hT2[gfp]; pole-4 and rtel-1/hT2[gfp]; pole-4; rfs-1/hT2 [gfp] from which we could isolate homozygous triple mutants and stain for the presence of RAD-51. Compared to rtel-1; pole-4 double mutants, both rtel-1; mus-81; pole-4 and rtel-1; pole-4; rfs-1 triple mutant animals exhibited statistically fewer (Dehe´ and Gaillard, 2017), whereas RFS-1 is a RAD-51 paralog that is required for RAD-51 loading speciﬁcally at stalled replica- tion forks, but not at sites of fork collapse or DNA DSBs (Ward et al., 2007). If the RAD-51 foci in rtel-1; pole-4 are dependent on RFS-1, this would suggest that damage sites represent stalled/blocked replication forks rather than collapsed forks or Cell Reports 31, 107675, May 26, 2020 5 A ti l Article Figure 4. Mitotic RAD-51 Foci in rtel-1; pole- 4 Double Mutants Are Dependent on Both MUS-81 and RFS-1 (A) Representative images of mitotic nuclei stained with anti-RAD-51 in the indicated genotypes. (B) Quantiﬁcation of the average number of RAD- 51 foci per mitotic nucleus in the described ge- notypes is shown (p < 0.0001). Error bars represent standard deviation (SD) of the mean. Proliferative Failure and Impaired DNA Replication in Rtel1-Pole4 Double Knockout Cells More importantly, the combined loss of POLE4 and RTEL1 was associated with a striking increase in nuclear blebbing and micronuclei formation, suggestive of extensive chromosomal instability(Figure6C).Inadditiontothis,atypicalnuclearstructures and mitotic bridges were observed that are suggestive of incom- plete DNA replication (Figures S5A, S5B, and S5C). Loss of RTEL1 and POLE4 Leads to Replication Stress and Reduced Fork Extension Rates 0 20 40 60 80 EdU intensity (a.u.) n.s. RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE RTEL1 f/f DAPI EdU POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE DAPI EdU A B C 0 1 2 3 4 0 5 10 Cumulative PDLs number of passages POLE4 +/+ Ad-GFP POLE4 +/+ Ad-CRE POLE4 -/- Ad-GFP POLE4 -/- Ad-CRE * * Figure 5. Reduce EdU Incorporatio Primary Mouse Em (A) Cumulative pop RTEL1F/F POLE4+/+ MEFs infected with CRE or empty GFP analysis 72 h after inf to a standard 3T3 pro SD of triplicate expe (B) Bar graphs sho (arbitrary units) of RTEL1F/F POLE4/ Cells were analyzed after infection with G represent mean ± S (p < 0.0001; n.s., (C) Representative i RTEL1F/F POLE4+/+ MEFs infected or n Scale bars, 16 mm. tion of Rtel1 in replication fork e inter-origin distan asymmetry, whic fork stalling and due to dorman et al., 2007; Ibar et al., 2013). Co exhibit increased and enhanced with heightened increased fork as 2018). To understand combined loss o replication fork a we analyzed the replication dynamics of s cells. To this aim, we infected RTEL1F/ POLE4/ primary MEFs with adenoviru or empty GFP and, 72 h after CRE-med pulse-labeled cells with CldU and IdU a analysis, as previously described (Figure Consistent with previous studies, the loss tion in fork speed, increased fork asymm origin distance, whereas the loss of POL crease in fork extension rates due to r and increased inter-origin distance and 7B, 7C, and 7D) Strikingly, when we analyzed fork e Pole4 double knockout cells, we obse speed compared with both wild-type a suggestive of compromised fork elong loss of RTEL1 and POLE4. In agreeme more than 70% of newly activated rep asymmetry of newly incorporated nucle 0 20 40 60 80 EdU intensity (a.u.) n.s. Proliferative Failure and Impaired DNA Replication in Rtel1-Pole4 Double Knockout Cells RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE B ** * A 0 1 2 3 4 0 5 10 Cumulative PDLs number of passages POLE4 +/+ Ad-GFP POLE4 +/+ Ad-CRE POLE4 -/- Ad-GFP POLE4 -/- Ad-CRE * * Figure 5. Reduced Growth and Impaired EdU Incorporation in RTEL1F/F POLE4/ Primary Mouse Embryo Fibroblasts (MEFs) (A) Cumulative population doublings (PDLs) of RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with adenovirus expressing GFP- CRE or empty GFP. Cells were seeded for PDL analysis 72 h after infection and cultured according to a standard 3T3 protocol. Bars represent mean ± SD of triplicate experiments (*p < 0.001). (B) Bar graphs showing EdU intensity staining (arbitrary units) of RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ infected or not with CRE. Cells were analyzed for EdU incorporation 72h after infection with GFP-CRE or empty GFP. Bars represent mean ± SD of triplicate experiments (p < 0.0001; n.s., not signiﬁcant). (C) Representative images of EdU staining from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected or not with CRE recombinase. Scale bars, 16 mm. B B A 4 EdU intensity (a.u.) Cumulative PDLs (B) Bar graphs showing EdU intensity staining (arbitrary units) of RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ infected or not with CRE. Cells were analyzed for EdU incorporation 72h after infection with GFP-CRE or empty GFP. Bars represent mean ± SD of triplicate experiments (p < 0.0001; n.s., not signiﬁcant). (C) Representative images of EdU staining from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected or not with CRE recombinase. Scale bars, 16 mm. RTEL1 f/f RTEL1 f/f DAPI EdU POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE DAPI EdU C C tion of Rtel1 in MEFs leads to reduced replication fork extension rates, reduced inter-origin distance, and increased fork asymmetry, which are all suggestive of fork stalling and increased origin use due to dormant origin activation (Ge et al., 2007; Ibarra et al., 2008; Vannier et al., 2013). Conversely, Pole4/ cells exhibit increased inter-origin distances and enhanced fork speed associated with heightened replication stress and increased fork asymmetry (Bellelli et al., 2018). tion of Rtel1 in MEFs leads to reduced replication fork extension rates, reduced inter-origin distance, and increased fork asymmetry, which are all suggestive of fork stalling and increased origin use due to dormant origin activation (Ge et al., 2007; Ibarra et al., 2008; Vannier et al., 2013). Proliferative Failure and Impaired DNA Replication in Rtel1-Pole4 Double Knockout Cells More importantly, the combined loss of POLE4 and RTEL1 was associated with a striking increase in nuclear blebbing and micronuclei formation, suggestive of extensive chromosomal instability(Figure6C).Inadditiontothis,atypicalnuclearstructures and mitotic bridges were observed that are suggestive of incom- plete DNA replication (Figures S5A, S5B, and S5C). Loss of RTEL1 and POLE4 Leads to Replication Stress and Reduced Fork Extension Rates 0 20 40 60 80 EdU intensity (a.u.) n.s. RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE RTEL1 f/f DAPI EdU POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE DAPI EdU A B C 0 1 2 3 4 0 5 10 Cumulative PDLs number of passages POLE4 +/+ Ad-GFP POLE4 +/+ Ad-CRE POLE4 -/- Ad-GFP POLE4 -/- Ad-CRE * * we analyzed the repl cells. To this aim, w POLE4/ primary M or empty GFP and, 7 pulse-labeled cells w analysis, as previous Consistent with prev tion in fork speed, in origin distance, whe crease in fork exten and increased inter- 7B, 7C, and 7D) Strikingly, when w Pole4 double knock speed compared w suggestive of comp loss of RTEL1 and more than 70% of n asymmetry of newly Article Extensive DNA Damage and Chromosomal Instability upon Combined Loss of Rtel1 and Pole4 0 20 40 60 80 EdU intensity (a.u.) n.s. RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE RTEL1 f/f DAPI EdU POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE DAPI EdU A B C 0 1 2 3 4 0 5 10 Cumulative PDLs number of passages POLE4 +/+ Ad-GFP POLE4 +/+ Ad-CRE POLE4 -/- Ad-GFP POLE4 -/- Ad-CRE * * we analyzed the repl cells To this aim w Article Extensive DNA Damage and Chromosomal Instability upon Combined Loss of Rtel1 and Pole4 To further characterize the mechanism responsible for reduced growth and EdU incorporation in Rtel1-Pole4 double knockout mouse cells, we analyzed by immunoﬂuorescence the presence of markers of DNA damage, including gH2AX and 53BP1 foci, in Rtel1F/F POLE4+/+ and / MEFs infected or not with CRE (Panier and Boulton, 2014). In accordance with a failure to complete DNA replication,theabsenceofbothRTEL1 and POLE4leadtoastrong increase in both gH2AX and 53BP1 foci-positive cells (Figures 6A and 6B). Proliferative Failure and Impaired DNA Replication in Rtel1-Pole4 Double Knockout Cells Conversely, Pole4/ cells exhibit increased inter-origin distances and enhanced fork speed associated with heightened replication stress and increased fork asymmetry (Bellelli et al., 2018). EdU DAPI POLE4 -/- POLE4 -/- EdU To understand the consequences of the combined loss of RTEL1 and POLE4 on replication fork activation and elongation, we analyzed the replication dynamics of single and double mutant cells. To this aim, we infected RTEL1F/F POLE4+/+ or RTEL1F/F POLE4/ primary MEFs with adenovirus expressing GFP-CRE or empty GFP and, 72 h after CRE-mediated excision of RTEL1, pulse-labeled cells with CldU and IdU and performed DNA ﬁber analysis, as previously described (Figure 7A; Bellelli et al., 2018). Consistent with previous studies, the loss of RTEL1 led to a reduc- tion in fork speed, increased fork asymmetry, and reduced inter- origin distance, whereas the loss of POLE4 led to a signiﬁcant in- crease in fork extension rates due to reduced origin activation and increased inter-origin distance and fork asymmetry (Figures 7B, 7C, and 7D) EdU Extensive DNA Damage and Chromosomal Instability upon Combined Loss of Rtel1 and Pole4 To further characterize the mechanism responsible for reduced growth and EdU incorporation in Rtel1-Pole4 double knockout mouse cells, we analyzed by immunoﬂuorescence the presence of markers of DNA damage, including gH2AX and 53BP1 foci, in Rtel1F/F POLE4+/+ and / MEFs infected or not with CRE (Panier and Boulton, 2014). In accordance with a failure to complete DNA replication,theabsenceofbothRTEL1 and POLE4leadtoastrong increase in both gH2AX and 53BP1 foci-positive cells (Figures 6A and 6B). More importantly, the combined loss of POLE4 and RTEL1 was associated with a striking increase in nuclear blebbing and micronuclei formation, suggestive of extensive chromosomal instability(Figure6C).Inadditiontothis,atypicalnuclearstructures and mitotic bridges were observed that are suggestive of incom- plete DNA replication (Figures S5A, S5B, and S5C). To further characterize the mechanism responsible for reduced growth and EdU incorporation in Rtel1-Pole4 double knockout mouse cells, we analyzed by immunoﬂuorescence the presence of markers of DNA damage, including gH2AX and 53BP1 foci, in Rtel1F/F POLE4+/+ and / MEFs infected or not with CRE (Panier and Boulton, 2014). In accordance with a failure to complete DNA replication,theabsenceofbothRTEL1 and POLE4leadtoastrong increase in both gH2AX and 53BP1 foci-positive cells (Figures 6A and 6B). Proliferative Failure and Impaired DNA Replication in Rtel1-Pole4 Double Knockout Cells More importantly, the combined loss of POLE4 and RTEL1 was associated with a striking increase in nuclear blebbing and micronuclei formation, suggestive of extensive chromosomal instability(Figure6C).Inadditiontothis,atypicalnuclearstructures and mitotic bridges were observed that are suggestive of incom- plete DNA replication (Figures S5A, S5B, and S5C). Strikingly, when we analyzed fork extension rates in Rtel1- Pole4 double knockout cells, we observed a reduction in fork speed compared with both wild-type and Rtel1-null-only cells, suggestive of compromised fork elongation upon concomitant loss of RTEL1 and POLE4. In agreement with this hypothesis, more than 70% of newly activated replication origins featured asymmetry of newly incorporated nucleotide tracks (Figures 7B and 7C). However, distinct from that observed in Rtel1-null- only cells, double mutant cells did not exhibit a signiﬁcant Proliferative Failure and Impaired DNA Replication in Rtel1-Pole4 Double Knockout Cells Of note, both Pole4/ and Rtel1F/F cells showed a variable degree of reduced cellular proliferation, which complicate the identiﬁcation of a speciﬁc synthetic inter- action. In addition to this, the concomitant loss of Pole4 and Rtel1 was associated with an overall reduction in EdU incorpora- tion, similarly to that observed in rtel-1; pole4 mutant worms, suggesting a cooperative function for Pol epsilon and RTEL1 in promoting genome-wide replication in both worms and mamma- lian cells in culture (Figures 5B and 5C). mitotic RAD-51 foci (Figures 4A and 4B), indicating that in rtel-1; pole-4 mutants, RAD-51 foci formation is partially dependent on both MUS-81 and RFS-1. Overall, RAD-51 foci were more signif- icantly reduced in rtel-1; pole-4; rfs-1 than in rtel-1; mus-81; pole- 4, suggesting a greater dependence on RFS-1 than on MUS-81. Given that MUS-81 and HIM-9/XPF-1 exhibit redundancy in generating recombination substrates at inter-strand cross-links (Ward et al., 2007), we speculate that both MUS-81 and HIM-9/ XPF-1 might independently process replication intermediates present in rtel-1; pole-4 animals, explaining the partial depen- mitotic RAD-51 foci (Figures 4A and 4B), indicating that in rtel-1; pole-4 mutants, RAD-51 foci formation is partially dependent on both MUS-81 and RFS-1. Overall, RAD-51 foci were more signif- icantly reduced in rtel-1; pole-4; rfs-1 than in rtel-1; mus-81; pole- 4, suggesting a greater dependence on RFS-1 than on MUS-81. Given that MUS-81 and HIM-9/XPF-1 exhibit redundancy in generating recombination substrates at inter-strand cross-links (Ward et al., 2007), we speculate that both MUS-81 and HIM-9/ XPF-1 might independently process replication intermediates present in rtel-1; pole-4 animals, explaining the partial depen- 6 Cell Reports 31, 107675, May 26, 2020 ll OPEN ACCESS ll OPEN ACCESS Extensive DNA Damage and Chromosomal Instability upon Combined Loss of Rtel1 and Pole4 To further characterize the mechanism responsible for reduced growth and EdU incorporation in Rtel1-Pole4 double knockout mouse cells, we analyzed by immunoﬂuorescence the presence of markers of DNA damage, including gH2AX and 53BP1 foci, in Rtel1F/F POLE4+/+ and / MEFs infected or not with CRE (Panier and Boulton, 2014). In accordance with a failure to complete DNA replication,theabsenceofbothRTEL1 and POLE4leadtoastrong increase in both gH2AX and 53BP1 foci-positive cells (Figures 6A and 6B). 8 Cell Reports 31, 107675, May 26, 2020 DISCUSSION Here, we uncover a synthetic lethal interaction between the RTEL1 helicase and DNA Pol epsilon in the nematode C. elegans. RNAi-mediated loss of Pol epsilon complex subunits pole-1, pole-2, and pole-3 conferred reduced viability in rtel-1 mutant worms associated with elevated replication stress, as shown by RPA and RAD-51 foci accumulation, in both mitotic and meiotic zones. The viability of a strain lacking the fourth sub- unit of Pol Epsilon, pole-4, allowed us to genetically combine the loss of rtel-1 and pole-4 in C. elegans, which revealed a complete loss of viability upon removal of both RTEL-1 and POLE-4 in nematodes. We establish that this synthetic lethal interaction is also conserved in vertebrates and that, in both worms and mouse cells, the combined loss of Rtel1 and Pole4 confers extensive genome instability and cessation of DNA replication. We previously showed that RTEL1 binds to the proliferating cell nuclear antigen (PCNA) by its PIP-box domain and that this func- tion is required for replication, potentially though problematic se- quences, such as G4-DNA structures (Vannier et al., 2013). How- ever, the lack of synthetic lethality between pole-4 and dog1/ FANCJ, which is required for G4-DNA stability in worms (Cheung et al., 2002; Youds et al., 2008), likely excludes that G4-DNA is a source of synthetic lethality in rtel-1; pole-4 double mutants. In addition to this, it was recently shown that RTEL1 is required for the bypass of DNA-protein cross-links as well as non-covalent nucleoprotein complexes (Sparks et al., 2019). This function of RTEL1 appears to be PIP-box independent and suggests addi- tional mechanisms of RTEL1 recruitment at the replication fork. If and how this newly described function of RTEL1 is required for genome-wide replication fork progression remains to be addressed. Importantly, a synthetic lethal interaction was also observed be- tween rtel-1 and other DNA replication genes required for the initi- ation of DNA replication, such as Topbp1 and psf2-psf3, which are components of the GINS complex. Topbp1 is the homolog of budding yeast Dpb11, which binds to phosphorylated Sld2 and Sld3 upon CDK activation and promotes pre-loading complex for- mation by recruiting the GINS complex in concert with Pol epsilon (Muramatsu et al., 2010). DISCUSSION The fact that Dpb11, Psf2-Psf3 (in the context of GINS), and Pol epsilon directly bind to each other and are required for a speciﬁc step of DNA replication initiation strongly points toa direct and mechanistic connection between RTEL1 and replication fork progression in nematodes. Consistent with this hy- pothesis, we did not observe any evidence of a synthetic lethal interaction between RTEL1 and DNA Pol delta. In summary, through the identiﬁcation of a novel genetic inter- action between RTEL1 and Pol epsilon in nematodes, we reveal an essential role for RTEL1 in DNA replication under conditions of reduced origin ﬁring and unveil a fundamental requirement for the strict coordination between origin activation and fork elonga- tion in the maintenance of genome stability in metazoans. Loss of RTEL1 and POLE4 Leads to Replication Stress and Reduced Fork Extension Rates Loss of RTEL1 and POLE4 Leads to Replication Stress and Reduced Fork Extension Rates We previously showed that RTEL1 is involved in telomere and genome-wide replication (Vannier et al., 2013). Conditional dele- Cell Reports 31, 107675, May 26, 2020 7 Article ll OPEN ACCESS Art (legend on next page) 8 Cell Reports 31, 107675, May 26, 2020 Article ll OPEN ACCESS reduction in inter-origin distance, which is suggestive of a failure to efﬁciently activate dormant replication origins (Figure 7D). tion fork extension rates, which are all hallmarks of replication stress (Zeman and Cimprich, 2014; Te´ cher et al., 2017). Howev- er, in contrast to that observed in a Pole4 wild-type background, where the loss of Rtel1 leads to reduced inter-origin distance due to dormant origin activation, double knockout cells failed to efﬁ- ciently activate dormant origins. Figure 6. RTEL1-POLE4 Double Knockout Cells Accumulate DNA Damage and Genomic Instability (A) Top: bar graphs showing percentage of cells with more than 10 gH2AX foci from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. Cells were analyzed 72 h after infection. Bars represent mean ± SD of triplicate experiments (p < 0.001, **p < 0.0001). Bottom: representative immunoﬂuorescence staining from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. (B) Top: bar graphs showing percentage of cells with more than 5 53BP1 foci from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. Cells were analyzed 72 h after infection. Bars represent mean ± SD of triplicate experiments. (p < 0.05, p < 0.01). Bottom: representative immunoﬂuorescence staining from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. (C) Left: bar graphs showing percentage of cells with micronuclei; bars represent mean ± SD of triplicate experiments (p < 0.001). Right: representative images of micronuclei in cells of the indicated genotype. Scale bar, 10 mm. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: In agreement with a profound replication defect, RAD-51 foci accumulate in rtel-1; pole-4 mutants and were partially depen- dent on MUS-81, a structure-speciﬁc endonuclease previously reported to process stalled replication intermediates to promote HR-dependent rescue of collapsed replication forks (Dehe´ and Gaillard, 2017). RAD-51 foci were also dependent on the Rad51 paralog RFS-1, which we previously showed to be exclu- sively required for RAD-51 loading at stalled replication forks (Ward et al., 2007). In agreement with a profound replication defect, RAD-51 foci accumulate in rtel-1; pole-4 mutants and were partially depen- dent on MUS-81, a structure-speciﬁc endonuclease previously reported to process stalled replication intermediates to promote HR-dependent rescue of collapsed replication forks (Dehe´ and Gaillard, 2017). RAD-51 foci were also dependent on the Rad51 paralog RFS-1, which we previously showed to be exclu- sively required for RAD-51 loading at stalled replication forks (Ward et al., 2007). d KEY RESOURCES TABLE d RESOURCE AVAILABILITY d RESOURCE AVAILABILITY B Lead Contact B Materials Availability B Data and Code Availabilit B Data and Code Availability d EXPERIMENTAL MODEL AND SU B Mouse strains and cell lines B C. elegans strains Similar to the observations in worms, the combined loss of Pole4 and Rtel1 in MEFs led to a block to cellular proliferation, which was associated with DNA damage, genome instability, and incomplete DNA replication. At the molecular level, the loss of RTEL1 in a Pole4 knockout background led to a substan- tial increase in replication fork asymmetry with reduced replica- d METHOD DETAILS B RNA interference and plate phenotype scoring B Immunoﬂuorescence analysis in C.elegans B Assay for poly G/C tract deletions B Immunoﬂuorescence analysis in C.elegans B Assay for poly-G/C-tract deletions B Mouse Embryonic Fibroblasts (MEFs) isolation and culture (C) Left: bar graphs showing percentage of cells with micronuclei; bars represent mean ± SD of triplicate experiments (p < 0.001). Right: representative images of micronuclei in cells of the indicated genotype. Scale bar, 10 mm. Cell Reports 31, 107675, May 26, 2020 9 Article ll OPEN ACCESS 0 1 2 3 4 5 fork speed (Kb/min) 0 50 100 150 200 Mean IOD (Kb) 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. STAR+METHODS 51 0 20 40 60 80 0 20 40 60 80 Left (kb) 36 % n. 75 0 20 40 60 0 20 40 60 80 Left (kb) 71% n. 65 RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-CRE RTEL1 f/f POLE4 +/+ Ad-CRE RTEL1 f/f POLE4 +/+ Ad-GFP C D 0 50 100 150 200 Mean IOD (Kb) 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. 51 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 36 % n. 75 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 71% n. 65 RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-C Ad-GFP Ad-CRE RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-CRE RTEL1 f/f POLE4 +/+ Ad-CRE RTEL1 f/f POLE4 +/+ Ad-GFP C D * 0 50 100 150 200 Mean IOD (Kb) 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. 51 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 36 % n. 75 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 71% n. 65 RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-CRE RTEL1 f/f POLE4 +/+ Ad-CRE RTEL1 f/f POLE4 +/+ Ad-GFP C D * Figure 7. Impaired Fork Progression and Increased Fork Stalling in RTEL1-POLE4 Double Knockout Cells (A) Representative DNA ﬁber immunoﬂuorescence from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. (B) Bar graphs showing replication fork speed (measured as IdU track length/min) from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with CRE. p < 0.0001. (C). Analysis of replication fork symmetry from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. Total number of newly established replication forks analyzed is indicated. (D) Bar graphs showing inter-origin distance measurements from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with CRE recombinase (p < 0.01, *p < 0.001). Error bars represent standard deviation (SD) of the mean. STAR+METHODS Scale bars, 10 mm. 0 50 100 150 200 Mean IOD (Kb) 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. 51 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 36 % n. 75 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 71% n. 65 RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-CRE RTEL1 f/f POLE4 +/+ Ad-CRE RTEL1 f/f POLE4 +/+ Ad-GFP C D * Figure 7. Impaired Fork Progression and Increased Fork Stalling in RTEL1-POLE4 Double Knockout Cells (A) Representative DNA ﬁber immunoﬂuorescence from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. (B) Bar graphs showing replication fork speed (measured as IdU track length/min) from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with CRE. **p < 0.0001. (C). Analysis of replication fork symmetry from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. Total number of newly established replication forks analyzed is indicated. (D) Bar graphs showing inter-origin distance measurements from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with CRE recombinase (p < 0.01, *p < 0.001). Error bars represent standard deviation (SD) of the mean. Scale bars, 10 mm. 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. 51 RTEL1 f/f POLE4 -/- Ad-GFP D 0 50 100 150 200 Mean IOD (Kb) RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE * C D 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. 51 RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 +/+ Ad-GFP 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% L ft (kb) RTEL1 f/f POLE4 +/+ Ad-GFP 1 Mean IOD (Kb) 80 % 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 71% n. STAR+METHODS 51 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 36 % n. 75 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 71% n. 65 RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-CRE RTEL1 f/f POLE4 +/+ Ad-CRE RTEL1 f/f POLE4 +/+ Ad-GFP A B C RTEL1 f/f POLE4 +/+ Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP D ** * 7. Impaired Fork Progression and Increased Fork Stalling in RTEL1-POLE4 Double Knockout Cells resentative DNA ﬁber immunoﬂuorescence from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. graphs showing replication fork speed (measured as IdU track length/min) from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with p < 0.0001. alysis of replication fork symmetry from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. Total number of newly shed replication forks analyzed is indicated. graphs showing inter-origin distance measurements from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with CRE recombinase .01, p < 0.001). Error bars represent standard deviation (SD) of the mean. Scale bars, 10 mm. fork speed (Kb/min) B RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP 0 1 2 3 4 5 RTEL1 f/f POLE4 +/+ POLE4 -/- Ad-GFP Ad-CRE Ad-GFP Ad-CRE * fork speed (Kb/min) A B RTEL1 f/f POLE4 +/+ Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP B A RTEL1 f/f POLE4 +/+ Ad-GFP fork speed (Kb/min) RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 -/- Ad-GFP 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. 51 RTEL1 f/f POLE4 -/- Ad-GFP RTEL1 f/f POLE4 +/+ Ad-GFP C D 0 50 100 150 200 Mean IOD (Kb) 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) n. 72 5% 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 29% n. REFERENCES Vannier, J.B., Pavicic-Kaltenbrunner, V., Petalcorin, M.I., Ding, H., and Boul- ton, S.J. (2012). RTEL1 dismantles T loops and counteracts telomeric G4- DNA to maintain telomere integrity. Cell 149, 795–806. Barber, L.J., Youds, J.L., Ward, J.D., McIlwraith, M.J., O’Neil, N.J., Petalcorin, M.I., Martin, J.S., Collis, S.J., Cantor, S.B., Auclair, M., et al. (2008). RTEL1 maintains genomic stability by suppressing homologous recombination. Cell 135, 261–271. Barber, L.J., Youds, J.L., Ward, J.D., McIlwraith, M.J., O’Neil, N.J., Petalcorin, M.I., Martin, J.S., Collis, S.J., Cantor, S.B., Auclair, M., et al. (2008). RTEL1 maintains genomic stability by suppressing homologous recombination. Cell 135, 261–271. Vannier, J.B., Sandhu, S., Petalcorin, M.I., Wu, X., Nabi, Z., Ding, H., and Boul- ton, S.J. (2013). RTEL1 is a replisome-associated helicase that promotes telo- mere and genome-wide replication. Science 342, 239–242. Bell, S.P., and Labib, K. (2016). Chromosome Duplication in Saccharomyces cerevisiae. Genetics 203, 1027–1067. Ward, J.D., Barber, L.J., Petalcorin, M.I., Yanowitz, J., and Boulton, S.J. (2007). Replication blocking lesions present a unique substrate for homolo- gous recombination. EMBO J. 26, 3384–3396. Bellelli, R., Borel, V., Logan, C., Svendsen, J., Cox, D.E., Nye, E., Metcalfe, K., O’Connell, S.M., Stamp, G., Flynn, H.R., et al. (2018). Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis. Mol. Cell 70, 707–721.e7. Youds, J.L., O’Neil, N.J., and Rose, A.M. (2006). Homologous recombination is required for genome stability in the absence of DOG-1 in Caenorhabditis ele- gans. Genetics 173, 697–708. Bhat, K.P., and Cortez, D. (2018). RPA and RAD51: fork reversal, fork protec- tion, and genome stability. Nat. Struct. Mol. Biol. 25, 446–453. Wicky, C., Alpi, A., Passanante, M., Rose, A., Gartner, A., and M€uller, F. (2004). Multiple genetic pathways involving the Caenorhabditis elegans Bloom’s syn- drome genes him-6, rad-51, and top-3 are needed to maintain genome stabil- ity in the germ line. Mol. Cell. Biol. 24, 5016–5017. Burgers, P.M.J., and Kunkel, T.A. (2017). Eukaryotic DNA Replication Fork. Annu. Rev. Biochem. 86, 417–438. Cheung, I., Schertzer, M., Rose, A., and Lansdorp, P.M. (2002). Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA. Nat. Genet. 31, 405–409. Youds, J.L., Barber, L.J., Ward, J.D., Collis, S.J., O’Neil, N.J., Boulton, S.J., and Rose, A.M. (2008). DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair. Mol. Cell. Biol. 28, 1470–1479. Dehe´ , P.M., and Gaillard, P.H.L. (2017). Control of structure-speciﬁc endonu- cleases to maintain genome stability. Nat. Rev. Mol. Cell Biol. 18, 315–330. AUTHOR CONTRIBUTIONS Panier, S., and Boulton, S.J. (2014). Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18. S.J.B. conceived the project. J.Y. performed the RNAi screening in C. elegans and experiments in worms with help from J.S. and V.T.-P. R.B. performed ex- periments in mammalian cells with help from V.B. R.B., J.Y., and S.J.B. wrote the paper. Saito, T.T., Youds, J.L., Boulton, S.J., and Colaia´ covo, M.P. (2009). Caeno- rhabditis elegans HIM-18/SLX-4 interacts with SLX-1 and XPF-1 and maintains genomic integrity in the germline by processing recombination intermediates. PLoS Genet 5, e1000735. genomic integrity in the germline by processing recombination intermediates. PLoS Genet 5, e1000735. STAR+METHODS 65 RTEL1 f/f POLE4 -/- Ad-CRE E 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 36 % n. 75 Left (kb) RTEL1 f/f POLE4 +/+ Ad-CRE 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 36 % n. 75 0 20 40 60 80 0 20 40 60 80 Right (kb) Left (kb) 71% n. 65 RTEL1 f/f POLE4 -/- Ad-CRE RTEL1 f/f POLE4 +/+ Ad-CRE Figure 7. Impaired Fork Progression and Increased Fork Stalling in RTEL1-POLE4 Double Knockout Cells Figure 7. Impaired Fork Progression and Increased Fork Stalling in RTEL1 POLE4 Double Knockout Cells (A) Representative DNA ﬁber immunoﬂuorescence from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. (B) Bar graphs showing replication fork speed (measured as IdU track length/min) from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with CRE. p < 0.0001. (C). Analysis of replication fork symmetry from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs infected with GFP-CRE or empty GFP. Total number of newly established replication forks analyzed is indicated. (D) Bar graphs showing inter-origin distance measurements from RTEL1F/F POLE4+/+ and RTEL1F/F POLE4/ MEFs transduced or not with CRE recombinase (p < 0.01, ***p < 0.001). Error bars represent standard deviation (SD) of the mean. Scale bars, 10 mm. 10 Cell Reports 31, 107675, May 26, 2020 Article ll OPEN ACCESS B Cre-mediated recombination Ge, X.Q., Jackson, D.A., and Blow, J.J. (2007). Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes Dev. 21, 3331–3341. B Immunoﬂuorescence staining B DNA ﬁber stretching assay B DNA ﬁber stretching assay d QUANTIFICATION AND STATISTICAL ANALYSES Hustedt, N., A´ lvarez-Quilo´ n, A., McEwan, A., Yuan, J.Y., Cho, T., Koob, L., Hart, T., and Durocher, D. (2019). A consensus set of genetic vulnerabilities to ATR inhibition. Open Biol. 9, 190156. d QUANTIFICATION AND STATISTICAL ANALYSES d QUANTIFICATION AND STATISTICAL ANALYSES ACKNOWLEDGMENTS We would like to thank the Francis Crick Biological Resource facility for assis- tance with mouse breeding and maintenance and Joe Brock for help with ﬁgure generation. R.B. and V.B. are funded by the Francis Crick Institute. Boul- ton lab work is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC0010048), the UK Medical Research Council (FC0010048), and the Wellcome Trust (FC0010048); a European Research Council (ERC) Advanced Investigator Grant (TelMetab); and Well- come Trust senior investigator and collaborative grants. Leo´ n-Ortiz, A.M., Svendsen, J., and Boulton, S.J. (2014). Metabolism of DNA secondary structures at the eukaryotic replication fork. DNA Repair (Amst.) 19, 152–162. Muramatsu, S., Hirai, K., Tak, Y.S., Kamimura, Y., and Araki, H. (2010). CDK- dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon, and GINS in budding yeast. Genes Dev. 24, 602–612. Navas, T.A., Zhou, Z., and Elledge, S.J. (1995). DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint. Cell 80, 29–39. DECLARATION OF INTERESTS Sarek, G., Vannier, J.B., Panier, S., Petrini, J.H.J., and Boulton, S.J. (2015). TRF2 recruits RTEL1 to telomeres in S phase to promote t-loop unwinding. Mol. Cell 57, 622–635. S.J.B. is scientiﬁc co-founder and SVP Science Strategy at Artios Pharma Ltd., Babraham, UK. Sparks, J.L., Chistol, G., Gao, A.O., Ra¨ schle, M., Larsen, N.B., Mann, M., Duxin, J.P., and Walter, J.C. (2019). The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair. Cell 176, 167–181.e21. Received: February 20, 2020 Revised: March 28, 2020 Accepted: April 30, 2020 Published: May 26, 2020 Te´ cher, H., Koundrioukoff, S., Nicolas, A., and Debatisse, M. (2017). The impact of replication stress on replication dynamics and DNA damage in verte- brate cells. Nat. Rev. Genet. 18, 535–550. Published: May 26, 2020 SUPPLEMENTAL INFORMATION Ibarra, A., Schwob, E., and Me´ ndez, J. (2008). Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc. Natl. Acad. Sci. USA 105, 8956–8961. Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2020.107675. Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kana- pin, A., Le Bot, N., Moreno, S., Sohrmann, M., et al. (2003). Systematic func- tional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237. REFERENCES Youds, J.L., Mets, D.G., McIlwraith, M.J., Martin, J.S., Ward, J.D., ONeil, N.J., Rose, A.M., West, S.C., Meyer, B.J., and Boulton, S.J. (2010). RTEL-1 enforces meiotic crossover interference and homeostasis. Science 327, 1254–1258. Dungrawala, H., Rose, K.L., Bhat, K.P., Mohni, K.N., Glick, G.G., Couch, F.B., and Cortez, D. (2015). The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability. Mol. Cell 59, 998–1010. Zeman, M.K., and Cimprich, K.A. (2014). Causes and consequences of repli- cation stress. Nat. Cell Biol. 16, 2–9. Fragkos, M., Ganier, O., Coulombe, P., and Me´ chali, M. (2015). DNA replica- tion origin activation in space and time. Nat. Rev. Mol. Cell Biol. 16, 360–374. Cell Reports 31, 107675, May 26, 2020 11 Article ll OPEN ACCESS ll OPEN ACCESS STAR+METHODS KEY RESOURCES TABLE RESOURCE AVAILABILITY Lead Contact Further information and requests for reagents should be directed to and will be fulﬁlled by the Lead Contact, Simon Boulton (simon. boulton@crick.ac.uk). Lead Contact Further information and requests for reagents should be directed to and will be fulﬁlled by the Lead Contact, Simon Boulton (simon. boulton@crick.ac.uk). Materials Availability STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat Anti-Rat IgG (H+L) Antibody, Alexa Fluor 594 Conjugated Thermo Fisher Cat#A-11007; RRID: AB_141374 Rabbit Anti-Mouse IgG (H+L) Antibody, Alexa Fluor488 Conjugated Thermo Fisher Cat#A-11059; RRID: AB_142495 Goat Anti-Rabbit IgG (H+L) Antibody, Alexa Fluor488 Conjugated Thermo Fisher Cat#A-11034 Rabbit polyclonal anti-53BP1 Novus Biologicals Cat#NB100-304; RRID: AB_10003037 Mouse monoclonal gH2AX clone JBW301 Millipore Cat#05-63; RRID: AB_309864 Rat monoclonal anti-BrdU AbD Serotec Cat#OBT0030 Mouse monoclonal anti-BrdU Becton Dickinson Cat#347580 Chemicals, Peptides, and Recombinant Proteins Adenovirus Ad-Cre-GFP Vector Biolabs Cat#1700 Adenovirus Ad-GFP Vector Biolabs Cat#1060 CldU Sigma-Aldrich Cat#C6891 IdU Sigma-Aldrich Cat#I7125 EdU Thermo Fisher Scientiﬁc Cat#A10044 Benzonase Novagen Cat#71206-3 DAPI SIGMA Cat#10236276001 Critical Commercial Assays FiberPrep (DNA Extraction Kit) Genomic Vision Cat#EXTR-001 Lipofectamine 2000 Thermo Fisher Cat# 11668027 QIAprep Spin Miniprep Kit QIAGEN Cat#27106 Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit Thermo Fisher Cat#C10425 Experimental Models: Mouse Strains Pole4tm1(KOMP)Vlcg Bellelli et al., 2018 N/A RTEL1 f/f Vannier et al., 2012 N/A Experimental Models: Cell Lines Mouse Embryonic Fibroblasts RTEL1 f/f Pole4-+/+ This study N/A Mouse Embryonic Fibroblasts RTEL1 f/f Pole4/ This study N/A Human HEK293 cells The Francis Crick Institute Cell Services N/A Experimental Models: C. elegans strains C. elegans: WT, Bristol (N2) background CGC N2 FX1866 rtel-1(tm1866) Barber et al., 2008 DW663 FX4613 Y53F4B.3 pole-4(tm4613) CGC N/A FX1937 mus-81(tm1937) Barber et al., 2008 N/A DW238 rtel-1(tm1866) mus-81(tm1937)/hT2[bli-4(e937) let- ?(q782) qIs48](I;III) This study N/A VC13 dog-1(gk10) Cheung et al., 2002 N/A VC193 him-6(ok412), Wicky et al., 2004 N/A RB1279 rfs-1(ok1372) Ward et al., 2007 N/A CB1487 him-9(e1487) Saito et al., 2009 N/A Software and Algorithms Adobe Photoshop CC Adobe https://www.adobe.com/es/ / e1 Cell Reports 31, 107675, May 26, 2020 Continued REAGENT or RESOURCE SOURCE IDENTIFIER ImageJ NIH https://imagej.nih.gov/ij/ Volocity 6.3 PerkinElmer http://www.perkinelmer.com/lab- products-and-services/resources/ cellular-imaging-software- downloads.html GraphPad Prism 7 GraphPad https://www.graphpad.com/ Article ll OPEN ACCESS Article ll OPEN ACCESS GraphPad Prism 7 C. elegans strains The strains used in this work are listed in Key Resource Table and include FX1866 rtel-1(tm1866), FX4613 Y53F4B.3 pole-4(tm4613), FX1937 mus-81(tm1937), DW238 rtel-1(tm1866) mus-81(tm1937)/hT2[bli-4(e937) let-?(q782) qIs48](I;III), VC13 dog-1(gk10), VC193 him-6(ok412), RB1279 rfs-1(ok1372) and CB1487 him-9(e1487). rtel-1, pole-4 and mus-81 deletion strains were kindly provided by Shohei Mitani and the National Bio-resource Project. Strains were also obtained from the Caenorhabditis Genetics Centre and out- crossed to N2 multiple times before use. Strain maintenance and new strain construction was carried out by standard methods. Mouse strains and cell lines Mouse strains and cell lines used in the study are listed in Key Resource Table. Mouse Embryonic Fibroblasts were produced at em- bryonic day 13.5 from timed breeding between 8-12 weeks old RTEL1ﬂ/ﬂPole4+/ males and females. All animal experimentations were undertaken in compliance with UK Home Ofﬁce legislation (project license number 70/8527) under the Animals (Scientiﬁc Pro- cedures) Act 1986. Rtel1F/F Pole4+/+ and Pole4/ primary mouse embryonic ﬁbroblasts (MEFs) were cultured at 37C/ 5% CO2/ 5% O2 in Dulbecco’s modiﬁed Eagle’s medium (DMEM) (Invitrogen) supplemented with 15% fetal bovine serum (Sigma) and 1% peni- cillin-streptomycin (Invitrogen). 293 cells were cultured in 37C/ 5% CO2/ 5% O2 in Dulbecco’s modiﬁed Eagle’s medium (DMEM) (Invitrogen) supplemented with 15% fetal bovine serum (Sigma) and 1% penicillin-streptomycin (Invitrogen). Human 293 cells were cultured in DMEM 10% FBS (SIGMA) at 37C/ 5% CO2. Data and Code Availability This study did not generate/analyze datasets/code. METHOD DETAILS RNA interference and plate phenotype scoring Materials Availability Mouse cell lines and C. elegans strains generated in this study are available upon request to the Lead Contact (simon.boulton@crick. ac.uk). DNA ﬁber stretching assay DNA ﬁber assay was performed as described in Bellelli et al. (2018). Brieﬂy, Rtel1F/F Pole4+/+ and Pole4/ MEFs infected with CRE recombinase or GFP expressing adenovirus, were pulse labeled with 20 mM CldU for 20 min and subsequently with 200 mM IdU for 20 min. Cells were trypsinized, washed in PBS and resuspended at a concentration of 5x 105 in PBS. 2.5 mL of cell suspension were spotted on clean glass slides and lysed with 7.5 mL of 0.5% SDS in 200 mM Tris-HCL, pH 7.4, 50 mM EDTA for 10 min at R.T. Slides were then tilted allowing a stream of DNA to run slowly down the slide, air-dried and then ﬁxed in methanol/acetic acid (3:1) for 15 min at R.T. After denaturation in HCl 2,5 M (30 min R.T.) slides were blocked in 1% BSA/PBS and incubated with rat anti-BrdU monoclonal antibody (1:1000 overnight; AbD Serotec) and mouse anti-BrdU monoclonal antibody (1:500 1h R.T.; Becton Dickinson). After washes in PBS, slides were incubated with Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 R.T.; Invitrogen) for 45 min and mounted in PBS/Glycerol 1:1. Fibers were then examined using Axio Imager.M2 (ZEISS) with 60x oil immersion objective and the Volocity 6.3 software. Immunoﬂuorescence staining For indirect immunoﬂuorescence staining, cells were seeded on coverslips and ﬁxed in 4% paraformaldehyde. After permeabilization with 0.5% Triton X-100 (5 min on ice), coverslips were blocked in 1% BSA/PBS and incubated with the following primary antibodies diluited in 0.5% BSA/PBS: anti-H2AX phosphorylated on Ser139 (gH2AX) (Millipore), 53BP1, (Novus Biologicals), for 1h at room temperature. Coverslips were then washed 3 times in PBS and incubated with Alexa Fluor 488 goat anti-rabbit or rabbit anti-mouse antibodies (Invitrogen) for 45 min at room temperature. After DAPI counterstaining, coverslips were mounted in Glycerol/PBS (1:1) and observed with Axio Imager.M2 (ZEISS) using the Volocity 6.3 software. For EdU immunoﬂuorescence analysis MEFs (passage 3) were labeled and processed using the Click-iT EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher). Cells were pulse labeled for 30 min with 10 mM EdU and ﬁxed in 4% paraformaldehyde, before being permeabilized in PBS-Triton 0.5% and washed in 1% BSA. Cells were then resuspended in Click-iT reaction cocktail containing Alexa Fluor 488 Azide and incubated for 30 min at R.T. After being washed, cells were ﬁnally counterstained for DNA content by DAPI (1 mg/ml) and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson). Cre-mediated recombination / / / Rtel1F/F Pole4+/+ and Pole4/ mouse primary cells were infected with adenovirus expressing the CRE recombinase together with a GFP marker to inactivate Rtel1 (Ad-CRE) or control adenovirus expressing only GFP (Ad-GFP). Samples were processed for analysis 72 hours after infection and loss of RTEL1 was veriﬁed by PCR and/or western blot (Sarek et al., 2015). Mouse Embryonic Fibroblasts (MEFs) isolation and culture ﬂ/ﬂ / RTEL1ﬂ/ﬂPole4+/ mice in C57BL/6 background were mated. Pregnant females at 13.5 days gestation were subjected to euthanasia under anesthesia, followed by uterine dissection to isolate individual embryos. After washing in PBS and removal of head (used for embryo genotyping) and internal organs (heart and liver), embryo bodies were minced with sterile razor blades and incubated in trypsin at 37C for 10 min, followed by gentle pipetting of the trypsin digest. Cell suspension was pelleted, resuspended and plated in 10 cm dishes (passage 0) in DMEM (Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 15% FBS (SIGMA) and 50mg/ml penicillin-streptomycin, 2mM L-glutamine. Once subconﬂuent, a standard 3T3 protocol was followed: every 3 days cells were trypsinized, counted using cellometer Auto 2000 (Nexcelom Bioscience) to determine the number of Population doublings (PD) and then re-plated at a ﬁxed density (8x105 cells per 100-mm dish) The accumulation of population doubling level (PDL) was calculated using the formula DPDL = log(nh/ni)/log2, where ni is the initial number of cells and nh is the cell number at each passage. Assay for poly-G/C-tract deletions The assay for poly-G/C-tract deletions was carried out as described in Youds et al. (2006). RNA interference and plate phenotype scoring Secondary RNAi screen and scoring data was collected by feeding worms with RNAi bacteria on 55mm MYOB plates. Clones of in- terest were located in a previously described RNAi library and streaked onto LB agar + Ampicillin 50ug/mL plates and grown over- night at 37 degrees. Single colonies were grown overnight at 37 degrees in 5mL of LB + Ampicillin 50ug/mL. Cultures were centri- fuged and resuspended in one-third of the original volume. For each RNAi plate, 50uL of RNAi bacteria was spotted onto MYOB plates containing 50ug/mL Ampicillin and 1mM IPTG. Plates were incubated at room temperature overnight before use. L1-stage worms were plated onto the RNAi plates and were transferred to fresh RNAi plates each day after the start of egg laying for 4 days. For scoring of double mutant strains and controls (under no RNAi conditions), L4-stage animals were individually plated and transferred onto fresh plates each day for 4 days. Unhatched eggs were scored 24 hours after removing the parent animal from the plate, and the total number of viable progeny and males (if scored) was counted after an additional 24-48 hours. Cell Reports 31, 107675, May 26, 2020 e2 Article ll OPEN ACCESS Immunoﬂuorescence analysis in C.elegans Germlines of young adult animals were extracted and ﬁxed in 4% PFA, and permeated by incubation with TBS containing 0.5% BSA and 0.1% Triton X-100. Rabbit anti-RPA and rabbit anti-RAD-51 primary antibodies were both used at 1:500 dilutions in TBS + 0.5% BSA and incubated overnight at 4 degrees. Alexa 488 goat anti-rabbit secondary antibody (Invitrogen) was used at 1:10000 dilution in TBS + 0.5% BSA. Germlines were also stained with DAPI (Sigma) and slides were mounted using Vectashield (Vector Labs). Slides were viewed on a Deltavision microscope (Appiled Precision) with 100X lens. Images were deconvolved using SoftWoRX software. RAD-51 foci in individual nuclei were counted using the Z stacks of images. QUANTIFICATION AND STATISTICAL ANALYSES Statistics, including statistical tests used, number of events quantiﬁed, standard deviation standard error of the mean, and statistical signiﬁcance are reported in ﬁgures and ﬁgure legends. Statistical analysis has been performed using GraphPad Prism7 software (GraphPad) and statistical signiﬁcance is determined by the value of p < 0.05. e3 Cell Reports 31, 107675, May 26, 2020
https://openalex.org/W3034832314	https://europepmc.org/articles/pmc7291629?pdf=render	English	null	Short-course Rifaximin therapy efficacy and lactulose hydrogen breath test in Chinese patients with diarrhea-predominant irritable bowel syndrome	BMC gastroenterology	2,020	cc-by	7,288	Zhuang et al. BMC Gastroenterology (2020) 20:187 https://doi.org/10.1186/s12876-020-01336-6 Zhuang et al. BMC Gastroenterology (2020) 20:187 https://doi.org/10.1186/s12876-020-01336-6 Open Access © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Short-course Rifaximin therapy efficacy and lactulose hydrogen breath test in Chinese patients with diarrhea-predominant irritable bowel syndrome Xiaojun Zhuang†, Zhenyi Tian†, Mei Luo and Lishou Xiong* Xiaojun Zhuang†, Zhenyi Tian†, Mei Luo and Lishou Xiong* Abstract Background: Gut microbiota alterations including small intestinal bacterial overgrowth (SIBO) might play a role in pathogenesis of irritable bowel syndrome (IBS). Rifaximin could effectively and safely improve IBS symptoms. The aim of this study was to investigate the effect of rifaximin on Gastrointestinal (GI) symptoms, quality of life (QOL) and SIBO eradication in Chinese IBS-D patients. Methods: This study included 78 IBS-D patients defined by the Rome IV criteria. Patients received 400 mg rifaximin twice daily for 2 weeks and 10-week follow-up. GI symptoms were assessed at week 0, 2, 4, 8 and 12. QOL and lactulose hydrogen breath test (LHBT) results were estimated at week 0 and 4. Results: All participants showed significant improvements in GI symptom subdomains after rifaximin treatment (all P < 0.05), which could maintain at least 10 weeks of follow-up. Additionally, QOL scores were increased with concomitant improvement of clinical symptoms (all P < 0.05). The 45 rifaximin-responsive patients (57.7%) achieved significantly greater GI-symptom improvement than non-responders (all P < 0.05). No GI symptoms were associated with SIBO (all P > 0.05). SIBO normalization after rifaximin treatment measured by LHBT was found in 44.4% (20/45) of patients with SIBO before treatment. Conclusion: A short course (2 weeks) of rifaximin improved GI symptoms and QOL in Chinese IBS-D patients whether they had SIBO or not. However, the efficacy of rifaximin could not be explained by the successful eradication of SIBO. Further studies on the therapeutic mechanisms of rifaximin in IBS are urgently needed. rds: Irritable bowel syndrome, Rifaximin therapy, Small intestinal bacterial overgrowth, Quality of life * Correspondence: xionglishou@163.com †Xiaojun Zhuang and Zhenyi Tian contributed equally to this work. Department of Gastroenterology and Hepatology, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China Background approximately 7–21%; it is 1–16% in China, but the prevalence differs depending on regions and diagnostic criteria [2, 3]. Dissatisfaction and comorbidities of trad- itional treatment are associated with a significant reduc- tion in the quality of life (QOL) and growing social, sanitary and economic burden worldwide [4–6]. Patients are stratified into four subtypes based on the predomin- ant bowel habit: constipation-predominant IBS (IBS-C), diarrhea-predominant IBS (IBS-D), mixed IBS (IBS-M) and unclassified IBS (IBS-U) [1]. Although the precise Irritable bowel syndrome (IBS) is one of the most com- mon functional bowel disorders, with a relapsing and re- mitting natural history characterized by abdominal pain that is associated with defecation or alterations in bowel habits [1]. The prevalence of IBS around the world is * Correspondence: xionglishou@163.com †Xiaojun Zhuang and Zhenyi Tian contributed equally to this work. Department of Gastroenterology and Hepatology, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China * Correspondence: xionglishou@163.com †Xiaojun Zhuang and Zhenyi Tian contributed equally to this work. Department of Gastroenterology and Hepatology, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China Zhuang et al. BMC Gastroenterology (2020) 20:187 Page 2 of 10 Page 2 of 10 discomfort, abdominal distension, abdominal pain, defecatory urgency, diarrhea and incomplete evacuation) and QOL (physical functioning, role-physical, bodily pain, general health, vitality, social functioning, role- emotional and mental health) in Chinese IBS patients. We hypothesized that rifaximin treatment could relieve GI symptoms and optimize QOL by normalizing SIBO a measured by the LHBT. etiology of IBS remains unknown, the possible mecha- nisms include visceral hypersensitivity, gut motility dysfunction, immunomodulation disturbances, gut microbiota alterations and an imbalance in brain-gut axis interactions [7–10]. In addition, new-onset IBS symptoms following acute infectious gastroenteritis might also suggest a microbial pathogenesis for IBS [11]. g gg p g Alterations in the quantity or composition of the gut microbiota with subsequent metabolic disturbances have been observed in patients with IBS. In a recent system- atic review, increased abundances of Enterobacteriaceae and Lactobacillaceae at the family level and Bacteroides at the genus level were found in patients with IBS com- pared with controls, whereas the abundance of the order uncultured Clostridiales I, and the genera Faecalibacter- ium and Bifidobacterium were decreased in IBS patients [10]. Moreover, we previously reported alterations in the abundance of predominant fermenting bacteria involved in the pathophysiology of IBS-D (such as Bacteroidales and Clostridiales) [12]. Furthermore, an association be- tween IBS and small intestinal bacterial overgrowth (SIBO) has been observed in some patients with IBS, al- though the causal relationship between SIBO and IBS remains to be elucidated [13–18]. SIBO might partly ex- plain IBS symptoms, such as bloating, abdominal pain and changes in bowel habits. A definite diagnosis of SIBO is characterized by greater than 105 microorgan- isms/ml with poly-microbial flora in cultures of duo- denal or jejunal fluid [19]. However, SIBO is diagnosed by various breath tests clinically, and the lactulose hydrogen breath testing (LHBT) is most commonly used, as intestinal samples are difficult to obtain [20– 22]. Gut microbiota alterations indicate that the manipu- lation of the composition of the intestinal microbiota with probiotics, prebiotics, antibiotics, dietary interven- tions and fecal microbiota transplantation may be useful treatment approaches [23]. Study subjects h Seventy-eight patients with IBS-D were recruited into this study by two gastroenterologists with expertise in IBS. The inclusion criteria were men or women aged 18 years and above who met the Rome IV criteria for IBS-D, symp- toms for more than 6 months, and patients with IBS symptoms as mentioned and normal appearance of the gastrointestinal mucosa. The exclusion criteria were clin- ical evidence of inflammatory bowel disease, a history of duodenal or gastric ulcers, diverticulitis or infectious gastroenteritis, abdominal surgery, cardiac, pulmonary, hepatic, renal or metabolic disease, use of antibiotics, pro- biotics, prebiotics, corticosteroids, proton-pump inhibi- tors, or IBS prescription medications within the last 4 weeks. A colonoscopy was performed on all patients to rule out organic disease. Rifaximin, as a gastrointestinal (GI)-specific broad- spectrum antibiotic, shows activity against both gram- positive and gram-negative, anaerobic and aerobic bacteria [24]. Since it displays low systemic absorption and no clin- ically significant interactions with other drugs, rifaximin may be a promising treatment for IBS, mainly due to its ability to act on IBS pathogenesis by modulating gut microbiota, altering bacterial metabolism, preserving epi- thelial function and reducing proinflammatory cytokine production [25–27]. Additionally, prior studies on rifaxi- min in nonconstipated IBS patients with SIBO indicated that rifaximin treatment is effective in improving IBS symptoms and eradicating SIBO [28–32]. However, there are few studies on the association of GI symptoms and QOL with LHBT results in the Chinese population. Ethics statement This study was conducted at the Department of Gastro- enterology and Hepatology, the First Affiliated Hospital of Sun Yat-sen University, from December 2016 to De- cember 2018. The protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, and all patients provided written in- formed consent. The ClinicalTrials.gov ID for the study is NCT02565654. Evaluation of SIBO by LHBT The LHBT was performed according to a standard protocol. Patients did not receive any antibiotics, probio- tics, prebiotics, or laxatives in the 4 weeks preceding the test. To minimize basal H2 excretion, IBS-D patients were asked to avoid foods containing complex carbohy- drates (bread, potato, and corn) and fiber in the previous evening and fasted for at least 12 h before the breath test. Cigarette smoking and physical exercise were not allowed for 2 h before and during the test. On the day of testing, patients washed their mouths with 20 ml of 0.05% chlorhexidine (Koutai, Shenzhen, China) to elim- inate the fermentation by oropharyngeal bacteria flora. LHBT was performed in IBS-D patients using a gas analyzer (GastroLyzer R Breath Hydrogen Monitor; Bed- font Science Ltd., UK). Immediately before the test, a sample of expired air was taken to assess the basal H2 concentration. Then, 10 g of lactulose dissolved in 100 ml of water was administered within 30 s, and the ex- pired air was sampled every 30 min over the next 3 con- secutive hours by a trained study coordinator. Statistical analysis incomplete evacuation; the severity of GI symptoms was rated using a 7-point Likert scale (0 = not at all, 1 = hardly, 2 = somewhat, 3 = moderately, 4 = a good deal, 5 = a great deal, and 6 = a very great deal). In addition, a QOL ques- tionnaire was completed by IBS-D patients at baseline and at the end of the 2-week posttreatment period. The SF-36 is a 36-item questionnaire that measures 8 domains rele- vant to patients with IBS: (1) Physical Functioning, (2) Role-physical, (3) Bodily pain, (4) General Health, (5) Vi- tality, (6) Social Functioning, (7) Role-Emotional, and (8) Mental Health. Finally, all patients received an LHBT at baseline and the end of 2 weeks after rifaximin treatment. All statistical analyses were performed using SPSS ver- sion 23.0 (SPSS, Inc., Chicago, IL, United States) and Graph Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, United States). Continuous data were analyzed using Student’s t-test or Mann-Whitney U-test where appropriate. Categorical data were analyzed using a chi- square test. Pearson correlation coefficient analysis was used to assess the relationship between GI symptoms and SIBO. All tests for significance were two-sided and P < 0.05 was considered statistically significant. Evaluation of SIBO by LHBT Table 1 Demographic and clinical characteristics of all included patients at baseline Table 1 Demographic and clinical characteristics of all included patients at baseline Clinical factors LHBT (+) LHBT (−) P value Age (mean, years) 32.13 ± 7.48 37.24 ± 9.95 0.016 Gender (M/F) 29/16 23/10 0.627 GI symptoms (mean) 17.04 ± 5.02 17.42 ± 4.40 0.724 Abdominal discomfort 2.31 ± 1.38 2.55 ± 1.23 0.432 Abdominal distension 1.84 ± 1.35 2.00 ± 1.17 0.589 Abdominal pain 3.07 ± 1.37 2.45 ± 1.37 0.056 Defecatory urgency 3.78 ± 1.31 3.79 ± 1.29 0.973 Diarrhea 4.11 ± 3.56 3.64 ± 1.06 0.460 Incomplete evacuation 2.38 ± 1.35 3.00 ± 1.39 0.053 Quality of life (mean) 506.97 ± 126.70 477.82 ± 105.95 0.273 Physical Functioning 94.78 ± 11.03 93.03 ± 6.49 0.384 Role-physical 61.11 ± 40.68 52.27 ± 40.68 0.347 Bodily pain 57.72 ± 20.48 57.27 ± 21.06 0.449 General Health 43.33 ± 19.86 43.55 ± 16.13 0.958 Vitality 57.11 ± 16.57 55.91 ± 19.86 0.778 Social Functioning 75.67 ± 20.94 68.32 ± 21.79 0.138 Role-Emotional 55.55 ± 42.05 44.44 ± 37.89 0.226 Mental Health 61.69 ± 16.79 63.03 ± 13.42 0.696 According to the literature and our previous results [12, 33, 34], LHBT was considered indicative of the pres- ence of SIBO when (i) a baseline value of H2 ≥20 ppm and/or (ii) a > 20 ppm increase in H2 over basal values occurred within 90 min of lactulose administration. Results Demographic and clinical characteristics of patients Seventy-eight patients (33.5 years [18–58], 52 [66.7%] male) with IBS-D were enrolled in this study, and all participants completed a 12-week follow-up. Though IBS-D patients were more often male, the difference in age between the LHBT-positive and LHBT-negative groups was nonsignificant. Table 1 summarizes the demographic and clinical characteristics of all IBS-D pa- tients. At baseline, 45 patients (29/16) with SIBO were younger than those (23/10) with a negative LHBT result (32.13 ± 7.48 vs 37.24 ± 9.95, P = 0.016). In addition, there was no significant difference in the GI symptoms and QOL scores between the LHBT-negative and LHBT-positive groups. Moreover, no GI symptoms were found to be associated with the presence of SIBO (Table 2). Study design and procedures All participants received 400 mg rifaximin (Xifaxan®, ALFASIGMA S.p.A., Bologna, Italy) twice daily for 2 weeks. Then, they were further followed-up for an add- itional 10 weeks after treatment cessation. For recruited patients, they were informed to not to take any other pre- biotics, probiotics and antibiotic but rifaximin throughout the observation period. All investigators were asked to complete GI symptom questionnaire and an IBS-relevant QOL questionnaire based on the Medical Outcomes Study (MOS) item short-form health survey (SF-36). The symptoms were recorded in a diary at baseline, the end of the treatment (week 2), end of the 2-week follow-up (week 4), end of the 6-week follow-up (week 8), and the end of the 10-week follow-up (week 12). The assessed symptoms were abdominal discomfort, abdominal distension, abdominal pain, diarrhea, defecatory urgency and The overall aim of this study was to explore whether rifaximin treatment improves GI symptoms (abdominal Page 3 of 10 Page 3 of 10 Page 3 of 10 Zhuang et al. BMC Gastroenterology (2020) 20:187 Zhuang et al. BMC Gastroenterology (2020) 20:187 Outcome evaluation The primary endpoint was to assess the improvement in GI symptoms and QOL after 2 weeks of rifaximin treat- ment in the Chinese population. The secondary end- point was to compare the LHBT results before and after treatment with rifaximin. We also explored the response rate to rifaximin treatment by analyzing the self- reported GI symptoms, and the response to treatment was defined as a more than 50% improvement in the global GI symptoms 2 weeks after the cessation of treat- ment. Finally, we sought to search for symptoms closely associated with SIBO. Zhuang et al. BMC Gastroenterology (2020) 20:187 Page 4 of 10 Table 3 SIBO rate in Chinese IBS-D patients pre- and post- rifaximin treatment LHBT 95% CI (%) P value Positive/N (%) Negative/N (%) Pre-treatment 45 (57.7) 33 (42.3) 46.5–68.9 <0.001 Post-treatment 25 (32.1) 53 (67.9) 21.5–42.6 Note: SIBO, Small intestinal bacterial overgrowth; IBS, Irritable bowel syndrome; IBS-D, diarrhea-predominant IBS Effect of rifaximin on SIBO Table 3 SIBO rate in Chinese IBS-D patients pre- and post- rifaximin treatment For the subjects with SIBO before treatment, 25 (44.4%) had a negative LHBT after 2 weeks of rifaximin treatment (week 4). Furthermore, patients who received rifaximin treatment more often tended to have a negative LHBT (45/33 [42.3%] vs 25/53 [67.9%], P = 0.001) and had re- duced hydrogen production. As shown in Table 3, there was significant difference in SIBO rate of Chinese IBS-D patients before and after rifaximin treatment (57.7, 95% CI, 46.5–68.9% vs 32.1, 95% CI: 21.5–42.6%; P <0.001). In addition, there was no significant difference in age and gender between patients with and without LHBT normalization after 2 weeks of rifaximin therapy. improvement in each of the six GI symptoms after treat- ment. Finally, IBS-D patients with SIBO or without SIBO at week 4 recorded similar GI symptoms scores, regardless of whether they succeeded in eradicating SIBO (Table 5). Adverse events No patient developed any adverse events during rifaxi- min administration, except for two patients who re- ported transient nausea during rifaximin treatment. Overall, the treatment was well tolerated. Table 2 Correlation analysis between SIBO and GI symptoms SIBO ρ P value Abdominal discomfort 0.081 0.483 Abdominal distension 0.083 0.468 Abdominal pain 0.231 0.052 Defecatory urgency 0.013 0.909 Diarrhea 0.064 0.578 Incomplete evacuation 0.199 0.081 Note: ρ, Spearman rank correlation coefficient; SIBO, small intestinal bacterial overgrowth; GI symptoms, gastrointestinal symptoms Table 2 Correlation analysis between SIBO and GI symptoms Effect of rifaximin on the QOL A symptomatic evaluation after 2 weeks of rifaximin treatment might show improvements before the LHBT normalizes (Fig. 1). The IBS symptoms of abdominal pain, abdominal discomfort, abdominal distension, diar- rhea, defecatory urgency and incomplete evacuation im- proved significantly after rifaximin treatment, and the symptom relief persisted for at least 10 weeks during the follow-up period (all P < 0.05). In addition, 45 (57.7%) patients experienced a clinical response accompanied by a global IBS symptoms score reduction of at least 50% (5.36 ± 3.27 vs 13.79 ± 5.21, P < 0.001). The response group reported a full recovery or greater improvement in their symptoms than the nonresponse group, showing a significant difference in every GI symptom (Table 4). However, the difference in age and gender between the response and nonresponse groups was not statistically significant. For the IBS-D patients with SIBO, the GI symptoms showed significant improvement in each of the six symptom scores and in the global score after SIBO eradication through rifaximin treatment. Never- theless, subjects without SIBO eradication exhibited a similar resolution in five GI symptoms, but not abdom- inal discomfort, suggesting that the effect of rifaximin in IBS-D is not explained by SIBO eradication. In contrast, LHBT-negative patients at baseline showed significant At baseline, all participants reported severely reduced QOL scores. Fortunately, total QOL scores significantly increased 2 weeks after the completion of treatment (week 4), indicating QOL improvement (Fig. 2). Compared to the nonresponse group, the response group reported sig- nificant alterations in five domains of QOL, with no sig- nificant difference in vitality, role-emotional and mental health (Table 4). Additionally, there was no significant dif- ference in any of the eight domain scores or global QOL score between LHBT-positive and LHBT-negative groups. For the LHBT-positive IBS-D patients at baseline, bodily pain and general health improved significantly regardless of whether SIBO was successfully eliminated after rifaxi- min treatment. In contrast, a significant increase in seven QOL domain scores was observed in LHBT-negative pa- tients at baseline with no significant difference in physical functioning after 2 weeks of rifaximin treatment. Finally, IBS-D patients with SIBO or without SIBO at week 4 re- corded similar QOL scores, regardless of whether they succeeded in eradicating SIBO (Table 5). Discussion The findings of this study suggest that a short course (2 weeks) of rifaximin therapy is safe and efficacious for the treatment of IBS-D patients as assessed using the ROME IV criteria. The GI symptom relief, QOL improvement and SIBO normalization after rifaximin treatment ob- served in our study imply that rifaximin is an effective option for the treatment of IBS-D. Furthermore, the ef- fectiveness of the short-course rifaximin treatment was sustained for at least 12 weeks after treatment. To our knowledge, this is the first study evaluating the effect of Zhuang et al. BMC Gastroenterology (2020) 20:187 Page 5 of 10 Zhuang et al. BMC Gastroenterology Fig. 1 GI symptoms scores in IBS-D patients at different times during the study. Note: P < 0.05, * P < 0.01, *** P < 0.001; IBS, irritable bowel syndrome; IBS-D, diarrhea-predominant IBS; GI symptoms, gastrointestinal symptoms Fig. 1 GI symptoms scores in IBS-D patients at different times during the study. Note: syndrome; IBS-D, diarrhea-predominant IBS; GI symptoms, gastrointestinal symptoms Fig. 1 GI symptoms scores in IBS-D patients at different times during the study. Note: P < 0.05, * P < 0.01, *** P < 0.001; IBS, irritable bowel syndrome; IBS-D, diarrhea-predominant IBS; GI symptoms, gastrointestinal symptoms rifaximin on GI symptoms and QOL based on SIBO in Chinese patients with IBS-D. study, 57.7% of the included patients had a positive LHBT, and 20 showed LHBT normalization after 2 weeks of rifax- imin treatment, with a SIBO eradication rate of 44.4%. In addition, the LHBT-positive subjects were younger than the LHBT-negative subjects. In contrast to our study, a re- cent meta-analysis involving 32 studies reported that the overall eradication rate according to an intention-to-treat analysis was 70.8% (95% CI: 61.4–78.2; I2 = 89.4%) and ac- cording to a per-protocol analysis was 72.9% (95% CI: 65.5–79.8; I2 = 87.5%) [32]. However, another meta- analysis of eight studies showed that the overall breath- test normalization rate with rifaximin was 49.5%, which is somewhat similar to the result of our study [38]. The marked discrepancy in rates of SIBO eradication might be related to geographical, dietary or ethnicity differences in the microbiomes of the study populations or the dose of As previously stated, the etiological and symptomatic manifestation of IBS and SIBO may overlap, and SIBO has been postulated to be a pathophysiological mechanism for IBS. Discussion Moreover, SIBO is in fact associated with IBS-like symptoms, such as bloating, abdominal pain, and a change in bowel habits. The frequency of SIBO among IBS pa- tients ranges between 4 and 78%, and the variations in prevalence of SIBO in previous studies might be attribut- able to differences in the geographical origins of the stud- ied populations, different criteria for the diagnosis of IBS and methods for the diagnosis of SIBO using different breath tests [33–35]. The response to rifaximin treatment in IBS-D patients has been shown to correlate with the normalization of the LHBT results [32, 36, 37]. In our Zhuang et al. BMC Gastroenterology (2020) 20:187 Page 6 of 10 Page 6 of 10 Table 4 Comparison between the Response and Non-Response Groups after rifaximin treatment at week 4 Clinical factors Response group (n = 45) Non-response group (n = 33) P value Age (mean, years) 34.82 ± 9.04 33.58 ± 8.84 0.544 Gender (M/F) 31/14 21/12 0.627 LHBT (+/−) 18/27 8/25 0.145 GI symptoms (mean) 5.36 ± 3.27 13.79 ± 5.21 < 0.001 Abdominal discomfort 0.87 ± 0.89 2.45 ± 1.23 < 0.001 Abdominal distension 0.49 ± 0.70 1.67 ± 1.29 < 0.001 Abdominal pain 0.91 ± 0.93 2.27 ± 1.53 < 0.001 Defecatory urgency 1.09 ± 1.13 2.73 ± 1.26 < 0.001 Diarrhea 1.02 ± 0.97 2.42 ± 1.23 < 0.001 Incomplete evacuation 0.98 ± 1.03 2.33 ± 1.22 < 0.001 Quality of life (mean) 616.34 ± 84.06 545.34 ± 94.70 0.001 Physical Functioning 97.33 ± 4.47 94.85 ± 5.93 0.048 Role-physical 82.78 ± 24.90 67.42 ± 36.70 0.031 Bodily pain 77.34 ± 13.70 62.36 ± 19.88 < 0.001 General Health 59.22 ± 16.52 45.64 ± 16.70 0.001 Vitality 67.33 ± 15.06 61.67 ± 14.23 0.094 Social Functioning 85.63 ± 10.52 76.67 ± 19.98 0.012 Role-Emotional 76.30 ± 32.28 72.73 ± 30.57 0.621 Mental Health 70.40 ± 16.30 64.00 ± 18.08 0.112 Table 4 Comparison between the Response and Non-Response Groups after rifaximin treatment at week 4 rifaximin. The findings of our study suggest that either SIBO plays a limited role in causing IBS-associated gut microbiota disturbances or that LHBT is not a good test to measure SIBO. There was no significant difference in GI symptoms and QOL scores between the LHBT-positive and LHBT- negative groups. Discussion After 2 weeks of rifaximin treatment, all individual and global symptoms displayed instant im- provement, and these effects lasted for at least 10 weeks during the follow-up period. However, not all patients showed a desirable response to rifaximin therapy accord- ing to the formal prespecified criteria for a response. In the LHBT-positive group, the response rate was rela- tively high (40.0%) compared with that in the LHBT- negative group (24.2%), but the difference was not statis- tically significant. More importantly, participants with LHBT normalization after treatment appeared to experi- ence symptomatic improvement in all of the six symp- toms, whereas those without SIBO eradication showed similar symptom relief, with the exception of abdominal discomfort. However, more severe diarrhea was recorded in subjects with LHBT normalization than those without LHBT normalization, which means that this nonabsorb- able antimicrobial agent did not completely reverse the gut microflora dysbiosis when eradicating SIBO. In addition, subjects with a negative LHBT at baseline also achieved individual and global GI symptom improve- ments that persisted after rifaximin intervention. In the well-known TARGET 1 and TARGET II studies, only 40% of patients responded to rifaximin, but treatment with rifaximin for 2 weeks provided significant relief of IBS symptoms including bloating, abdominal pain, and loose or watery stools [28]. The inconsistent response to rifaximin in various studies may be due to IBS hetero- geneity, and LHBT normalization might not be a good marker to assess the response to rifaximin. In contrast to our study, an open-label study from Europe reported an improvement in individual symptoms (abdominal pain, diarrhea, and bloating) as well global symptoms with 800 mg/day rifaximin for 2 weeks [39]. Recently, a study of retreatment with rifaximin showed a 33% re- sponse rate in the rifaximin group compared to 25% in the placebo group (P = 0.02), consistent with FDA guide- lines for the clinical assessment of IBS drugs in the TARGET 3 study [40]. Discussion Table 5 GI symptoms and QOL comparisons between IBS-D patients with and without SIBO eradication after rifaximin treatment Clinical factors LHBT (+) LHBT (−) P value GI symptoms (mean) 7.88 ± 6.15 8.95 ± 5.23 0.531 Abdominal discomfort 1.64 ± 1.29 1.20 ± 1.20 0.243 Abdominal distension 0.92 ± 1.04 0.90 ± 1.29 0.956 Abdominal pain 1.64 ± 1.35 1.45 ± 1.28 0.631 Defecatory urgency 1.32 ± 1.44 2.05 ± 1.28 0.078 Diarrhea 1.20 ± 1.29 1.95 ± 1.10 0.051 Incomplete evacuation 1.16 ± 1.11 1.55 ± 1.15 0.256 Quality of life (mean) 579.93 ± 106.11 599.05 ± 88.29 0.513 Physical Functioning 96.20 ± 4.63 98.25 ± 3.73 0.107 Role-physical 70.00 ± 35.36 81.25 ± 25.49 0.222 Bodily pain 71.92 ± 14.29 73.68 ± 15.41 0.697 General Health 53.60 ± 19.72 53.85 ± 16.69 0.963 Vitality 63.80 ± 14.74 64.00 ± 15.01 0.965 Social Functioning 84.57 ± 12.92 82.95 ± 13.41 0.685 Role-Emotional 72.00 ± 32.89 81.67 ± 33.29 0.336 Mental Health 67.84 ± 17.42 63.40 ± 18.55 0.417 Note: IBS, irritable bowel syndrome; IBS-D, diarrhea-predominant IBS; GI symptoms, gastrointestinal symptoms; QOL, quality of life; SIBO, small intestinal bacterial overgrowth Table 5 GI symptoms and QOL comparisons between IBS-D patients with and without SIBO eradication after rifaximin At baseline, all participants reported reduced QOL scores. Interestingly, the IBS-QOL overall and all subdo- main scores improved from baseline for up to 2 weeks posttreatment and were accompanied by symptom relief in the included patients. Indeed, responders had a signifi- cantly greater improvement in the overall QOL score than nonresponders, which implies that a sufficient improve- ment in patient clinical symptoms guarantees that their QOL improves. Furthermore, rifaximin treatment Zhuang et al. BMC Gastroenterology (2020) 20:187 Page 7 of 10 Zhuang et al. BMC Gastroenterology Fig. 2 QOL scores comparison in IBS-D patients pre- and post-rifaximin treatment. Note: P < 0.05, * P < 0.01, *** P < 0.001; IBS, irritable bowel syndrome; IBS-D, diarrhea-predominant IBS; QOL, Quality of life comparison in IBS-D patients pre- and post-rifaximin treatment. Note: P < 0.05, * P < 0.01, *** P < 0.001; IBS, irritable bowe diarrhea-predominant IBS; QOL, Quality of life increased improvement in QOL following repeat treat- ment with rifaximin is associated with a reduced chance of subsequent symptom relapse [42]. However, rifaximin was not effective in improving IBS symptoms and QOL in Gulf War veterans with non-constipated IBS [43]. Discussion Finally, LHBT-positive and LHBT-negative IBS-D patients did not differ significantly in their reported post rifaximin total QOL or subscale scores. significantly impacted bodily pain and general health in patients with a positive LHBT, regardless of whether SIBO was successfully eradicated. Interestingly, our findings in- dicate that treatment with rifaximin favorably improves the total QOL and seven subdomain scores in LHBT- negative patients with IBS-D, which is consistent with pre- viously reported data [41]. Similar effects have been seen in another study, the findings of which suggested that the Page 8 of 10 Page 8 of 10 Zhuang et al. BMC Gastroenterology (2020) 20:187 Zhuang et al. BMC Gastroenterology (2020) 20:187 is the first study to show that short-course rifaximin therapy is an appropriate treatment option for Chinese IBS-D patients. Rifaximin was approved by the US Food and Drug Ad- ministration in 2015 to treat adults with IBS-D [44]. Al- though the mechanism of action of rifaximin in IBS is complex, a leading hypothesis proposes that rifaximin modulates intestinal flora imbalances. Mounting evi- dence has shown that rifaximin treatment induces alter- ations in the abundance of specific bacterial populations rather than affecting the overall composition of the microbiota in the treated subjects and has no apparent detrimental effects on gut microbiota [45–47]. On the one hand, rifaximin shows a potent killing effect on common SIBO pathogens [48, 49]. On the other hand, rifaximin appears to increase the abundance of certain potentially beneficial bacteria, such as Faecalibacterium prausnitzii, but reduces the abundance of detrimental bacteria such as Clostridium. In addition to the direct antibiotic effects of rifaximin on gut microbiota, rifaxi- min impacts the function of the gut microbiota (i.e., me- tabolism, adherence and virulence) [50–52]. Alterations in certain lipid species, saturated and unsaturated fatty acids, and products of carbohydrate metabolism were found in several studies focused on rifaximin treatment for IBS; these alterations might have beneficial effects on various symptoms (improved barrier function of the small bowel and reduced visceral hyperalgesia) of GI- related disease. For example, a study from Bajaj et al. found that alteration of gut bacterial linkages with me- tabolites rather than significant change in microbial abundance after rifaximin therapy, which especially linked to ammonia, aromatic amino acids and oxidative stress [50]. Acknowledgments None. Acknowledgments None. Discussion Furthermore, rifaximin could inhibit bacterial interactions with the host to reduce detrimental bacterial colonization, infection and the activation of the host im- mune response to prevent mucosal inflammation by re- ducing the level of proinflammatory mediators [53]. In addition, rifaximin is able to reduce bacterial virulence and translocation, has anti-inflammatory properties by increasing the relative abundance of Faecalibacterium prausnitzi endowed with powerful anti-inflammatory ac- tivities [54]. Taken together, these results show that the beneficial effects and safety of rifaximin treatment might be partly accounted for by resetting the gut microenvir- onment and modulating the inflammatory environment. There are several limitations in this study It was con- Competing interests Competing interests Authors declare that they have no competing interests. There are several limitations in this study. It was con- ducted in a single center with a relatively small sample size and open-label design so that conclusions should be drawn cautiously. Further limitations of our study are no control group and the lack of randomization. Addition- ally, the validity and interpretation of the LHBT for the diagnosis of SIBO is an ongoing controversy. The great- est weakness of the study is that the potential mecha- nisms by which rifaximin beneficially affects IBS-D patients with definite SIBO were not elaborated compre- hensively. Nevertheless, it might be noteworthy that this p g Authors declare that they have no competing interests. Received: 13 March 2020 Accepted: 5 June 2020 Received: 13 March 2020 Accepted: 5 June 2020 Received: 13 March 2020 Accepted: 5 June 2020 Authors’ contributions LX was responsible for the study concept and design, critical revision of the manuscript for important intellectual content. XZ was responsible for the study concept and design, acquisition of data, analysis and interpretation of data and drafting of the manuscript. ZT and ML assisted in collecting data and conducting the statistical analysis. All authors have read and approved this version of the article. Neither the entire paper nor any part of its content has been published or has been accepted elsewhere. Funding This study was funded by the National Natural Science Foundation of China (81970471) and Alfasigma Pharmaceutical Company. The funders had a role in study design, decision to publish and preparation of the manuscript. No additional external funding was received for this study. Conclusion In conclusion, a short course of rifaximin treatment sig- nificantly improved the GI symptoms and QOL of Chin- ese IBS-D patients in this study, and 2-week rifaximin treatment led to the sustained improvement of IBS symptoms for at least 10 weeks, which is consistent with multiple previous large clinical trials of single and repeat treatment cycles. However, the efficacy of rifaximin could not be explained by the successful eradication of SIBO. More therapeutic mechanisms of rifaximin for IBS-D patients are warranted in further studies. Ethics approval and consent to participate This study was approved by the Medical Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University. All patients had signed informed consent. Availability of data and materials All data and materials are not available in this study, and are available from the corresponding author on reasonable requests. Abbreviations IBS: Irritable bowel syndrome; IBS-D: patients with diarrhea-predominant IBS; SIBO: Small intestinal bacterial overgrowth; LHBT: Lactulose hydrogen breath test; QOL: Quality of life References 1. Drossman DA, Hasler WL. Rome IV-functional GIdisorders: disorders of gut- brain interaction. Gastroenterology. 2016;150(6):1257–61. 2. Chey WD, Kurlander J, Eswaran S. Irritable bowel syndrome: a clinical review. JAMA. 2015;313(9):949–58. 3. Xiong LS, Chen MH, Chen HX, Xu AG, Wang WA, Hu PJ, et al. A population- based epidemiologic study of irritable bowel syndrome in South China: stratified randomized study by cluster sampling. Aliment Pharmacol Ther. 2004;19(11):1217–24. 1. Drossman DA, Hasler WL. Rome IV-functional GIdisorders: disorders of gut- brain interaction. Gastroenterology. 2016;150(6):1257–61. 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https://openalex.org/W3034402389	https://ieeexplore.ieee.org/ielx7/7782633/9120059/09115033.pdf	English	null	Kernel Regularization in Frequency Domain: Encoding High-Frequency Decay Property	IEEE control systems letters	2,020	cc-by	6,519	I. INTRODUCTION B LANCING model complexity and data ﬁt is one of the key issues in system identiﬁcation ﬁeld (e.g., [1, Ch. 16]). A new approach for this issue, which is called the kernel regu- larization method [2], [3], has attracted much attention in these days [4], [5]. In kernel-based identiﬁcation for linear systems, the unknown impulse response is estimated via regularized least squares. The advantage of such approach w.r.t. classic parametric methods is that the trade-off between data ﬁt and model complexity is ruled by a real parameter instead of a dis- crete value, thus allowing for more ﬂexibility. From the above background, many works on kernel regularization have been reported; e.g., kernel design [6], [7], kernel properties [8]–[10], hyperparameter tuning [11]–[13], input design [14]–[16], and so on. B This letter employs the high frequency decay rate to design the regularization term, and reformulates the regularized least squares problem in the frequency domain. This reformulation drastically reduces the computational burden. Let n and N be the length of impulse response and observed data, respectively. The proposed method requires O(N) memory and O(N) ﬂops to construct the model, while the standard kernel regulariza- tion requires O(n2) memory and O(n3) ﬂops. Note that O(n2) or O(n3) are too large for some applications, e.g., acoustic engineering. Note also that N ≪n2 in most cases, thus the proposed method signiﬁcantly reduces the computational burden. The main contributions of this letter are the following: The main contributions of this letter are the following: • It proposes a quadratic regularization based on a prior knowledge in the frequency domain, i.e., the rate of high frequency decay. One of the main advantages of the kernel regularization is that it can encode a prior knowledge on the systems. For instance, most of the previous methods encode the expo- nential decay of the impulse response in the regularization term, and this makes the estimated impulse responses decay • It shows that the linear equation can be solved in compu- tationally efﬁcient way under a mild condition. In more detail, this letter assumes that the input/output relation is given by the circular convolution. To the best of the author’s knowledge, these contribution are novel. Manuscript received March 16, 2020; revised May 14, 2020; accepted May 29, 2020. Date of publication June 11, 2020; date of current version June 29, 2020. IEEE CONTROL SYSTEMS LETTERS, VOL. 5, NO. 1, JANUARY 2021 367 Kernel Regularization in Frequency Domain: Encoding High-Frequency Decay Property Yusuke Fujimoto , Member, IEEE exponentially. By using such an appropriate prior knowledge, the identiﬁcation accuracy can be improved. Abstract—This letter discusses the kernel regularization in the frequency domain. In particular, this letter proposes a new kernel which encodes prior knowledge on the rate of high frequency decay. The proposed kernel has a simi- lar structure to the one of the ﬁrst order spline kernel. By exploiting the known properties of such kernel, the deter- minant and the inverse of the Gram matrix of the proposed kernel are given in closed form. One of the important advan- tages of the proposed kernel is the computational burden reduction. In fact, it turns out that the complexity is lin- ear in the dataset size N, while standard methods require O(n2) memory and O(n3) ﬂops, where n is the impulse response length usually satisfying N ≪n2 in regularization frameworks. In this letter, we focus on encoding the system proper- ties in the frequency domain, on which there are few works. For example, [17] and [7] discuss the identiﬁcation from the frequency viewpoint. However, their ideas are rather trans- forming a prior knowledge in the time domain into the frequency one. In contrast, this letter directly designs the regularization based on a prior knowledge in the frequency domain. A property that can be available as prior information is the high frequency decay rate. There are a lot of systems (such as mechanic or electronic systems) which are known to evidence such property. In addition, the high frequency decay rate is known in advance in some cases. In fact, if the relative degree of the underlying system is known to be d, then the high frequency decay rate is given by −20d [dB/decade]. Index Terms—System identiﬁcation, regularization, impulse response. ve Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ II. PROBLEM SETTING Problem 1: Assume that {u(t), y(t)}N−1 k=0 is given. Also assume that the system is known to show high frequency decay with −20d [dB/decade] for some known d ∈N. Estimate g(t) so that the model shows high frequency decay with −20d [dB/decade]. We consider a discrete-time linear time invariant dynamic system described as y(t) = t j=0 g(j)u(t −j) + w(t), (1) (1) To this end, this letter employed the kernel regularization technique. where y(t), u(t), g(t) and w(t) denote the output, input, impulse response and the measurement noise at time t, respectively. The measurement noise is an i.i.d. Gaussian random variable, and its mean and variance are zero and σ 2, respectively. The goal of this letter is to estimate the impulse response g(t) (t = 0, . . . , N −1) from the observed data {(u(t), y(t))}N−1 t=0 . For the simplicity of discussion on the Discrete Fourier Transform (DFT), we assume that N is even. The extension to the odd case is straightforward, thus it is omitted in this letter. where y(t), u(t), g(t) and w(t) denote the output, input, impulse response and the measurement noise at time t, respectively. The measurement noise is an i.i.d. Gaussian random variable, and its mean and variance are zero and σ 2, respectively. The goal of this letter is to estimate the impulse response g(t) (t = 0, . . . , N −1) from the observed data {(u(t), y(t))}N−1 t=0 . For the simplicity of discussion on the Discrete Fourier Transform (DFT), we assume that N is even. The extension to the odd case is straightforward, thus it is omitted in this letter. I. INTRODUCTION Now the problem discussed in this letter is set as follows. N 1 III. REGULARIZED LEAST SQUARES IN FREQUENCY DOMAIN Although the ﬁnal goal is to estimate g(t), this letter pro- poses to estimate G ﬁrst, and then reconstruct g(t) by the inverse Fourier transform. In particular, the regularized least squares method in the frequency domain is formulated in this section. Note that G must satisfy some constraints to make g a real vector. To make the regularized least squares uncon- strained, Section III-A introduces a speciﬁc parametrization of G. Then the regularized least squares in the frequency domain is formulated in Section III-B, and Section III-C pro- poses a regularization matrix and the corresponding kernel. Properties of the kernel are investigated in Section IV. Before setting the problem in more detail, we brieﬂy recall the N-point DFT and set some notation. Let y = [y(0), . . . , y(N −1)]⊤∈RN, (2) u = [u(0), . . . , u(N −1)]⊤∈RN, (3) g = [g(0), . . . , g(N −1)]⊤∈RN, (4) w = [w(0), . . . , w(N −1)]⊤∈RN. (5) y = [y(0), . . . , y(N −1)]⊤∈RN, (2) u = [u(0), . . . , u(N −1)]⊤∈RN, (3) g = [g(0), . . . , g(N −1)]⊤∈RN, (4) w = [w(0), . . . , w(N −1)]⊤∈RN. (5) I. INTRODUCTION This work was supported in part by JST ACT-X under Grant 19205777, and in part by JSPS KAKENHI under Grant 19K15017. Recommended by Senior Editor J.-F. Zhang. This letter is organized as follows. The problem setting is shown in Section II, and the regularized least squares in the frequency domain is shown in Section III. Some properties of the proposed kernel are given in Section IV. Based on these properties, an efﬁcient implementation is shown in Section V. y g The author is with the Department of Environmental Engineering, University of Kitakyushu, Kitakyushu 808-0135, Japan (e-mail: y-fujimoto@kitakyu-u.ac.jp). y j y jp) Digital Object Identiﬁer 10.1109/LCSYS.2020.3001879 Digital Object Identiﬁer 10.1109/LCSYS.2020.3001879 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creat IEEE CONTROL SYSTEMS LETTERS, VOL. 5, NO. 1, JANUARY 2021 368 Section VI shows a numerical demonstration to illustrate the properties of the proposed kernel. ignore some difﬁculties such as leakage, and is often employed in acoustic engineering (e.g., [18]). In the rest of this letter, we assume u(t −N) = u(t) and consider (11). Notation: The sets of natural, real and complex numbers are denoted by N, R and C. Re(z) and Im(z) denote the real and imaginary parts of a complex vector z, and z denotes the complex conjugate of z. The n × n identity matrix is denoted by In. For a vector a, ∥a∥2 W denotes a⊤Wa. The ℓ-th element of a vector a is denoted by aℓ. For a vector a ∈RN, diag(a) denotes the N × N diagonal matrix whose (ℓ, ℓ) element is aℓ. K ≻0 indicates that the matrix K is positive deﬁnite. Throughout this letter, i and s denote the imaginary unit and the complex frequency of the Laplace transform, respectively. Note also that G is the frequency response of the system. For discussions in the frequency domain, let ω(k) = 2π N k (k = 0, . . . , N−1). With a slight abuse of notation, the (k+1)-th ele- ment of G is denoted by G(ω(k)) to emphasize the dependence on the frequency. Then, if the system shows high frequency decay, \|G(ω(k))\| decays with −20d [dB/decade] where d is a natural number for sufﬁciently large ω(k). Remark 1: From the viewpoint of digital ﬁltering, d is the relative degree of the underlying analogue ﬁlter. A. Parametrization With Real and Imaginary Part Also let F ∈CN×N be the matrix whose (ℓ, m)-th element is given by Also let F ∈CN×N be the matrix whose (ℓ, m)-th element is given by As mentioned above, this letter considers the regularized least squares in the frequency domain. However, employing G as the optimization variable is not easy. This is because the impulse response, F−1G, must be a real vector, and thus G must satisfy some constraints. To make the optimization problem unconstrained, consider Fℓ,m = exp −2πi(ℓ−1)(m −1) N . (6) (6) (6) Then, the DFTs of y, u, g and w are given by Then, the DFTs of y, u, g and w are given by Y = Fy ∈CN, (7) U = Fu ∈CN, (8) G = Fg ∈CN, (9) W = Fw ∈CN. (10) Gre = ⎡ ⎣Re G1: N 2 +1 Im G2: N 2 ⎤ ⎦∈RN. (12) (12) (10) Here, Gℓ:ℓ+m denotes the m + 1 dimensional vector whose elements are the ℓ-th to (ℓ+ m)-th elements of G. Recall that the latter half of G is the complex conjugate of the former half. In this way, all the information of G is included in the real vector Gre, which is going to be our optimization variable. Note that reconstructing G from Gre is straightforward, i.e., the Here, Gℓ:ℓ+m denotes the m + 1 dimensional vector whose elements are the ℓ-th to (ℓ+ m)-th elements of G. Recall that the latter half of G is the complex conjugate of the former half. In this way, all the information of G is included in the real vector Gre, which is going to be our optimization variable. Note that reconstructing G from Gre is straightforward, i.e., the For later discussions, note that these vectors have the following properties. p p • The ﬁrst and ( N 2 + 1)-th elements are real values. p p • The ﬁrst and ( N 2 + 1)-th elements are real values. 2 • The latter half is the complex conjugate of the former half. For instance, Yℓ= YN+2−ℓfor ℓ= N 2 + 2, . . . , N. 2 • The latter half is the complex conjugate of the former half. For instance, Yℓ= YN+2−ℓfor ℓ= N 2 + 2, . . . , N. B. Regularized Least Squares g q From (14), the following regularized least squares method is employed to estimate Gre. kS(xℓ, xm) = η1 min(xℓ, xm). (23) kS(xℓ, xm) = η1 min(xℓ, xm). (23) From (14), the following regularized least squares method is employed to estimate Gre. kS(xℓ, xm) = H th d k l From (14), the following regularized least squares method is employed to estimate Gre. kS(xℓ, xm) = η1 min(xℓ, xm). (23) Hence the proposed kernel (22) is understood as the spline From (14), the following regularized least squares method is employed to estimate Gre. (23) Hence the proposed kernel (22) is understood as the spline kernel with the coordinate change is employed to estimate Gre. ˆGre = argmin G ∈RN ∥Yre −UreGre∥2 W + G⊤ reK−1Gre, K ≻0 (17) Hence the proposed kernel (22) is understood as the spline kernel with the coordinate change ˆGre = argmin Gre∈RN ∥Yre −UreGre∥2 W + G⊤ reK−1Gre, K ≻0 (17) Wℓ,m = ⎧ ⎨ ⎩ 1 ℓ= m = 1, N 2 + 1 0 ℓ̸= m 2 otherwise. , W ∈RN×N (18) xℓ= 1 ω(ℓ)2 + η2 d . (24) (24) (18) The Bayesian estimation framework is useful for intuitive understanding of the proposed kernel. Figs. 1 to 3 illustrate the variances of Gaussian process whose covariance functions correspond to the ﬁrst order spline kernel, TC kernel and HFD kernel, respectively. The vertical axes show the variance, and the horizontal axes show x, time and frequency, respectively. Fig. 1 shows that the variance with the ﬁrst order spline kernel increases linearly. Remark 2: The weight matrix W is introduced to make the ﬁrst term of (17) equal to the square error ∥Y −diag(U)G∥2. Recall that the latter half of Y and related vectors are the complex conjugate of the former half. Hence j-th element of Yre, where j = 2, . . . , N 2 , N 2 + 2, . . . , N appears in Y twice. 2 2 It should be noted that the optimization problem (17) is unconstrained. This is because we employ the parametrization introduced in Section III-A. From the above observation, ˆGre is reduced to The TC kernel deﬁned as kTC(tℓ, tm) = η1 min(exp(−η′ 2tℓ), exp(−η′ 2tm)), (25) (25) ˆGre = U⊤ reWUre + K−1−1 U⊤ reWYre. B. Regularized Least Squares (19) (19) which is the combination of the spline kernel and the coor- dinate change x = exp(−η′ 2t) where t denotes time, implies that the variance decays exponentially as shown in Fig. 2. If we employ the TC kernel for the prior distribution of the impulse response, the estimated impulse response also decays exponentially. FUJIMOTO: KERNEL REGULARIZATION IN FREQUENCY DOMAIN: ENCODING HIGH-FREQUENCY DECAY PROPERTY 369 Kim,ℓ,m = kHFD(ω(ℓ), ω(m)). (21) kHFD(ω(ℓ), ω(m)) = η1 min 1 ω(ℓ)2 + η2 d , 1 ω(m)2 + η2 d (22) the above construction, the resulting F−1G becomes a real vector for any Gre. Similarly, let Yre ∈RN and Wre ∈RN be the above construction, the resulting F−1G becomes a real vector for any Gre. Similarly, let Yre ∈RN and Wre ∈RN be Yre = ⎡ ⎣Re Y1: N 2 +1 Im Y2: N 2 ⎤ ⎦∈RN, Wre = ⎡ ⎣Re W1: N 2 +1 Im W2: N 2 ⎤ ⎦∈RN. (13) (22) The hyperparameter is [η1, η2]⊤and η1 > 0, η2 > 0. The kernel deﬁned by (22) is called High-Frequency Decay (HFD) The hyperparameter is [η1, η2]⊤and η1 > 0, η2 > 0. The kernel deﬁned by (22) is called High-Frequency Decay (HFD) kernel in the rest of this letter. Recall that Kre,ℓ,ℓregulates the real part of Gℓ= G(ω(ℓ−1)), while Kim,ℓ,ℓregulates the imaginary part of Gℓ+1 = G(ω(ℓ)). Equation (21) is based on these indexes. With these notations, the relation (11) is reduced to With these notations, the relation (11) is reduced to (14) Yre = UreGre + Wre, (14) where (ℓ, m) element of Ure ∈RN×N is given by Ure,ℓ,m = ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ U1 ℓ= m = 1 U N 2 +1 ℓ= m = N 2 + 1 Re(Uℓ) ℓ= 2, . . . , N 2 , m = ℓ −Im(Uℓ) ℓ= 2, . . . , N 2 , m = ℓ+ N 2 Im(Uℓ) ℓ= N 2 + 2, . . . , N, m = ℓ−N 2 Re(Uℓ) ℓ= N 2 + 2, . . . , N, m = ℓ, (15) Ure,ℓ,m = ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ U1 ℓ= m = 1 U N 2 +1 ℓ= m = N 2 + 1 Re(Uℓ) ℓ= 2, . . . , N 2 , m = ℓ −Im(Uℓ) ℓ= 2, . . . , N 2 , m = ℓ+ N 2 Im(Uℓ) ℓ= N 2 + 2, . . . , N, m = ℓ−N 2 Re(Uℓ) ℓ= N 2 + 2, . . . , N, m = ℓ, The derivation and properties of the HFD kernel are shown in Section IV. (15) (15) IV. PROPERTIES OF PROPOSED KERNEL This section discusses some properties about the proposed kernel given by (22). which comes from which comes from This section discus kernel given by (22). which comes from Yk = [Re(Uk)Re(Gk) −Im(Uk)Im(Gk)] + i[Re(Uk)Im(Gk) + Im(Uk)Re(Gk)]. (16) Regularized Least Squares A. Relation With First Order Spline Kernel The HFD kernel is derived from the ﬁrst order spline kernel. The ﬁrst order spline kernel is deﬁned as Yk = [Re(Uk)Re(Gk) −Im(Uk)Im(Gk)] + i[Re(Uk)Im(Gk) + Im(Uk)Re(Gk Yk = [Re(Uk)Re(Gk) −Im(Uk)Im(Gk)] + i[Re(Uk)Im(Gk) + Im(Uk)Re(Gk)]. (16) A Th Yk = [Re(Uk)Re(Gk) −Im(Uk)Im(Gk)] + i[Re(Uk)Im(Gk) + Im(Uk)Re(Gk)]. (16) Regularized Least Squares A. Relation With First Order Spline Kernel The HFD kernel is derived from the ﬁrst order spline kernel. The ﬁrst order spline kernel is deﬁned as A. Parametrization With Real and Imaginary Part • When the input u(t) is periodic and u(t −N) = u(t), 2 • The latter half is the complex conjugate of the former half. For instance, Yℓ= YN+2−ℓfor ℓ= N 2 + 2, . . . , N. • When the input u(t) is periodic and u(t −N) = u(t), g g p Note that reconstructing G from Gre is straightforward, i.e., the ⎡0 ⎤ + 2 • When the input u(t) is periodic and u(t −N) = u(t), g G Gre g , , former half of G is given by Gre1: N 2 +1+i ⎡ ⎢⎣ 01×( N 2 −1) I N 2 −1 01×( N 2 −1) ⎤ ⎥⎦Gre N 2 +2:N, where 01×( N 2 −1) indicates the 1 × ( N 2 −1) zero matrix. With Y = diag(U)G + W. (11) (11) The convolution under the assumption u(t −N) = u(t) is called circular convolution. The circular convolution can ⎣ 2 ⎦ where 01×( N 2 −1) indicates the 1 × ( N 2 −1) zero matrix. With C. Design of Regularization Matrix Now let us consider how to design K. Recall the following two points: • The optimization variables are the real and imaginary parts of the frequency response. • The optimization variables are the real and imaginary parts of the frequency response. As shown in Fig. 3, the variance with the HFD kernel decays slower than the TC kernel. Recall that the gain of the ﬁrst order delay system P(s) = K s+α is given by • The system is known to show high frequency decay. • The system is known to show high frequency decay. Based on these observations, this letter proposes the following regularization matrix: • The system is known to show high frequency decay. Based on these observations, this letter proposes the following regularization matrix: \|P(iω)\|2 = K2 ω2 + α2 . (26) K = Kre 0 0 Kim ∈RN×N, (20) (26) (20) (26) and (20) indicate that the variances of real and imaginary parts of the frequency response function decay at the same rate as a d-th order delay system, and η1, η2 correspond to K2, α2. where (ℓ, m) elements of Kre ∈R( N 2 +1)×( N 2 +1) and Kim ∈ R( N 2 −1)×( N 2 −1) are given by Kre,ℓ,m = kHFD(ω(ℓ−1), ω(m −1)), IEEE CONTROL SYSTEMS LETTERS, VOL. 5, NO. 1, JANUARY 2021 370 Fig. 1. Illustration of variance with ﬁrst order spline kernel. det(Kim) = η N 2 −1 1 G N 2 −1 N 2 −2 j=1 (G(j) −G(j + 1)). (32) (32) Fig. 1. Illustration of variance with ﬁrst order spline kernel. Fig. 2. Illustration of variance with TC kernel. Fig. 3. Illustration of variance with HFD kernel. The inverse of K is given by The inverse of K is given by K−1 = K−1 re 0 0 K−1 im , (33) (33) Fig. 1. Illustration of variance with ﬁrst order spline kernel. Fig. 1. Illustration of variance with ﬁrst order spline kernel. where the (ℓ, m)-th elements of K−1 re and K−1 im are given by K−1 re,ℓ,m = ⎧ ⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎩ 1 η1 G(N/2−1) G(N/2)(G(N/2−1)−G(N/2)) ℓ= m = N/2 + 1, 1 η1 G(ℓ−2)−G(ℓ) (G(ℓ−2)−G(ℓ−1))(G(ℓ−1)−G(ℓ)) ℓ= m = 2, . . . C. Design of Regularization Matrix , N 2 , 1 η1 1 G(0)−G(1) ℓ= m = 1, 0 \|ℓ−m\| > 1, −1 η1 1 max(G(ℓ−1),G(m−1))−min(G(ℓ−1),G(m−1)) otherwise (34) K−1 im,ℓ,m = ⎧ ⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎩ 1 η1 G(N/2−2) G(N/2−1)(G(N/2−2)−G(N/2−1)) ℓ= m = N/2 −1, 1 η1 G(ℓ−1)−G(ℓ+1) (G(ℓ)−G(ℓ+1))(G(ℓ−1)−G(ℓ)) ℓ= m = 2, . . . , N 2 , 1 η1 1 G(1)−G(2) ℓ= m = 1, 0 \|ℓ−m\| > 1, −1 η1 1 max(G(ℓ),G(m))−min(G(ℓ),G(m)) otherwise. (35) 2 (34) Fig. 2. Illustration of variance with TC kernel. Fig. 2. Illustration of variance with TC kernel. Proof: Due to the space limitation, only the proof for Kre is shown. The extension to Kim is straightforward and thus omitted in this letter. N N Let T ∈R( N 2 +1)×( N 2 +1) be the matrix whose all anti-diagonal l 1 d h h l Th T i Let T ∈R( N 2 +1)×( N 2 +1) be the matrix whose all anti-diagonal elements are 1 and the other elements are zero. Then, T is a permutation matrix which ﬂips the rows of the matrix up to down. Note that T is the orthogonal matrix and T⊤T = I N 2 +1, and is of course nonsingular. Note also that T is symmetric. This implies that det(T)2 = det(T) det(T−1) = 1. Fig. 3. Illustration of variance with HFD kernel. B. Determinant and Inverse Matrix Since the structure of (21) is the same as the one of ﬁrst order spline kernel, the determinant and the inverse matrix of K can be computed in closed form. To this end, the following lemma plays an important role. Consider K′ = TKreT. This matrix has exactly the same structure as the Gram matrix of the ﬁrst order spline kernel with xj = G( N 2 + 1 −j). Hence det(K′) and K′−1 are given by Lemma 1, and Lemma 1 (Chen et al. [8]): Let 0 < x1 < · · · < xn and KS ∈Rn×n be the matrix whose (ℓ, m)-th element is given by (23). Then, det(Kre) = det(T) det(K′) det(T) = det(K′), (36) K−1 re = TK′−1T, (37) (36) (37) (36) (37) ( ) (37) det(KS) = ηn 1x1 n−1 j=1 (xj+1 −xj), (27) gives the determinant and the inverse matrix of Kre. (27) The main point of this theorem is that the inverse of K is tridiagonal and the number of non-zero element is at most 3N −2. Thanks to this sparsity, the computationally efﬁcient implementation of (19) is available. and the (ℓ, m) element of the inverse matrix of KS is given by K−1 S,ℓ,m = ⎧ ⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎩ 1 η1 x2 x1(x2−x1) ℓ= m = 1, 1 η1 xℓ+1−xℓ−1 (xℓ+1−xℓ)(xℓ−xℓ−1) ℓ= m = 2, . . . , n −1, 1 η1 1 xn−xn−1 ℓ= m = n, 0 \|ℓ−m\| > 1, −1 η1 1 max(xℓ,xm)−min(xℓ,xm) otherwise. . (28) ℓ= m = 1, ℓ= m = 2, . . . , n −1, ℓ= m = n, . (28 V. COMPUTATIONALLY EFFICIENT IMPLEMENTATION . (28) . (28) This section discusses the implementation of (19), and hyperparameter tuning. For ease of notation, let VI. NUMERICAL DEMONSTRATION VI. NUMERICAL DEMONSTRATION otherwise In this section, a numerical example is shown to demonstrate the effectiveness of the proposed kernel.1 These equations show that UjU⊤ j′ = 0 if j ̸= j, and the statement has been proven. The target discrete-time system is constructed from P(s) = 10(s+10) s2+2s+101. Here, P(s) is discretized by zero-order hold where the sampling rate is 3 times of the bandwidth of P(s). The input sequence u(t) is generated from i.i.d Gaussian random variable, with N = 3000. The output is generated by the circular convolution, i.e., the above sequence is added to the system twice, and the latter half of the output is recorded. The variance of the measurement noise is set so that the Signal-to-Noise Ratio becomes 20. The candidates of the hyperparameters ηi (i = 1, 2) are 50 logarithmically equidis- tant points from 10−8 to 105 obtained via MATLAB command logspace, hence the number of candidate hyperparameters couples is 2500. Note that this result holds since we consider the regularized least squares in the frequency domain. In the time domain, such a special structure does not appear in general. p pp g Corollary 1: The matrix U⊤ reWUre + K−1 is symmetric and tridiagonal. Corollary 1 gives an important observation for an efﬁcient computation of ˆGre. Recall that ˆGre in (19) is the solution of the linear equation U⊤ reWUre + K−1 ˆGre = U⊤ reWYre. (40) (40) Since (U⊤ reWUre +K−1) is tridiagonal, the TriDiagonal Matrix Algorithm (TDMA), also known as Thomas algorithm, can be employed to compute ˆGre. For the notational convenience, con- sider a linear equation Ax = b where A ∈RN×N is tridiagonal and its (ℓ, m)-th element is denoted by Aℓ,m. Then, TDMA is given as Algorithm 1 [19]. Since (U⊤ reWUre +K−1) is tridiagonal, the TriDiagonal Matrix Algorithm (TDMA), also known as Thomas algorithm, can be employed to compute ˆGre. For the notational convenience, con- sider a linear equation Ax = b where A ∈RN×N is tridiagonal and its (ℓ, m)-th element is denoted by Aℓ,m. Then, TDMA is given as Algorithm 1 [19]. Fig. 4 shows the estimated result with the procedure described in Section V. The horizontal axis shows the frequency [×π rad/sample], and the vertical axes show the gain [dB] and the phase [rad], respectively. 1It is difﬁcult to estimate the high frequency decay rate of the randomly generated systems employed in e.g., [3]. Statistical analysis with randomly generated systems is one of the future tasks. VI. NUMERICAL DEMONSTRATION The thick solid, thin solid, and the broken lines are the estimated model with the HFD kernel, the one with the TC kernel, and the true system. Hyperparameters of both the HFD kernel and the TC kernel are tuned by the procedure described in Section V-B. TDMA consists of two loops and the intermediate variables are Pj and Qj (j = 1, . . . , N −1). Hence TDMA only requires O(N) memory and O(N) ﬂops. This is much lower than the standard kernel regularization which requires O(N2) memory and O(N3) ﬂops. The estimated model with the HFD kernel decays with −20 [dB/decade] as expected. The model with the TC kernel also shows a good high frequency decay, but it is not smooth. This is because the TC kernel only considers the smoothness in the time domain. FUJIMOTO: KERNEL REGULARIZATION IN FREQUENCY DOMAIN: ENCODING HIGH-FREQUENCY DECAY PROPE , N 2 , N 2 + 2, . . . , N. Now consider Uj, j ̸= 1, ( N 2 + 1). When j ≤N 2 , the ℓ-th element of Uj is given as j = 2, . . . , N. Similarly, U N 2 +1 satisﬁes U N 2 +1U⊤ j = 0 for j = 1, . . . , N 2 , N 2 + 2, . . . , N. Step 5 Select η∗= argminηj E(ηj) as the hyperparameter. This procedure requires O(Nm) ﬂops, hence it can be com- puted efﬁciently. j 2 2 Now consider Uj, j ̸= 1, ( N 2 + 1). When j ≤N 2 , the ℓ-th element of Uj is given as Note that if we design the candidate {η1, . . . , ηm} to be grid points on a speciﬁc space, the above procedure is almost the same as the conventional exhaustive grid search used in the machine learning ﬁeld. Uj,ℓ= ⎧ ⎨ ⎩ Re Uj ℓ= j −Im Uj ℓ= j + N 2 0 otherwise , (38) and when j ≥N 2 + 2, Uj,ℓ= ⎧ ⎨ ⎩ Im Uj ℓ= j −N 2 Re Uj ℓ= j 0 otherwise . (39) Uj,ℓ= ⎧ ⎨ ⎩ Re Uj ℓ= j −Im Uj ℓ= j + N 2 0 otherwise , (38) and when j ≥N 2 + 2, Uj,ℓ= ⎧ ⎨ ⎩ Im Uj ℓ= j −N 2 Re Uj ℓ= j 0 otherwise . (39) (38) It should be noted that the candidates should be densely placed to improve the identiﬁcation accuracy, which may increase the execution time. More efﬁcient hyperparameter tuning is a future task. Uj,ℓ= ⎧ ⎨ ⎩ Im Uj ℓ= j −N 2 Re Uj ℓ= j 0 otherwise . (39) (39) A. Solving Linear Equation To investigate the sparsity of the matrix which appears in (19), the following theorem is useful. G(j) = 1 ω(j)2 + η2 d . (29) (29) (29) Theorem 2: Consider Ure and W deﬁned by (15) and (18). Then, U⊤ reWUre is a diagonal matrix. Theorem 2: Consider Ure and W deﬁned by (15) and (18). Then, U⊤ reWUre is a diagonal matrix. The following theorem is obtained in a straightforward manner from Lemma 1. Proof: Note that W is a diagonal matrix. This indicates that the statement is proven by showing that all rows of Ure are orthogonal to each other. Theorem 1: For K deﬁned by (20) and (21), we have det(K) = det(Kre) det(Kim), (30) det(Kre) = η N 2 +1 1 G N 2 N 2 −1 j=0 (G(j) −G(j + 1)), (31) (30) Let Uj be the j th row of Ure. From (15), U1 is the only vector which has non-zero element in the ﬁrst column. Because the rest of U1 are all zero, this implies that U1U⊤ j = 0 for (31) FUJIMOTO: KERNEL REGULARIZATION IN FREQUENCY DOMAIN: ENCODING HIGH-FREQUENCY DECAY PROPERTY FUJIMOTO: KERNEL REGULARIZATION IN FREQUENCY DOMAIN: ENCODING HIGH-FREQUENCY DECAY PROPE 371 Algorithm 1 TDMA (Thomas Algorithm) Require: A ∈RN×N, b ∈RN Ensure: x ∈RN PN−1 ←−AN,N−1 AN,N , QN−1 ← bN AN,N for j = N −1 : −1 : 2 do Pj−1 ←− Aj,j−1 Aj,j+Aj+1,jPj , Qj−1 = bj−Aj+1,jQj Aj,j+Aj+1,jPj end for x1 ← b1−A1,2Q1 A1,1+A1,2P1 for j = 1 : N −1 do xj+1 ←Pjxj + Qj end for Algorithm 1 TDMA (Thomas Algorithm) Require: A ∈RN×N, b ∈RN Ensure: x ∈RN PN−1 ←−AN,N−1 AN,N , QN−1 ← bN AN,N for j = N −1 : −1 : 2 do Pj−1 ←− Aj,j−1 Aj,j+Aj+1,jPj , Qj−1 = bj−Aj+1,jQj Aj,j+Aj+1,jPj end for x1 ← b1−A1,2Q1 A1,1+A1,2P1 for j = 1 : N −1 do xj+1 ←Pjxj + Qj end for Algorithm 1 TDMA (Thomas Algorithm) Require: A ∈RN×N, b ∈RN Ensure: x ∈RN PN−1 ←−AN,N−1 AN,N , QN−1 ← bN AN,N for j = N −1 : −1 : 2 do Pj−1 ←− Aj,j−1 Aj,j+Aj+1,jPj , Qj−1 = bj−Aj+1,jQj Aj,j+Aj+1,jPj end for x1 ← b1−A1,2Q1 A1,1+A1,2P1 for j = 1 : N −1 do xj+1 ←Pjxj + Qj end for Algorithm 1 TDMA (Thomas Algorithm) Algorithm 1 TDMA (Thomas Algorithm) One simple method to exploit the fast optimization of (19) is to use validation data. Assume that we can use {u(t), yv(t)}N−1 t=0 , One simple method to exploit the fast optimization of (19) is to use validation data. Assume that we can use {u(t), yv(t)}N−1 t=0 , where the input is the same as the original experiment. The only difference between y(t) and yv(t) is the realization of the measurement noise. Then, the following procedure can select an appropriate hyperparameter. Step 1 Prepare the candidates of the hyperparameter {η1, . . . , ηm}. N 1 j Step 1 Prepare the candidates of the hyperparameter {η1, . . . , ηm}. N 1 j Step 2 Estimate g(t) from {u(t), y(t)}N−1 t=0 and ηj. Step 3 Compute the predictive output ˆyj(k) from the circu- lar convolution. Step 3 Compute the predictive output ˆyj(k) from the circu- lar convolution. Step 4 Compute the prediction error E(ηj) = N−1 t=0 (ˆyj(t) −yv(t))2. j Step 4 Compute the prediction error E(ηj) = N−1 t=0 (ˆyj(t) −yv(t))2. j = 2, . . . , N. Similarly, U N 2 +1 satisﬁes U N 2 +1U⊤ j = 0 for j = 1, . . . VII. CONCLUSION This letter proposes a new kernel regularization method that exploits a prior knowledge in the frequency domain, i.e., the high frequency decay property. The proposed kernel has the same structure as the ﬁrst order spline kernel, hence the determinant and inverse matrix of its Gram matrix are given in closed form. Thanks to the problem setting in the frequency domain and the kernel structure, the linear equation to be solved is described by a sparse matrix. This sparsity enables an efﬁcient implementation, with O(N) memory and O(N) ﬂops. p y p Efﬁcient implementations for the hyperparameter tuning and the input design are future tasks. Fig. 4. Gain plots of estimated models. Fig. 4. Gain plots of estimated models. REFERENCES Fig. 5. Estimated impulse response with proposed kernel. [1] L. Ljung, System Identiﬁcation: Theory for the User, 2nd ed. Upper Saddle River, NJ, USA: Prentice-Hall, 1999. [2] G. Pillonetto and G. De Nicolao, “A new kernel-based approach for linear system identiﬁcation,” Automatica, vol. 46, no. 1, pp. 81–93, 2010. [3] T. Chen, H. Ohlsson, and L. Ljung, “On the estimation of transfer func- tions, regularizations and Gaussian processes—Revisited,” Automatica, vol. 48, no. 8, pp. 1525–1535, 2012. [4] G. Pillonetto, F. Dinuzzo, T. Chen, G. De Nicolao, and L. Ljung, “Kernel methods in system identiﬁcation, machine learning and function estimation: A survey,” Automatica, vol. 50, no. 3, pp. 657–682, 2014. [5] F. Dinuzzo, “Kernels for linear time invariant system identiﬁcation,” SIAM J. Control Optim., vol. 53, no. 5, pp. 3299–3317, 2015. [6] T. Chen, “On kernel design for regularized LTI system identiﬁcation,” Automatica, vol. 90, pp. 109–122, Apr. 2018. Fig. 5. Estimated impulse response with proposed kernel. [7] M. A. H. Darwish, J. Lataire, and R. Tóth, “Bayesian frequency domain indentiﬁcation of LTI systems with OBFs kernel,” in Proc. 20th IFAC World Congr., 2017, pp. 6412–6417. [8] T. Chen, T. Ardeshiri, F. P. Carli, A. Chiuso, L. Ljung, and G. Pillonetto, “Maximum entropy properties of discrete-time ﬁrst-order stable spline kernel,” Automatica, vol. 66, pp. 34–38, Apr. 2016. The square errors between the true impulse response and the estimated ones are 8.0 × 10−3 and 8.3 × 10−3 for the HFD and TC kernel, respectively. Hence, the accuracies of the model with these kernels are almost the same. The time required to solve the linear equations are 1.6 × 10−2 [s] and 2.9 × 10−1 [s] for the HFD and TC kernel, respectively. VII. CONCLUSION The linear equation with the TC kernel is solved by the MATLAB command mldivide. Here, the scripts are run with Intel Core i9-7980XE (2.60 GHz), 64.0 GB RAM, Windows 10 Pro, and MATLAB 2019a. The computational time becomes much faster by the proposed kernel. [9] T. Chen, “Continuous-time DC kernel—A stable generalized ﬁrst- order spline kernel,” IEEE Trans. Autom. Control, vol. 63, no. 12, pp. 4442–4447, Oct. 2018. [10] F. P. Carli, T. Chen, and L. Ljung, “Maximum entropy kernels for system identiﬁcation,” IEEE Trans. Autom. Control, vol. 62, no. 3, pp. 1471–1477, Jun. 2017. [11] G. Pillonetto and A. Chiuso, “Tuning complexity in kernel-based lin- ear system identiﬁcation: The robustness of the marginal likelihood,” in Proc. Eur. Control Conf., 2014, pp. 2386–2391. [12] B. Mu, T. Chen, and L. Ljung, “On asymptotic properties of hyperpa- rameter estimators for kernel-based regularization methods,” Automatica, vol. 94, pp. 381–395, Aug. 2018. Fig. 5 shows the true impulse response and the estimated impulse responses with the proposed and TC kernels. The hor- izontal axis shows the time, and the vertical axis shows the impulse response. The thick solid, thin solid, and the broken lines are the estimated model with the HFD kernel, the one with the TC kernel, and the true system. The estimate with the TC kernel shows high frequency oscillation around t = 500 to t = 1000. The estimate with the proposed kernel, on the other hand, shows smooth behavior on this domain. However, we can see oscillations in the estimate with the proposed kernel near t = 3000. This is because the proposed kernel does not consider the exponential decay in the time domain. This indicates that the accuracy would be further improved by combining both the prior knowledge in time and frequency domains. [13] B. Mu, T. Chen, and L. Ljung, “Asymptotic properties of generalized cross validation estimators for regularized system identiﬁcation,” IFAC PapersOnLine, vol. 51, no. 15, pp. 203–208, 2018. [14] Y. Fujimoto and T. Sugie, “Informative input design for kernel-based system identiﬁcaiton,” Automatica, vol. 89, pp. 37–43, Mar. 2018. [15] Y. Fujimoto, I. Maruta, and T. Sugie, “Input design for kernel-based system identiﬁcation from the viewpoint of frequency response,” IEEE Trans. Autom. Control, vol. 63, no. 10, pp. 3075–3082, Sep. 2018. [16] B. Mu and T. Chen, “On input design for regularized LTI system iden- tiﬁcation: Power-constrained input,” Automatica, vol. 97, pp. 327–338, Nov. 2018. B. Hyperparameter Tuning ˆ Although the solution ˆGre can be computed efﬁciently, the hyperparameter η is not easy to compute so fast. The widely used methods for the hyperparameter tuning are empirical Bayes, SURE, or generalized cross validation. However, these methods require more than O(N3) computations in general. IEEE CONTROL SYSTEMS LETTERS, VOL. 5, NO. 1, JANUARY 2021 IEEE CONTROL SYSTEMS LETTERS, VOL. 5, NO. 1, JANUARY 2021 372 Fig. 4. Gain plots of estimated models. Fig. 5. Estimated impulse response with proposed kernel. Fi 4 G i l t f ti t d d l VII. CONCLUSION [17] J. Lataire and T. Chen, “Transfer function and transient estimation by Gaussian process regression in the frequency domain,” Automatica, vol. 72, pp. 217–229, Oct. 2016. [18] J. P. Paulo, C. R. Martins, and J. L. B. Coelho, “A hybrid MLS technique for room impulse response estimation,” Appl. Acoust., vol. 70, no. 4, pp. 556–562, 2009. [19] M. Andrecut, Introductory Numerical Analysis: Lecture Note. Parkland, FL, USA: Universal Publ., 2000.
https://openalex.org/W4220698767	https://www.researchsquare.com/article/rs-307052/latest.pdf	English	null	The difference of lipid profiles between psoriasis with arthritis and psoriasis without arthritis and sex-specific downregulation of methotrexate on the apolipoprotein B/apolipoprotein A-1 ratio	Arthritis research & therapy	2,022	cc-by	8,062	The difference of lipid profiles between psoriasis with arthritis and psoriasis without arthritis and sex-specific downregulation of methotrexate on the apolipoprotein B/apolipoprotein A-1 ratio Bing Wang The difference of lipid profiles between psoriasis with arthritis and psoriasis without arthritis and sex-specific downregulation of methotrexate on the apolipoprotein B/apolipoprotein A-1 ratio Bing Wang The difference of lipid profiles between psoriasis with arthritis and psoriasis without arthritis and sex-specific downregulation of methotrexate on the apolipoprotein B/apolipoprotein A-1 ratio The difference of lipid profiles between psoriasis with arthritis and psoriasis without arthritis and sex-specific downregulation of methotrexate on the apolipoprotein B/apolipoprotein A-1 ratio Bing Wang Huashan Hospital Fudan University Hui Deng Shanghai Sixth Peoples Hospital Yao Hu Huashan Hospital Fudan University Ling Han Huashan Hospital Fudan University Qiong Huang Huashan Hospital Fudan University Xu Fang Huashan Hospital Fudan University Ke Yang Huashan Hospital Fudan University Siyuan Wu Huashan Hospital Fudan University Zhizhong Zheng Huashan Hospital Fudan University Yawalkar Nikhil Inselspital University Hospital Bern: Inselspital Universitatsspital Bern Zhenghua Zhang Huashan Hospital Fudan University Kexiang Yan (  ykx2292002@aliyun.com ) Huashan Hospital Fudan University Siyuan Wu Huashan Hospital Fudan University Zhizhong Zheng Huashan Hospital Fudan University Yawalkar Nikhil Inselspital University Hospital Bern: Inselspital Universitatsspital Bern Zhenghua Zhang Huashan Hospital Fudan University Kexiang Yan (  ykx2292002@aliyun.com ) Huashan Hospital Fudan University Introduction Psoriasis is a common chronic inflammatory disease characterized by keratinocyte abnormalities and immune dysfunctions. It affects about 2–3% of the world population.1 About 5.8–30% psoriatic patients without arthritis (PsO) will develop psoriatic arthritis (PsA).2,3 Epidemiological and clinical studies have consistently shown that psoriasis is associated with an increased cardiovascular risk.4 The inflammatory cytokines found in psoriatic lesions may cause insulin resistance and trigger endothelial cell dysfunction leading to atherosclerosis and ultimately resulting in stroke or myocardial infarction.5 Compelling evidence further suggests that pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α may alter the function of hepatocytes and arterial smooth muscular cells to induce alternated lipoprotein compositions, enhance expression of cellular adhesion molecules, and increase lipid deposition on arterial walls. All of these features can contribute to the development of arterial plaques.6 Cytokines may then destabilize the plaque by promoting the rupture of fragile neo-vessels and increasing the expression of the plaque’s fibrous cap. This cascade of events may ultimately lead to plaque rupture and the formation of life-threatening thrombi.7 Psoriasis is significantly associated with a higher prevalence and incidence of dyslipidemia8—a known risk factor for cardiovascular disease. Dyslipidemia is a broad term that describes any abnormality of plasma lipids including perturbations in plasma lipid levels or abnormalities in lipid composition. Multiple measurements of dyslipidemia are significantly affected and include raised triglycerides, raised LDL cholesterol, lowered HDL cholesterol, raised cholesterol, and raised lipoproteins. 9–11 Due to the remarkable heterogeneity of defining dyslipidemia, there is a lack of sex- and age-matched healthy controls. Moreover, adjustments were not made for confounding factors in most studies. Thus, the association between psoriasis and dyslipidemia still remains unclear. Arthritis is an important determinant for psoriatic patients to develop severe vascular events in Taiwan.12 Moreover, some arthritic patients on low dose methotrexate (MTX) have altered blood lipids versus those not taking MTX.13 In this prospective, cross-sectional study, we compared the differences in lipid profiles and cardiovascular risk parameters between PsA patients and sex- and age-matched healthy controls; between PsO patients and sex- and age- matched healthy controls; and between male and female psoriatic patients. Research article Keywords: psoriatic arthritis, psoriasis, apolipoproteins, lipid profiles, methotrexate DOI: https://doi.org/10.21203/rs.3.rs-307052/v1 DOI: https://doi.org/10.21203/rs.3.rs-307052/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Arthritis Research & Therapy on January 7th, 2022. See the published version at https://doi.org/10.1186/s13075-021-02715-4. of this preprint was published at Arthritis Research & Therapy on January 7th, 2022. See the published version at 075-021-02715-4. Page 1/11 Page 1/11 Page 1/11 Summary The ApoB/ApoA1 ratio is a strong risk factor for cardiovascular disease. The ApoB/ApoA1 ratio was significantly higher in male and PsA patients compared to female and PsO patients, respectively.. Methotrexate significantly decreased the ratio of ApoB to ApoA1 in male patients with psoriasis. The ApoB/ApoA1 ratio was significantly higher in male and PsA patients compared to female and PsO pat poA1 ratio was significantly higher in male and PsA patients compared to female and PsO patients, respectively.. Methotrexate significantly decreased the ratio of ApoB to ApoA1 in male patients with psoriasis. Abstract Background: Methotrexate (MTX) has a protective effect against cardiovascular diseases (CVD), but the mechanism is unclear. of MTX on lipid profiles and the difference between psoriasis without arthritis (PsO) and psoriatic arthritis (PsA). Objective: To investigate the effect of MTX on lipid profiles and the difference between psoriasis without arthritis (PsO) and psoriatic arthritis (PsA). bjective: To investigate the effect of MTX on lipid profiles and the difference between psoriasis without arthritis (P Methods: In this prospective study, we recruited 288 psoriatic patients (136 PsA and 152 PsO) who completed 12 weeks of MTX treatment. Total cholesterol (TC), triglycerides (TG), lipoprotein A [LP(a)], high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein (LDL), apolipoprotein A1 (ApoA1), and ApoB were measured. Results: Compared with sex and age-matched healthy controls, psoriatic patients had significantly (p<0.0001) higher levels of proatherogenic lipids and lower levels of anti-atherogenic lipids. PsA patients had a higher ApoB/ApoA1 ratio than PsO patients(p<0.05). Stepwise regression analysis found a positive correlation between the inflammatory marker hCRP and the psoriasis area severiy index (PASI), ApoB/ApoA1 ratio, BMI, and smoking. ApoB was positively associated with concomitant arthritis, diabetes and hypertension. MTX decreased the levels of pro-atherogenic and anti-atherogenic lipids. However, a significant reduction of the ApoB/ApoA1 ratio by MTX was only observed in male patients. Conclusion: PsA patients had a significantly higher percentage of concomitant disease than PsO. The decrease of MTX on CVD might be related with sex. Trial Registration: ChiCTR2000036192 Conclusion: PsA patients had a significantly higher percentage of concomitant disease than PsO. The decrease of MTX on CVD might be related with sex. Trial Registration: ChiCTR2000036192 Introduction Furthermore, we analyzed the effect of MTX on lipid profiles and further analyzed the influencing factors on lipid profiles and cardiovascular risk parameters after adjustments for sex, age, age at disease duration, disease duration, Psoriasis Area Severity Index (PASI), body mass area (BSA) scores at baseline, smoking, alcohol, height, weight, body mass index (BMI), hypertension, and diabetes. Assessments of lipid profiles and disease characteristics Two certified dermatologists graded the severity and extent of psoriasis using the Psoriasis Area Severity Index (PASI) and body surface area (BSA) scores. Lipid profiles at baseline and 12 weeks for MTX treatment and fasting blood glucose at baseline were measured using conventional laboratory techniques at Huashan Hospital. Sex, age, age at disease onset, smoking, alcohol, hypertension, diabetes, height, weight, and body mass index (BMI) were recorded. Treatment The initial oral MTX dose was 7.5-10 mg once weekly. The dose was increased by 2.5 mg every 2 to 4 weeks to a maximum of 15 mg weekly depending on the patient’s clinical response, side effects, and hematology/chemistry tests. If liver enzyme elevations were >2- and <3-fold, then the MTX dose was reduced by 2.5 mg weekly and administered once 2-4 weeks later. MTX treatment was stopped if the liver enzyme elevations were >3-fold.14 Statistical Analysis Data are expressed as the means±standard deviations (SDs). Statistical analyses were performed using the Mann-Whitney test, unpaired t-test, unpaired t-test with Welch correction, paired t test, χ2 test, or Fisher’s exact test as appropriate. Stepwise multiple regression analysis was performed after adjustments for sex, age, age at disease onset, disease duration, height, weight, body mass index (BMI), hypertension, diabetes, smoking, alcohol consumption, and PASI/BSA scores at baseline. Data analyses were performed using Graph Pad Prism version 5 (Graph Pad Software Inc) and SPSS ver. 23.0 software (SPSS Inc., Chicago, IL, USA). A P-value of <.05 (or P<.025 after multiple test correction) was considered to be statistically significant. Methods Patients This single-center prospective trial was performed in the Department of Dermatology, Huashan Hospital, Fudan University between December 2, 2015 to December 2, 2019. In total, 288 psoriatic patients who received oral MTX treatment for 12 weeks and 288 sex- and age-matched healthy controls without prior medication from a medical examination center were recruited. The medical ethics committee of Huashan Hospital at Fudan University reviewed and approved the protocol (approval #MTX201501); all patients provided written informed consent. Patients aged ≥18 years were recruited from the outpatient population. The diagnosis of psoriasis and psoriatic arthritis (n= 136) was based on typical clinical and/or histopathological criteria and the Classification Criteria for Psoriatic Arthritis (CASPAR classification criteria), respectively. Patients who received systemic treatments (acitretin, cyclosporin, glucocorticoids) for arthritis or psoriasis at 1 month were excluded. The topical treatments had been stopped for more than 1 week before the beginning of study. The therapeutic regimen followed the European guidelines on contraindications and restrictions on methotrexate. None of the patients used lipid-lowering drugs. Page 2/11 Page 2/11 The improvement of skin lesions in psoriasis without arthritis (PsO) was superior to that of psoriasis with arthritis (PsA) by methotrexate Table 1 summarizes the baseline and clinical characteristics according to psoriasis subtype and sex. PsA patients had a significantly older age (p < .0001), age at disease onset (p = .0052), longer disease duration (p = .0061), and higher percentage of hypercholesterolemia (p = .0087) than PsO patients. The mean PASI score at 12 W in PsO patients (p = .0354) was significantly lower than that in PsA patients although there was no difference in PASI score at baseline. The mean PASI (p = .0236) and BSA (p = .0105) change from baseline was significantly higher in PsO patients than that in PsA patients. In addition, the percentage of hypertension in PsA patients was significantly higher than that in PsO patients (p = .0029). The improvement of skin lesions in psoriasis without arthritis (PsO) was superior to that of psoriasis with arthritis (PsA) by methotrexate Page 3/11 Table 1 Table 1 Differences in baseline characteristics between psoriasis with and without psoriatic arthritis a Characteristic PsA (n = 136) PsO (n = 152) p-valueb Male (n = 189) Female (n = 99) p-valueb Age, mean (SD), y 50.45 (13.12) 42.87 (15.63) < .0001 46.79 (14.72) 45.79 (15.46) .5627 Age at disease onset, mean (SD), y 36.00 (16.07) 31.35 (15.83) .0052 35.15 (14.98) 30.50 (17.68) .0021 Disease duration, mean (SD), y 14.51 (10.22) 11.52 (9.89) .0061 11.65 (9.27) 15.39 (11.26) .0089 Height, mean (SD), m 1.67 (0.08) 1.68 (0.08) .8273 1.71 (0.06) 1.60 (0.06) < .0001 Weight, mean (SD), kg 70.21 (12.28) 69.04 (13.28) .4500 73.20 (11.43) 62.81 (12.56) < .0001 BMI 25.01 (3.44) 24.48 (3.62) .2158 24.85 (3.22) 24.49 (4.09) .0865 PASI at baseline 14.04 (8.37) 13.78 (5.47) .7491 14.74 (7.25) 12.29 (6.16) .0045 PASI at 12W 4.58 (4.69) 3.55 (3.52) .0354 4.41 (4.48) 3.31 (3.29) .0288 Mean PASI change from baseline, mean (SD) 63.33 (31.50) 70.82 (28.28) .0236 66.12 (30.27) 69.51 (29.57) .2990 BSA at baseline 26.91 (22.64) 26.22 (17.42) .7713 27.83 (20.08) 24.08 (19.77) .0676 BSA at 12W 9.55 (16.80) 6.75 (12.20) .0518 9.11 (16.28) 6.07 (10.45) .0125 Mean BSA change from baseline, mean (SD) 60.75 (42.16) 71.38 (38.12) .0105 65.11 (37.95) 68.74 (44.72) .0813 Cumulative dose of MTX, mean (SD), mg 138.1 (19.85) 138.4 (20.00) .8869 139.8 (19.36) 135.3 (20.66) .0314 Hypertension 60 (44.1) 41(27.0) .0029 71 (37.6) 30 (30.3) .2434 Diabetes 28 (20.6) 25 (16.4) .4466 32 (16.9) 21 (21.2) .4240 Smoking 36 (26.5) 52 (34.2) .1613 86 (45.5) 2 (2.0) < .0001 Alcohol consumption 36 (26.5) 44 (28.9) .6933 74 (39.2) 6 (6.1) < .0001 Hypercholesterolemia (> 5.9 mmol/L) 23 (16.9) 10 (6.6) .0087 14 (7.4) 19 (19.2) .0056 Hypertriglyceridemia (> 1.8 mmol/L) 51 (37.5) 42 (27.6) .0786 61 (32.3) 32 (32.3) 1.000 Increased LDL (> 3.7mmol/L) 28 (20.6) 21 (13.8) .1574 35 (18.5) 14 (14.1) .4106 Hyperlipoproteinemia (a) (> 300mg/L) 18 (13.2) 19 (12.5) .8620 21 (11.1) 16 (16.2) .2662 Arthritis 86 (45.5) 50 (50.5) 0.4569 Male 86 (63.2) 103 (67.8) 0.4569 Abbreviation: BMI, body mass index (calculated as weight in kilograms divided by height in meters squared); BSA, body surface area; PASI, Psoriasis Area Severity Index. a Data are presented as number (percentage) of patients unless otherwise indicated. b Mann-Whitney test, unpaired t test, or Fisher exact test were used when appropriate. The improvement of skin lesions in psoriasis without arthritis (PsO) was superior to that of psoriasis with arthritis (PsA) by methotrexate P < .05 is considered to be statistically significant. Male patients had significantly older age at disease onset and a lower percentage of hypertryglyceridemia (p = .0056) than female (p = .0021). Several metrics were significantly higher in male patients than female patients: mean height (p < .0001), weight (p < .0001), PASI score at baseline (p = .0045), PASI score at 12W (p = .0288), BSA score at 12W (p = .0125), cumulative dose of MTX (p = .0314), smoking (p < .0001), and alcohol consumption (p < .0001). Table 2 shows the difference in blood lipid profiles (ApoA1, ApoB, TC, LDL, HDL-C, Lp(a), TG) and cardiovascular risk parameters (ApoB/ApoA1, TC/HDL-C, LDL/HDL-C, LDL/ApoB) between PsA patients and sex- and age-matched healthy controls; between PsO patients and sex- and age-matched healthy controls; and between PsA patients and PsO patients. The mean values of serum ApoB (p < .0001), LDL (p = .0115) levels, ApoB/ApoA1 ratio (p < .0001), TC/HDL ratio (p = .0227), and LDL/HDL ratio (p = .0009) were significantly higher in PsA patients than those in sex- and age-matched healthy controls. However, the mean value of serum ApoA1 (p < .0001) were significantly lower in PsA patients than that in sex- and age-matched healthy controls. The mean values of serum ApoB (p < .0001), TG (p = .0098) levels, ApoB/ApoA1 ratio (p < .0001), and LDL/HDL ratio (p = .002) were significantly higher in PsO patients than those in sex- and age-matched healthy controls. The mean value of serum ApoA1 (p < .0001) level and LDL/ ApoB ratio (p < .0001) were significantly lower in PsO patients than those in sex- and age-matched healthy controls. The mean values of serum ApoB (p = .0013), TC(p = .0013), LDL (p = .0229) levels, and ApoB/ ApoA1 ratio (p = .0208) were significantly higher in PsA patients than those in PsO patients. ssociation of the effect of MTX on lipid profiles and cardiovascular risk parameters with psoriasis subtype and se Table 3 shows that MTX significantly decreased the levels of serum ApoB (p = .0003), TC (p = .0007), TG (p = .041), HDL-C (p = .037), Lp(a) (p = .0055), ApoB/ApoA1 ratio (p = .0076), and increased LDL/ApoB ratio (p = .025) in PsA patients. MTX significantly decreased the levels of serum ApoB (p < .0001), TC (p < .0001), HDL-C (p = .011), LDL (p = .0001), Lp(a) (p < .0001), and ApoB/ApoA1p = .0011). MTX increased the LDL/ApoB ratio (p = .0464) in PsO patients. Table 3 shows that MTX significantly decreased the levels of serum ApoB (p = .0003), TC (p = .0007), TG (p = .041), HDL-C (p = .037), Lp(a) (p = .0055), ApoB/ApoA1 ratio (p = .0076), and increased LDL/ApoB ratio (p = .025) in PsA patients. MTX significantly decreased the levels of serum ApoB (p < .0001), TC (p < .0001), HDL-C (p = .011), LDL (p = .0001), Lp(a) (p < .0001), and ApoB/ApoA1p = .0011). MTX increased the LDL/ApoB ratio (p = .0464) in PsO patients. Table 4 summarizes the differences and the effect of MTX on lipid profiles and cardiovascular risk parameters (ApoB/ApoA1, TC/HDL-C, LDL/HDL-C, LDL/ApoB) in male and female patients. Female patients had significantly higher levels of serum ApoA1(p < .0001), TC (p = .0241), and HDL-C(p < .0001). Women had lower ApoB/ApoA1 ratio (p = .0004), TC /HDL ratio (p < .0001), and LDL/HDL ratio (p < .0001) than men. MTX significantly decreased the levels of serum ApoA1(p = .0002), ApoB ( p < .0001), TC(p < .0001), HDL-C (p = .0029), LDL (p = .0001), Lp(a) (p = .0076), and ApoB/ApoA1 ratio (p = .0001); it increased the LDL/ApoB ratio (p = .0019) in men. MTX significantly decreased the levels of serum ApoB (p = .0214), TC (p = .0083) and Lp(a) (p = .0014) in women poA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; a), lipoprotein A; PsA: psoriatic arthritis; PsO, psoriasis without arthritis; TC, total cholesterol; TG, triglyceride; Paired t test, Wilcoxon matched pairs test, Mann-Whitney test, unpaired t test, or unpaired t test with Welch correction were used when appropriate. P < .025 is considered to be statistically significant. Abbreviation: ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; LDL: low-density lipoprotein; Lp(a), lipoprotein A; PsA: psoriatic arthritis; PsO, psoriasis without arthritis; TC, total cholesterol; TG, triglyceride; The improvement of skin lesions in psoriasis without arthritis (PsO) was superior to that of psoriasis with arthritis (PsA) by methotrexate Page 4/11 Table 2 The difference in blood lipid and cardiovascular risk parameters between psoriasis with and without psoriatic arthritis and healthy controls PsA (n = 136) HC 1(n = 136) PsA vs HC1 p-value PsO (n = 152) HC2 (n = 152) PsO vs HC2 p-value PsA vs PsO p-value ApoA1, g/L 1.08 (0.18) 1.21 (0.17) < .0001 1.07 (0.18) 1.20 (0.16) < .0001 .4496 ApoB, g/L 0.76 (0.15) 0.65 (0.13) < .0001 0.70 (0.17) 0.60 (0.12) < .0001 .0013 TC, mmol/L 4.95 (0.89) 4.82 (0.82) .1939 4.61 (0.89) 4.56 (0.71) .6033 .0013 TG, mmol/L 1.74 (1.13) 1.45 (1.07) .0347 1.57 (1.02) 1.29 (0.86) .0098 .1894 HDL-C, mmol/L 1.23 (0.32) 1.26 (0.26) .3967 1.21 (0.29) 1.27 (0.27) .037 .4731 LDL, mmol/L 3.06 (0.77) 2.83 (0.70) .0115 2.84 (0.81) 2.67 (0.65) .0405 .0229 Lp(a), mg/L 147.9 (158.0) 121.5 (138.8) .1443 139.6 (175.2) 125.0 (150.0) .4364 .6747 ApoB: ApoA1 ratio 0.72 (0.19) 0.55 (0.13) < .0001 0.67 (0.19) 0.51 (0.13) < .0001 .0208 TC: HDL ratio 4.21 (1.07) 3.94 (0.92) .0227 3.98 (1.03) 3.73 (0.92) .0261 .0603 LDL: HDL ratio 2.62 (0.82) 2.31 (0.67) .0009 2.47 (0.85) 2.19 (0.71) .0022 .1309 LDL: ApoB ratio 4.03 (0.59) 4.37 (0.69) < .0001 4.08 (0.56) 4.41 (0.55) < .0001 .4869 Abbreviation: ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; LDL: low-density lipoprotein; Lp(a), lipoprotein A; PsA: psoriatic arthritis; PsO, psoriasis without arthritis; TC, total cholesterol; TG, triglyceride; Mann-Whitney test, unpaired t test, unpaired t test with Welch correction were used when appropriate. P < .025 is considered to be statistically significant. ciation of the effect of MTX on lipid profiles and cardiovascular risk parameters with psoriasis subtype and sex Mann-Whitney test, unpaired t test, unpaired t test with Welch correction were used when appropriate. P < .025 is considered to be statistically significant. ein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; LDL: low-density sA: psoriatic arthritis; PsO, psoriasis without arthritis; TC, total cholesterol; TG, triglyceride; ssociation of the effect of MTX on lipid profiles and cardiovascular risk parameters with psoriasis subtype and se Table 3 The effect of methotrexate on lipid profiles and cardiovascular risk parameters in psoriatic patients with and without arthritis PsA (n = 136, 0W) PsA (n = 136, 12W) PsA (0W vs 12W) p-value PsO (n = 152,0W) PsO (n = 152,12W) PsO (0W vs 12W) p-value ApoA1, g/L 1.08 (0.18) 1.07 (0.17) .1026 1.07 (0.18) 1.03 (0.15) .0001 ApoB, g/L 0.76 (0.15) 0.72 (0.15) .0003 0.70 (0.17) 0.64 (0.16) < .0001 TC, mmol/L 4.95 (0.89) 4.75 (0.89) .0007 4.61 (0.89) 4.39 (0.90) < .0001 TG, mmol/L 1.74 (1.13) 1.61 (0.96) .0410 1.57 (1.02) 1.63 (1.86) .6930 HDL-C, mmol/L 1.23 (0.32) 1.20 (0.30) .0370 1.21 (0.29) 1.17 (0.27) .0110 LDL, mmol/L 3.06 (0.77) 2.96 (0.76) .0632 2.84 (0.81) 2.68 (0.81) .0001 LPA, mg/L 147.9 (158.0) 137.1 (149.5) .0055 139.6 (175.2) 127.7 (173.9) < .0001 ApoB: ApoA1 ratio 0.72 (0.19) 0.69 (0.17) .0076 0.67 (0.19) 0.64 (0.18) .0011 TC: HDL ratio 4.21 (1.07) 4.13 (0.98) .1303 3.98 (1.03) 3.90 (0.18) .1121 LDL: HDL ratio 2.62 (0.82) 2.59 (0.79) .5030 2.47 (0.85) 2.40 (0.86) .0551 LDL: ApoB ratio 4.03 (0.59) 4.11 (0.58) .0250 4.08 (0.56) 4.14 (0.59) .0464 Abbreviation: ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; LDL: low-density lipoprotein; Lp(a), lipoprotein A; PsA: psoriatic arthritis; PsO, psoriasis without arthritis; TC, total cholesterol; TG, triglyceride; Paired t test, Wilcoxon matched pairs test, Mann-Whitney test, unpaired t test, or unpaired t test with Welch correction were used when appropriate. As shown in Table 5, stepwise regression analysis demonstrated that serum hCRP level was positively correlated with PASI score at baseline (p = .000), ApoB/ApoA1 ratio (p = .011), BMI (p = .003), and smoking (p = .015). Serum ApoA1 level was positively related with age (p = .006), negatively associated with sex (p = .015), weight (p = .004), hCRP (p = .006). Serum HDL level was negatively correlated with sex (p = .000) and weight (p = .014). Serum ApoB level was positively associated with diastolic blood pressure (p = .044), the concomitant of arthritis (p = .001) and diabetes (p = .006). Serum TC level was negatively related with sex (p = .031), and positively correlated with age (p = .000), diastolic blood pressure (p = .015), and the concomitant of arthritis (p = .002). Serum LDL level was positively associated with diastolic blood pressure (p = .003), and age at disease onset (p = .014). Serum TG level was related with diabetes (p = .000), and BMI (p = .001). ApoB/ApoA1 ratio was positively correlated with the concomitant of arthritis (p = .019), hCRP (p = .036), diastolic blood pressure (p = .014), and weight (p = .001). TC/HDL-C ratio was positively associated with sex (p = .006), diabetes (p = .008), diastolic blood pressure (p = .006), and BMI (p = .000). LDL/HDL ratio was positively related with diastolic blood pressure (p = .001), and weight (p = .000). LDL/ApoB was negatively correlated with the concomitant of hypertension (p = .021). Paired t test, or Wilcoxon matched pairs test were used when appropriate. P < .025 is considered to be statistically significant. Abbreviation: ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; LDL: low-density lipoprotein; Lp(a), lipoprotein A; TC, total cholesterol; TG, triglyceride; The association between the inflammatory marker hCRP, lipid profiles and cardiovascular risk parameters in psoriatic patients Abbreviation: ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprot lipoprotein; Lp(a), lipoprotein A; TC, total cholesterol; TG, triglyceride; ssociation of the effect of MTX on lipid profiles and cardiovascular risk parameters with psoriasis subtype and se P < .025 is considered to be statistically significant Page 5/11 Page 5/11 Table 4 The effect of methotrexate on lipid profiles and cardiovascular risk parameters in male and female psoriatic patients Male (n = 189, 0W) Male (n = 189, 12W) Male (0W vs 12W) p-value Female (n = 99,0W) Female (n = 99,12W) Female (0W vs 12W ) p-value Male vs Female p-value ApoA1, g/L 1.03 (0.16) 1.00 (0.13) .0002 1.16 (0.19) 1.14 (0.17) .1195 < .0001 ApoB, g/L 0.73 (0.17) 0.67 (0.15) < .0001 0.72 (0.17) 0.69 (0.19) .0214 .7756 TC, mmol/L 4.68 (0.85) 4.45 (0.83) < .0001 4.94 (0.98) 4.76 (1.03) .0083 .0241 TG, mmol/L 1.69 (1.06) 1.68 (1.66) .1256 1.57 (1.10) 1.50 (1.14) .1126 .3815 HDL-C, mmol/L 1.14 (0.26) 1.10 (0.23) .0029 1.37 (0.32) 1.34 (0.31) .1519 < .0001 LDL, mmol/L 2.94 (0.79) 2.79 (0.74) .0001 2.95 (0.81) 2.87 (0.89) .1342 .8775 Lp(a), mg/L 129.6 (148.6) 119.7 (143.9) .0076 170.1 (195.7) 156.0 (191.8) .0014 .0399 ApoB: ApoA1 ratio 0.72 (0.19) 0.69 (0.17) .0001 0.64 (0.17) 0.62 (0.17) .0762 .0004 TC: HDL-C ratio 4.26 (1.04) 4.18 (1.01) .0512 3.76 (1.00) 3.68 (0.97) .234 < .0001 LDL: HDL-C ratio 2.68 (0.84) 2.62 (0.82) .1057 2.27 (0.77) 2.23 (0.79) .4272 < .0001 LDL: ApoB ratio 4.04 (0.56) 4.13 (0.56) .0019 4.09 (0.61) 4.13 (0.64) .4161 .2722 Abbreviation: ApoA1 apolipoprotein A1; ApoB apolipoprotein B; HC healthy controls; HDL-C high-density lipoprotein-cholesterol; LDL: low-density Abbreviation: ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; LDL: low-density lipoprotein; Lp(a), lipoprotein A; TC, total cholesterol; TG, triglyceride; Paired t test, or Wilcoxon matched pairs test were used when appropriate. P < .025 is considered to be statistically significant. ssociation of the effect of MTX on lipid profiles and cardiovascular risk parameters with psoriasis subtype and se he association between the inflammatory marker hCRP, lipid profiles and cardiovascular risk parameters in psoriat Page 6/11 Table 5 d di Table 5 The association between inflammatory marker hCRP and lipid profiles and cardiovascular risk parameters and clinic characteristics of patients with psoria Univariate analysis stepwise regression analysis Predictors p-value B 95%CI for B p-value B 95%CI for B hCRP PASI score at baseline .000 0.142 (0.084-0.200) .000 0.144 (0.083–0.205) BMI .001 0.203 (0.079–0.328) .003 0.195 (0.065–0.324) ApoB/ApoA1 .000 4.009 (1.777–6.241) .011 3.153 (0.746–5.56) Smoking .022 0.821 (0.119–1.522) .015 0.841 (0.165–1.518) ApoA1 Sex .000 -0.128 (-0.129–0.086) .001 -0.091 (-0.142–0.039) Age .000 0.003 (0.002–0.004) .006 0.002 (0.001–0.004) Weight .000 -0.005 (-0.006–0.003) .004 -0.003 (-0.005–0.001) hCRP .003 -0.012 (-0.02–0.004) .006 -0.011 (-0.018–0.003) HDL Sex .000 -0.228 (-0.297–0.159) .000 -0.008 (-0.011–0.005) Weight .000 -0.010 (-0.013–0.008) .014 -0.096 (-0.173–0.019) ApoB diabstolic blood pressure .003 0.003 (0.001–0.005) .044 0.002 (0.000-0.004) arthritis .001 0.063 (0.025–0.101) .001 0.084 (0.036–0.131) Diabetes .001 0.081 (0.032–0.130) .006 0.090 (0.026–0.154) TC Sex .024 -0.252 (-0.471–0.033) .031 -0.253 (0.483–0.023) Age .000 0.017 (0.011–0.024) .000 0.016 (0.009–0.024) Diastolic blood pressure .013 0.013 (0.003–0.023) .015 0.012 (0.002–0.021) Arthritis .001 0.339 (0.133–0.545) .002 0.225 (0.002–0.448) LDL Diastolic blood pressure .001 0.015 (0.006–0.024) .003 0.014 (0.005–0.023) Age at disease onset .010 0.008 (0.002–0.013) .014 0.008 (0.002–0.015) TG Diabetes .000 0.657 (0.344–0.970) .000 0.604 (0.025–0.095) BMI .000 0.068 (0.033–0.103) .001 0.060 (0.025–0.095) ApoB/ApoA1 Arthritis .021 0.052 (0.008–0.096) .019 0.066 (0.011–0.120) hCRP .000 0.015 (0.007–0.023) .036 0.009 (0.001–0.018) Diastolic blood pressure .000 0.004 (0.002–0.006) .014 0.003 (0.001–0.005) Weight .000 0.005 (0.004–0.007) .001 0.004 (0.001–0.006) TC/HDL-C Sex .000 0.504 (0.253–0.754) .006 0.016 (0.005–0.027) Diabetes .001 0.516 (0.207–0.825) .008 0.460 (0.121–0.799) Diastolic blood pressure .000 0.024 (0.013–0.036) .006 0.016 (0.005–0.027) BMI .000 0.109 (0.077–0.142) .000 0.087 (0.050–0.125) LDL/HDL-C Diastolic blood pressure .000 0.021 (0.012–0.030) .001 0.019 (0.008–0.030) Weight .000 0.025 (0.018–0.032) .000 0.020 (0.010–0.030) LDL/ApoB Hypertension .016 -0.172 (-0.310–0.033) .021 -0.168 (-0.310–0.026) Abbreviation: ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; HC, healthy controls; HDL-C, high-density lipoprotein-cholesterol; LDL: low-density lipoprotein; Lp(a), lipoprotein A; TC, total cholesterol; TG, triglyceride; Discussion In particular, PsA and severe psoriasis were associated with a greater odds of hypertension.22 Psoriatic patients also have increased renin- angiotensin system activity, vascular damage, and oxidative stress linked to psoriasis and hypertension.23–25 In addition, we found that PsA patients had significantly higher levels of ApoB, TC, and LDL and a higher ApoB/ApoA1 ratio than PsO patients, which is in accordance with a previous report that PsA patients had higher burdens of specific comorbid disease than PsO patients. 26 Serum ApoB and TC levels were positively associated with arthritis after adjusting for sex, age, weight, BMI, disease duration, PASI and BSA score, diabetes, and hypertension. Our results on a higher ApoB/ApoA1 ratio in PsA differ from a recent study, which reported a higher ApoA/ApoB ratio in PsA compared to PsO. 27 Discrepancy in the study population might explain these results, since only age- but not sex-matched healthy controls were investigated in the former study. Indeed, our study found sex was also an import factor influencing lipid profiles. Male patients had significantly lower levels of TC, especially anti-atherogenic lipid profiles such as high- density lipoprotein HDL-C and ApoA1 than female patients. The ratio of ApoB/ApoA1 was significantly higher in male patients than in female patients, which was consistent with the epidemiology of cardiac and vascular disease whereby the age-adjusted CVD mortality and morbidity rates are highest in men than in women.28 The effects of MTX on lipid profiles and CVD are controversial. One meta-analysis showed that use of MTX predicted a reduction of 20% in the risk of cardiovascular events. 29 However, another study found that there was no effect of MTX on the levels of HDL, LDL, TG, TC, IL-1β, IL-6 and CRP. Low-dose MTX did not result in fewer cardiovascular events among patients with stable atherosclerosis than placebo. But this study did not stratify the patients by sex. 30 In our study, when patients were stratified by PsO and PsA, MTX significantly downregulated the levels of serum ApoB, TC, HDL-C, Lp(a), as well as the ratios of ApoB/ApoA1 and LDL/ApoB in patients with PsO and PsA. Furthermore, MTX differentially reduced the levels of serum ApoA1 and LDL in patients with PsO, and decreased serum TG level in patients with PsA. Discussion Our results demonstrate that PsA and PsO patients have significantly higher levels of pro-atherogenic lipids such as ApoB and LDL and lower levels of anti- atherogenic lipids such as ApoA1 than sex and age-matched healthy controls. The ratios of ApoB/ApoA1 and LDL/HDL-C were also significantly higher in PsA and PsO patients than in healthy controls. The ApoB/ApoA1 ratio is a strong and new risk factor for cardiovascular disease (CVD).15 Data from Indian patients with acute myocardial infarction (AMI) demonstrated that the ApoB/ApoA1 ratio was a better discriminator of coronary artery disease (CAD) risk than Page 7/11 Page 7/11 other conventional lipid ratios including the ratio of TC/HDL-C and LDL/HDL-C.16 Moreover, the ApoB/ApoA1 ratio was associated with femoral artery atherosclerosis as measured both as intima-media thickness and plaque occurrence;17 and carotid artery atherosclerosis measured by intima-media thickness. 18 A higher ApoB/ApoA ratio implies that more cholesterol is likely to be deposited in the arterial wall thereby provoking the atherogenesis.19 Our results are in accordance with previous observation that psoriatic patients had a higher prevalence of ischemic heart disease (3.3% vs 1.8%, OR 1.87) and vascular cerebral accidents (1.8% vs 1.2%, OR 1.55).20 other conventional lipid ratios including the ratio of TC/HDL-C and LDL/HDL-C.16 Moreover, the ApoB/ApoA1 ratio was associated with femoral artery atherosclerosis as measured both as intima-media thickness and plaque occurrence;17 and carotid artery atherosclerosis measured by intima-media thickness. 18 A higher ApoB/ApoA ratio implies that more cholesterol is likely to be deposited in the arterial wall thereby provoking the atherogenesis.19 Our results are in accordance with previous observation that psoriatic patients had a higher prevalence of ischemic heart disease (3.3% vs 1.8%, OR 1.87) and vascular cerebral accidents (1.8% vs 1.2%, OR 1.55).20 We report a significantly higher percentage of hypertension and hypercholesterolemia in PsA patients than PsO patients probably because the PsA cohort was older at disease onset with longer disease duration.21 Previous studies demonstrated that psoriatic patients have a greater prevalence and incidence of hypertension. Discussion When patients were stratified by sex, MTX significantly downregulated the levels of serum ApoB, TC, and Lp(a) in both male and female patients with psoriasis, and differentially reduced the levels of serum ApoA1, HDL-C, LDL, and ratios of ApoB/ApoA1 and LDL/ApoB in male patients with psoriasis, but not in female patients. Therefore, the decrease of ApoB, TC and Lp(a) by MTX was not related with sex and psoriasis subtype. However, the effect of MTX on some lipid profiles (ApoA1, HDL-C, LDL, TG) was associated with sex and psoriasis subtype. The mechanism remains to be clarified. Our another study also found MTX treatement significantly reduced the levels of serum hCRP and blood pressure in male patients, not in female patints. This indicates that the downregulation of MTX on lipid profiles was related with anti-inflammation. It has been reported that androgens can enhance the anti-inflammatory effects of MTX, which might be the main reason why the anti-inflammatory effect and downregulation of lipid profiles of MTX were more obvious in male patients than in female patients.31 The association between inflammation and lipid profiles was further analyzed in this study. Our results demonstrated that the inflammatory biomarker hCRP was not only positively correlated with disease severity (PASI score) at baseline, but was also positively associated with ApoB/ApoA1. In addition, high BMI and smoking is known to increase serum hCRP level. This might be one reason why men had significantly lower levels of anti-atherogenic lipid profiles. The ratios of ApoB/ApoA1, TC/HDL, LDL/HDL-C, LDL/ApoB were all regarded as cardiovascular risk parameters. But only ApoB/ApoA1 was positively correlated with inflammatory marker hCRP and the concomitant of arthritis, and they were all associated with diastolic blood pressure or the concomitant of hypertension, weight or BMI in a stepwise regression analysis. In addition, TC/HDL-C ratio was positively related with the concomitant of diabetes. Therefore, ApoB/ApoA1 ratio is the best biomarker to predict the risk of CVD. 32 In addition, we further analyzed the association of lipid profiles with clinic characteristics of psoriasis and concomitant disease. Our data showed that ApoA1 level was negatively associated with hCRP, not HDL level. This indicates that ApoA1 is more sensitive to inflammation than HDL-C. Discussion Another study also found that serum ApoA1 level showed a strong negative correlation with some markers of systemic inflammation including serum CRP and interleukin (IL)-8 levels and blood neutrophil count.33 ApoB is the best marker to predict concomitant disease, which was positively correlated with diastolic blood pressure, the concomitant of diabetes and arthritis. ApoB is a key component of atherogenic lipids and a more accurate measure of cardiovascular risk than LDL or non HDL-C. ApoB was reported to aggravate arthritis by eliciting the production of TNF-α, IL-1β, and IL-6 through p38 mitogen-activated protein kinase and NF-κB pathways. ApoB is an important independent immune-modulator that links lipid metabolism with local and systemic inflammatory responses.34 TC and LDL were positively associated with diastolic blood pressure, and TG was positively correlated with diabetes. It has been reported that TC might correlate to arterial stiffness35 and increase the lifetime risk of coronary heart disease mortality. 36 Diabetes was reported to reduce HDL cholesterol, a predominance of small dense LDL particles, and elevated triglyceride levels. 37 Therefore, the levels of lipid profiles were related with psoriasis and its concomitant disease. Conclusions In conclusion, inflammation in psoriasis can increase serum hCRP levels, resulting in the reduction of anti-atherogenic lipid ApoA1 and increase of pro- atherogenic lipid ApoB. ApoB and ApoB/ApoA1 ratio were the best biomarkers to predict inflammation and concomitant disease, especially CVD. MTX decreased pro-atherogenic lipid profiles as well as anti-atherogenic lipid profiles. The ratio of ApoB to ApoA1 was significantly downregulated in male patients with psoriasis after MTX treatment, indicating that the role of MTX on CVD and other concomitant disease might be related to sex. Page 8/11 Page 8/11 Bing Wang Bing Wang Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing- original draft, Writing-review & editing Hui Deng Data curation, Formal analysis, Methodology, Resources, Software, Validation, Visualization, Writing-original draft ata curation, Formal analysis, Methodology, Resources, Software, Validation, Visualization, Writing-original draft H Zhenghua Zhang Data curation, Funding acquisition, Investigation, Project administration, Software, Supervision, Validation, Visualization, Writing-review & editing k i kexiang yan Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing original draft, Writing-review & editing Acknowledgements： We thank Jianfeng Luo for statistical assistance in this study. Acknowledgements： We thank Jianfeng Luo for statistical assistance in this study. Authors' information ：Bing Wang, Hui Deng, and Yao Hu contribute equally to this article. Authors' information ：Bing Wang, Hui Deng, and Yao Hu contribute equally to this article. Corresponding authors: Kexiang Yan and Zhenghua Zhang Corresponding authors: Kexiang Yan and Zhenghua Zhang Institute of Dermatology and Department of Dermatology, Huashan Hospital, Fudan University, No. 12 Middle Wulumuqi Road, Shanghai 200040, China Tel :+ 86 13501748188 Tel.:+ 86 13501748188 Fax: +86 21 52887782 Email: ykx2292002@aliyun.com and verzhang@foxmail.com Declarations Declarations Ethical Approval and Consent to participate：MTX201501 Consent for publication：Yes Availability of supporting data ：Yes Competing interests：None declared. Funding：This study was financially supported by the National Natural Science Fou of SHDC (Nos. SHDC2020CR6022 and SHDC2020CR1014B). Authors' contributions： Bing Wang Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Pro original draft, Writing-review & editing Hui Deng Data curation, Formal analysis, Methodology, Resources, Software, Validation, Vis Yao Hu Data curation, Formal analysis, Methodology, Resources, Software, Validation, Vis Ling Han Data curation, Resources, Validation, Visualization Qiong Huang Data curation, Resources, Validation, Visualization Xu Fang Conceptualization, Data curation, Resources, Validation, Visualization Ke Yang Data curation, Resources, Validation, Visualization Siyuan Wu Data curation, Resources, Validation, Visualization Zhizhong Zheng Conceptualization, Investigation, Supervision Yawalkar Nikhil Writing-review & editing Zhenghua Zhang Data curation, Funding acquisition, Investigation, Project administration, Software kexiang yan Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, P original draft, Writing-review & editing Acknowledgements： We thank Jianfeng Luo for statistical assistance in this study Authors' information ：Bing Wang, Hui Deng, and Yao Hu contribute equally to this Declarations Ethical Approval and Consent to participate：MTX201501 Consent for publication：Yes Availability of supporting data ：Yes Competing interests：None declared. Funding：This study was financially supported by the National Natural S of SHDC (Nos. SHDC2020CR6022 and SHDC2020CR1014B). Authors' contributions： Bing Wang Conceptualization, Data curation, Formal analysis, Investigation, Metho original draft, Writing-review & editing Hui Deng Data curation, Formal analysis, Methodology, Resources, Software, Vali Yao Hu Data curation, Formal analysis, Methodology, Resources, Software, Vali Ling Han Data curation, Resources, Validation, Visualization Qiong Huang Data curation, Resources, Validation, Visualization Xu Fang Conceptualization, Data curation, Resources, Validation, Visualization Ke Yang Data curation, Resources, Validation, Visualization Siyuan Wu Data curation, Resources, Validation, Visualization Zhizhong Zheng Conceptualization, Investigation, Supervision Yawalkar Nikhil Writing-review & editing Zhenghua Zhang Data curation, Funding acquisition, Investigation, Project administration kexiang yan Data curation, Formal analysis, Funding acquisition, Investigation, Meth original draft, Writing-review & editing Acknowledgements： We thank Jianfeng Luo for statistical assistance i Authors' information ：Bing Wang, Hui Deng, and Yao Hu contribute eq Corresponding authors: Kexiang Yan and Zhenghua Zhang Institute of Dermatology and Department of Dermatology, Huashan Ho Tel.:+ 86 13501748188 Fax: +86 21 52887782 Email: ykx2292002@aliyun.com and verzhang@foxmail.com Abbreviations Competing interests：None declared. Funding：This study was financially supported by the National Natural Science Foundation of China (Nos. 81773322, 81673054), and Clinical Research Plan of SHDC (Nos. SHDC2020CR6022 and SHDC2020CR1014B). A th ' t ib ti ： Abbreviations ApoA1: apolipoprotein A1 ApoA1: apolipoprotein A1 ApoB: apolipoprotein B BMI: body mass index BSA: body surface area CVD: cardiovascular disease HC: healthy control Page 9/11 Page 9/11 HDL-C: high-density lipoprotein-cholesterol Interleukin: IL TG: triglyceride TNF: tumor necrosis factor References Evaluation of the atherogenic tendency of lipids and lipoprotein content and their relationships with oxidant- antioxidant system in patients with psoriasis. Clin Chim Acta. 2003;328:71–82. 11. Vanizor Kural B, Orem A, Cimsit G, et al. Evaluation of the atherogenic tendency of lipids and lipoprotein content and their relationships with oxidant- antioxidant system in patients with psoriasis. Clin Chim Acta. 2003;328:71–82. 12. Chin YY, Yu HS, Li WC, et al. 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https://openalex.org/W2951599433	https://hal.science/hal-02293213/file/GOFS.pdf	French	null	Impact des représentations du sang menstruel sur le choix contraceptif des femmes	Gynécologie obstétrique fertilité & sénologie	2,019	cc-by	9,723	Impact des représentations du sang menstruel sur le choix contraceptif des femmes S. Fernandez-Sala, R. Rousseau-Durand, P.-E. Morange, J. Chiaroni, B. Courbiere To cite this version: S. Fernandez-Sala, R. Rousseau-Durand, P.-E. Morange, J. Chiaroni, B. Courbiere. Impact des représentations du sang menstruel sur le choix contraceptif des femmes. Gynécologie Obstétrique Fertilité & Sénologie, 2019, 47 (9), pp.662-671. 10.1016/j.gofs.2019.06.001. hal-02293213 Distributed under a Creative Commons Attribution 4.0 International License of menstrual blood self-representation on contraceptive choice of women S. Fernandez-Sala a,, R. Rousseau-Durand a, P.-E. Morange b,c, J. Chiaroni d,e, B. C a Aix Marseille universite´, De´partement Universitaire de Me´decine Ge´ne´rale, 13005, Ma b Laboratoire d’he´matologie, hoˆpital de La Timone, AP–HM, 13005 Marseille, France g p c Aix Marseille universite´, Inserm, Inra, C2VN, 13005 Marseille, France d E´tablissement franc¸ais du sang PACA Corse, biologie des groupes sanguins, Marseille, France e Aix Marseille universite´, CNRS, EFS, ADES, « Biologie des Groupes Sanguins », 13005 Marseille, France e Aix Marseille universite´, CNRS, EFS, ADES, « Biologie des Groupes Sanguins », 13005 Marseille, France f AP–HM, Poˆle Femmes–Parents-Enfants, Service de Gyne´cologie-Obste´trique et Centre d’AMP,Hoˆpital de La Conception, 13005 Marseille, France g Aix Marseille Universite´, Avignon Universite´, CNRS, IRD, IMBE, 13005, Marseille, France f AP–HM, Poˆle Femmes–Parents-Enfants, Service de Gyne´cologie-Obste´trique et Centre d’AMP,Hoˆpital de La Conception, 13005 Marseille, France g Aix Marseille Universite´, Avignon Universite´, CNRS, IRD, IMBE, 13005, Marseille, France Auteur correspondant. Adresse e-mail : dr.sylviefernandez@gmail.com (S. Fernandez-Sala). R E´ S U M E´ Objectifs. – E´ tudier les repre´sentations psychiques et le ve´cu du sang menstruel chez des femmes et leur impact sur le choix d’une me´thode contraceptive, avec ou sans he´morragie de privation. Me´thodes. – E´tude qualitative par entretiens semi-dirige´s aupre`s de femmes majeures sous contraception. Mots cle´s : Me´narche Menstruations Ame´norrhe´e Contraception Expe´rience Re´sultats. – Vingt-trois entretiens ont e´te´ re´alise´s parmi des femmes d’aˆge et de classes socioe´cono- miques varie´s. Trois the`mes ont e´te´ aborde´s : l’expe´rience de la me´narche, la repre´sentation et le ve´cu du sang menstruel, et la repre´sentation et le ve´cu de l’ame´norrhe´e induite par une contraception. La me´narche a e´te´ une expe´rience ne´gative et de´cisive dans les repre´sentations et le ve´cu du sang menstruel pour la majorite´ d’entre elles. Concernant le saignement menstruel, deux proﬁls de groupes de femmes ont e´te´ observe´s. Celles ayant une repre´sentation positive du sang menstruel le conside´raient comme ne´cessaire a` la puriﬁcation de leur corps ainsi qu’a` la procre´ation et e´taient re´ticentes a` l’ide´e d’une ame´norrhe´e induite par leur contraception. Celles ayant une repre´sentation ne´gative du sang menstruel le conside´raient comme une source de souffrance physique et morale et acceptaient plus facilement l’ide´e d’avoir une ame´norrhe´e induite par leur contraception, l’ame´norrhe´e e´tant conside´re´e alors comme un traitement ou une libe´ration. Certains facteurs e´volutifs tels que l’aˆge et la situation familiale des femmes inﬂuenc¸aient leurs repre´sentations du sang menstruel et de l’ame´norrhe´e induite. Conclusion. – Le choix d’une ame´norrhe´e induite ou non par la contraception semble eˆtre propre a` Conclusion. – Le choix d’une ame´norrhe´e induite ou non par la contraception semble eˆtre propre a` chaque femme avec des repre´sentations psychiques du sang menstruel inde´pendantes de la classe socioe´conomique. Les re´sultats de cette e´tude mettent en e´vidence l’effet de la repre´sentation et du ve´cu du sang menstruel d’une femme sur son choix contraceptif. ´ ´ HAL Id: hal-02293213 https://hal.science/hal-02293213v1 Submitted on 20 Sep 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1. Introduction Ce n’est qu’a` partir de la deuxie`me moitie´ du XXe sie`cle, avec la de´couverte du roˆle des œstroge`nes, de la progeste´rone, de l’ovulation et des hormones hypotahlamo-hypophysaires, que les menstruations ont pu eˆtre explique´es par le cycle hormonal lie´ a` l’ovulation et aux modiﬁcations de l’endome`tre, apportant une explication scientiﬁque a` la survenue d’un saignement cyclique menstruel chez les femmes. Au cours de l’antiquite´, le sang menstruel avait une fonction puriﬁcatrice, repre´sentant un signe de bonne sante´ et maintenait l’e´quilibre entre sante´ et maladie [1]. L’ame´norrhe´e e´tait a` l’origine de multiples maladies, de douleurs, de ste´rilite´ ou de de´ce`s. Le sang menstruel e´tait le symbole de la fonction procre´atrice de la femme, de sa capacite´ a` se reproduire, soit selon Hippocrate, de par sa fonction de semence fe´minine, soit selon Aristote, de par sa fonction nourricie`re, permettant le de´veloppement de l’embryon cre´e par la seule semence de l’homme. Paradoxalement, la femme menstrue´e e´tait crainte. Le sang menstruel, impur, e´tait conside´re´ comme une marque de l’infe´riorite´ « naturelle » des femmes par rapport aux hommes, et Pine l’ancien conside´rait que les femmes menstrue´es e´tait nocives pour leur environnement (hommes, animaux, plantes et aliments) [2]. Au cours du Moyen Aˆge, le sang menstruel avait toujours ce caracte`re ambigu entre son roˆle be´ne´ﬁque, utile a` la procre´ation et ne´faste du fait de son impurete´ contagieuse. Pour le corps me´dical, lors de la procre´ation, le sang menstruel e´tait en partie ne´cessaire a` l’alimentation du fœtus dans l’ute´rus et en partie transforme´ en lait puis mis en re´serve dans les seins a` l’intention du nouveau-ne´. Selon le Le´vitique, l’acte sexuel e´tait re´pre´hensible durant les menstruations et les croyances populaires conside´raient que les enfants roux et la le`pre e´tait les re´sultats de cette transgression, peurs corrobore´es par le discours me´dical de l’e´poque [3]. Durant le XIIIe sie`cle, Barthe´lemy l’Anglais re´actualisa les vieilles traditions d’Aristote, de Galien et de Pline l’Ancien, expliquant que les menstrues des femmes e´taient une e´vacuation de superﬂuite´s nocives, pouvant pro- voquer des le´sions mentales par les vapeurs qu’elles ge´ne`raient [4]. La femme e´tait alors toujours conside´re´e comme une version imparfaite de l’homme. Du XVIe au XVIIIe sie`cle, la grande majorite´ des me´decins conside´raient que l’e´coulement de sang menstruel e´tait ne´cessaire a` la bonne sante´ physique et psychique de la femme et pratiquaient des e´vacuations sanguines puriﬁcatrice via les saigne´es [5]. A B S T R A C T Ce n’est qu’en 1973, graˆce a` la circulaire Fontanet que les e´tablissements scolaires ont e´te´ oblige´s d’inclure au programme des cours sur la me´narche, la puberte´, la sexualite´ et la reproduction. A` la ﬁn du XXe sie`cle, la menstruation e´tait ainsi conside´re´e comme une condition de bonne sante´, me´canisme puriﬁant le corps des de´bris d’ovule et de muqueuse, mais e´galement comme un acce`s a` la fe´minite´, « pas de femmes sans re`gles ». Au de´but du XXIe sie`cle, pour beaucoup de femmes, la menstruation repre´sente un e´chec de la procre´ation et un signe de fe´condite´. De fac¸on caricaturale, la mission de la femme sur terre a longtemps e´te´ d’enfanter et pour cela elle doit eˆtre parfaitement re´gle´e. Ainsi, toute perturbation, retard ou absence des menstruations, e´tait conside´re´e comme une menace pour la sante´ et un e´chec personnel ayant des implications sociales. A B S T R A C T Keywords: Menarche Menstruations Amenorrhea Contraception Experience Objectives. – To study the psychic self-representations and experiences of menstrual blood in women and their impact on the choice of a contraceptive method, with or without amenorrhea. Methods. – Qualitative study based on semi-structured interviews with French women over age 18, under contraception. Results. – Twenty-three interviews were conducted with women of various ages and socio-economic classes. Three themes have been studied: the menarche experience, the representation and experience of menstrual blood, and the representation and experience of amenorrhea induced by contraception. Menarche has been a negative experience for most of them, and menarche is known to inﬂuence menstrual self-representation. About menstrual bleeding, two proﬁles of women could be described. Those with a positive self-representation of menstrual blood considered it necessary for the puriﬁcation of their bodies as well as for procreation and were reluctant to the idea of amenorrhea induced by their contraception. Those with a negative representation of menstrual blood considered it as a source of physical and mental suffering and accepted the idea of having amenorrhea induced by their contraception, amenorrhea being considered as a treatment or a release. Conclusion. – The choice of a contraception with or without a induced-amenorrhea seems to be speciﬁc to every woman and depends on there self-psychic representation of menstrual blood, independently from their socio-economic class. The results of this study highlighted the effect of women’s psychic representations and experience of menstrual blood on their contraceptive choice. 2019 El i M SAS All i h d l’homme, ou` les menstruations symbolisaient cette diffe´rence [7]. Au cours du XIXe sie`cle, les principaux de´bats me´dicaux concernant le sang menstruel e´taient relatifs a` l’ame´norrhe´e durant la grossesse, a` l’allaitement durant la menstruation et a` la nature de la femme menstrue´e. En 1878, des scientiﬁques afﬁrmaient la nocivite´ de la femme ayant ses menstrues sur les aliments [8]. La femme e´tait de´crite comme un eˆtre de nature essentiellement mauvaise et la criminalite´ fe´minine intimement lie´e a` la menstruation [9]. Une classiﬁcation des psychoses menstruelles regroupant la kleptomanie, la pyromanie, la dipsomanie, le de´lire religieux, la nymphomanie et la monomanie homicide a e´te´ e´tablie en 1890 [5]. Jusqu’a` la ﬁn du XIXe sie`cle, on pensait que l’ovulation avait lieu pendant la menstruation. 1. Introduction les caracte´ristiques des femmes interroge´es : l’aˆge, le nombre d’anne´es d’e´tude effectue´es, la couverture me´dicale, un ante´- ce´dent de grossesse non de´sire´e (avortement ou naissance non de´sire´e). Les origines ge´ographiques des femmes ont e´te´ conserve´es dans le descriptif de notre e´chantillon, car nous avons juge´ que celles-ci pouvaient eˆtre importantes dans le ve´cu ´ ´ i l d l ´ h d i transge´ne´rationnel de la me´narche et des menstruations ; p [ ] La contraception a comme particularite´ d’eˆtre a` la fois un traitement pre´ventif en e´vitant une grossesse non de´sire´e et curatif en cas de dysme´norrhe´e et/ou de me´norragies . Sa prescription ne´cessite une e´ducation the´rapeutique (adaptation des comporte- ments face a` une situation donne´e). En 2007, les femmes Franc¸aises en aˆge de procre´er repre´sentaient 23 % de la population ge´ne´rale. En 2012, 96,9 % des femmes entre 15 et 49 ans vivant en France me´tropolitaine, sexuellement actives avec des hommes, non ste´riles, ni enceintes et ne voulant pas d’enfant utilisaient une me´thode contraceptive [14]. D’apre`s l’INED, en 2007, le taux de grossesses « non pre´vues » (avortements, naissances non de´sire´es) e´tait estime´ a` 36 % du total des grossesses [15] et deux grossesses non pre´vues sur trois e´taient survenues alors qu’une me´thode contraceptive e´tait utilise´e [16]. Il apparaissait donc que l’efﬁcacite´ biologique d’un contraceptif ne serait pas le seul crite`re a` prendre en compte. En France, il existe une norme sociale contraceptive (usage du pre´servatif, puis de la pilule oestro-progestative lorsque la relation est stabilise´e et enﬁn dispositif intra-ute´rin une fois le nombre d’enfant de´sire´ atteint) ayant comme conse´quence la prescription de me´thodes contraceptives parfois inadapte´es aux conditions sociales, affectives et sexuelles des utilisatrices [17,18]. Si les mœurs e´voluent, les repre´sentations des menstruations demeurent encore un sujet peu aborde´ dans la sphe`re familiale et sociale. Les repre´sentations des menstruations, notamment dans la contraception, sont e´galement tre`s peu aborde´es par le corps me´dical . L’objectif de notre e´tude a e´te´ d’e´tudier l’impact des repre´sentations et le ve´cu de la me´narche, du sang menstruel et de l’ame´norrhe´e induite sur le choix contraceptif des femmes. 1. Introduction Au cours de l’e´poque moderne, le sang menstruel e´tait conside´re´ comme tabou a` cause de son lien avec la procre´ation, tout comme l’e´tait la semence masculine. Ce tabou a renforce´ l’ambivalence des perceptions de la femme re´gle´e, a` la fois impure et sacre´e [6]. Au tournant du XVIIIe sie`cle la femme se voit attribuer un sexe biologiquement diffe´rent de celui de Des anne´es 1960 a` nos jours, l’activisme menstruel donne une autre vision sur la nature de la pense´e et des actions fe´ministes [10]. Depuis les anne´es 2000, nous avons assiste´ a` une ve´ritable « re´volution menstruelle », avec un de´bat autour du sang menstruel, que ce soit en politique (de´bat sur le conge´ menstruel, remboursement des serviettes hygie´niques par la se´curite´ sociale), sur les re´seaux sociaux avec des artistes militantes exposant leur sang menstruel (ex. : photographies « beauty in blood » de Jen Lewis) ou me´dicalement, par le biais de l’ame´norrhe´e induite de fac¸on intentionnelle par certaines me´thodes contraceptives comme les dispositifs intra-ute´rin au le´vonorgestrel ou les pilules microprogestatives continues au de´sogestrel. L’activisme menstruel se positionne sur la sante´, l’environnement et le droit des consommateurs [10]. En France, les protections hygie´niques ont longtemps e´te´ taxe´es » a` 20 % et la TVA a e´te´ baisse´e a` 5,5 % en De´cembre 2015 suite a` une pe´tition mene´e par un collectif fe´ministe [11]. Et bien que les produits soient plus suˆrs, et ne pre´sentent pas de danger grave et imme´diat pour la sante´ des utilisatrices, il n’existe pas de re´glementation ou de normes ﬁxant la teneur maximale en substances chimiques pre´sentes dans les produits hygie´niques (allerge`nes, conservateurs, re´sidus de guide d’entretien pre´alablement e´labore´ et valide´ par le comite´ d’e´thique d’Aix-Marseille universite´ (2017-05-07-001) en vue d’e´tudier : pesticides, herbicide, dioxines, produits polluants organiques et compose´s organiques haloge´ne´s etc.) [10,12]. Les proce´de´s de fabrication des serviettes et tampons hygie´niques jetables (blanchiment et ste´rilisation) seraient des plus polluants avec une absence de recyclage (en moyenne 10 000–15 000 produits menstruels utilise´s dans la vie d’une femme, pour une de´gradation de 500 ans par produit) [13]. Les militantes radicales de l’activisme menstruel se positionne e´galement sur la nature du corps sexue´ de la femme de´tachant les menstruations du genre fe´minin, e´voquant les transsexuelles, les intersexue´es, les femmes ayant une ame´norrhe´e primaire. [7,10]. 3. Re´sultats Vingt-trois entretiens, nume´rote´s de E1 a` E23, ont e´te´ re´alise´s entre juillet 2015 et juillet 2017 (Tableau 1). Ces entretiens ont dure´ entre 14 et 60 minutes, avec une moyenne de 33 minutes. Les volontaires avaient entre 18 et 56 ans, pour une moyenne de 33, 8 ans. 2. Mate´riels et me´thodes Sur notre e´chantillon, onze femmes avaient moins de 35 ans. Ce groupe e´ tait compose´ de neuf femmes d’origine caucasienne, une femme ne´ e en France de parents africains et une femme africaine. Parmi elles, trois femmes be´ ne´ ﬁciaient d’une couver- ture me´dicale spe´ciﬁque. Trois femmes n’avaient pas le baccalaure´ at, quatre avaient effectue´ deux anne´ es d’e´ tude apre` s le baccalaure´ at et quatre avaient effectue´ trois ans ou plus apre` s le baccalaure´ at. Seule la me´thodologie qualitative permet d’e´tudier les repre´- sentations du sang menstruel et de l’ame´norrhe´e induite et leurs impacts sur le comportement contraceptif des femmes. Une e´tude qualitative par entretiens semi-dirige´s a e´te´ mene´e aupre`s de femmes sous contraception dans la re´gion Marseillaise. Les crite`res d’inclusion ont e´te´ des femmes majeures, sous contraception hormonale ou non. Les crite`res d’exclusion ont e´te´ les femmes mineures, sans contraception, enceintes ou me´nopau- se´es. Les entretiens ont e´te´ re´alise´s sur la base du volontariat. Aﬁn d’obtenir un e´chantillon varie´ de femmes sur le plan socio- e´conomique, nous avons effectue´ des entretiens dans divers lieux (3e, 4e, 5e, 6e, 7e, 15e arrondissement de Marseille, Vitrolles et Martigues). Ces entretiens ont e´te´ effectue´s a` l’occasion d’un rendez-vous chez le me´decin, en cabinet libe´ral de me´decine ge´ne´rale ou de gyne´cologie, en maison de´partementale de la solidarite´, ou a` l’occasion d’une hospitalisation dans un service de gyne´cologie. Concernant les femmes recrute´es par le biais de notre entourage, les entretiens ont e´te´ re´alise´s selon leurs convenances (sur leur lieu de travail, dans un parc, a` leur domicile, a` celui de l’enqueˆteur ou encore par visioconfe´rence). Tous les entretiens ont e´te´ nume´rote´s et anonymise´s, permettant d’instaurer plus facilement un climat de conﬁance favorable aux conﬁdences. Les entretiens ont e´te´ re´alise´s par la meˆme enqueˆtrice a` l’aide d’une Douze femmes avaient 35 ans ou plus. Le groupe e´tait compose´ de huit femmes d’origine caucasienne, une femme ne´e en France de parents africains, deux femmes africaines et femmes ne´e aux Antilles. Parmi elles, trois femmes be´ne´ﬁciaient d’une couverture me´dicale spe´ciﬁque, six femmes n’avaient pas le baccalaure´at, trois femmes avaient effectue´ deux anne´es apre`s le baccalaure´at et trois femmes avaient effectue´ 3 ans ou plus apre`s le baccalaure´at. 1. Introduction le ve´cu et les repre´sentations de la me´narche : l’aˆge, l’informa- tion rec¸ue pre´alablement ou lors de la me´narche, le ve´cu et e´ventuellement le changement de statut social ressenti ; le ve´cu et les repre´sentations du sang menstruel : la connaissance du fonctionnement biologique des menstruations et des he´morragies de privation et le ve´cu des saignements dans le quotidien ; le ve´cu et les repre´sentations de l’ame´norrhe´e induite e´ventuel- lement par une contraception ; les crite`res de choix dans une contraception : l’ame´norrhe´e induite ou la survenue d’une he´morragie de privation ; nous avons choisi comme facteur de confusion l’aˆge et la classe socioe´conomique ; le seuil d’aˆge a e´te´ e´tabli a` 35 ans : aˆge cle´ de survenue de facteur de risques cardiovasculaires et auquel la contraception estro- progestative doit eˆtre re´e´value´e ; la classe socioe´conomique a e´te´ de´termine´e par l’afﬁliation ou non a` une couverture me´dicale universelle ou une aide a` la comple´mentaire sante´. Le nombre d’entretiens ne´cessaire a` l’obtention de la saturation des donne´es conﬁrme´e par deux entretiens supple´mentaires a e´te´ de 23. 3.1. Le ve´cu et les repre´sentations de la me´narche Les femmes interroge´es ont de´clare´ n’avoir pre´sente´ aucun empressement a` eˆtre menstrue´es. E11, 27 ans, me´narche a` 11 ans : « J’e´tais pas dans l’expectation de les avoir ni, ha vivement, non pas du tout ». Au contraire, la me´narche a e´te´ ve´cue comme un e´ve`nement brutal, angoissant, douloureux, honteux et/ou contrai- gnant. E13, 28 ans, me´narche a` 11 ans : « J’e´tais pas preˆte, j’e´tais pas Tableau 1 Caracte´rist q Entretien Mode de recrutement Aˆge CMU ACS E´tudes Profession Origine culturelle Ante´ce´dent de grossesse non de´sire´e Dure´e de l’entretien E1 Gyne´cologue. Vitrolles 41 ans – BAC-BAC+2 Cadre Caucasienne – 21 minutes E2 Gyne´cologue. Vitrolles 35 ans – BAC-BAC+2 Responsable cuisine collective Caucasienne Oui 16 minutes E3 Entourage. Vitrolles 21 ans – BAC-BAC+2 Bibliothe´caire Caucasienne – 21 minutes E4 Ge´ne´raliste. Marseille 6e arrondissement 18 ans CMU < BAC Serveuse Caucasienne – 29 minutes E5 Ge´ne´raliste. Marseille 6e arrondissement 23 ans – BAC + 3 E´ tudiante en me´decine Caucasienne – 24 minutes E6 Ge´ne´raliste. Marseille 6e arrondissement 26 ans – BAC + 3 Attache´e de presse Caucasienne – 14 minutes E7 Entourage. Martigues 52 ans – BAC + 3 Secre´taire me´dicale Caucasienne – 37 minutes E8 Entourage. Marseille 4e arrondissement 22 ans CMU < BAC Aucune Caucasienne Oui 39 minutes E9 Entourage. Vitrolles 56 ans – BAC + 3 Directrice d’e´cole Caucasienne – 60 minutes E10 Entourage. Vitrolles 21 ans – BAC-BAC+2 E´ tudiante en arts plastiques Caucasienne – 45 minutes E11 Ge´ne´raliste. Marseille 6e arrondissement 27 ans – BAC + 3 Docteur en gestion Caucasienne – 51 minutes E12 Ge´ne´raliste. Marseille 6e arrondissement 38 ans ACS < BAC Aucune Caucasienne Oui 29 minutes E13 Entourage. Marseille 7e arrondissement 28 ans – BAC + 3 E´ tudiante en orthophonie Caucasienne – 27 minutes E14 Gyne´cologie. Marseille 15e arrondissement 44 ans – BAC-BAC+2 Secre´taire me´dicale Caucasienne – 29 minutes E15 Gyne´cologie. Marseille 15e arrondissement 36 ans – < BAC Aide-soignante Africaine – 54 minutes E16 Entourage. Marseille 5e arrondissement 48 ans – BAC + 3 Inﬁrmie`re Caucasienne Oui 53 minutes E17 MDS. Marseille 3e arrondissement 26 ans – BAC-BAC+2 E´ tudiante en droit Africaine Oui 29 minutes E18 MDS. Marseille 3e arrondissement 21 ans CMU < BAC Serveuse Africaine Oui 26 minutes E19 MDS. Marseille 6e arrondissement 49 ans CMU < BAC Aucune Caucasienne – 22 minutes E20 MDS. 3.1. Le ve´cu et les repre´sentations de la me´narche Marseille 6e arrondissement 48 ans CMU < BAC Aide a` la personne Antillaise Oui 40 minutes E21 MDS. Marseille 6e arrondissement 35 ans – < BAC Agent d’entretien Africaine Oui 32 minutes E22 MDS. Marseille 6e arrondissement 24 ans – BAC-BAC+2 Etude en droit Caucasienne – 25 minutes E23 MDS. Marseille 6e arrondissement 40 ans – < BAC Non pre´cise´ Africaine Oui 37 minutes BAC : baccalaure´at ; CMU : couverture me´dicale universelle ; ACS : aide comple´mentaire sante´ ; MDS : maison de la solidarite´. preˆte du tout, [Rires], pas du tout ». Une seule femme a parle´ de sa me´narche en des termes positifs. E18, 21 ans, me´narche a` 13 ans : « je me sentais. . .plus le´ger ». Pour certaines femmes, la me´narche a mate´rialise´ un changement de statut social, ressenti ou impose´ par le regard des autres. E12, 38 ans, me´narche a` 14 ans : « Mes Nana c’e´tait mon petit accessoire de femme dans mon sac a` main, j’e´tais devenue une femme ». E13, me´narche a` 11 ans : « Je me suis dit qu’on devait penser de moi que j’e´tais une femme, [. . .] C’e´tait vraiment d’eˆtre vue de´ja` un peu comme un objet sexuel [. . .] Mais je me sentais pas du tout comme c¸a, j’e´tais un be´be´ ». « Quatorze femmes ont rapporte´ qu’elles connaissaient l’existence des menstruations avant leur me´narche, connaissance allant de la simple information, E4, 18 ans, me´narche a` 9 ans « Je savais que les re`gles existaient on m’en avait de´ja` parle´. . . », a` une ve´ritable pre´paration, E17, 26 ans, me´narche a` 14 ans : « Moi j’ai eu le temps d’eˆtre bien pre´pare´e quand meˆme. J’ai eu aucune surprise ». Trois femmes ont exprime´ qu’elles ignoraient tous des menstruations avant leur me´narche, E7, 52 ans, me´narche a` 9 ans et demi « J’ai pas compris parce que moi j’e´tais pas avertie du tout [. . .] c’e´tait.. c¸a fait peur quand on est petite et qu’on est pas bien avertie ». La source de l’information, avant ou pendant la me´narche, avait e´te´ pour 9 femmes la me`re, pour 3 femmes la sœur, pour 4 femmes le programme scolaire ou les camarades d’e´cole de´ja` menstrue´es, pour 1 femme le pe`re, pour 1 femme la voisine. 3.1. Le ve´cu et les repre´sentations de la me´narche La connaissance de l’existence des menstruations associe´e a` une pre´paration a` la me´narche ont e´te´ des facteurs apaisants pour les femmes qui en ont be´ne´ﬁcie´es (Tableau 2). preˆte du tout, [Rires], pas du tout ». Une seule femme a parle´ de sa me´narche en des termes positifs. E18, 21 ans, me´narche a` 13 ans : « je me sentais. . .plus le´ger ». Pour certaines femmes, la me´narche a mate´rialise´ un changement de statut social, ressenti ou impose´ par le regard des autres. E12, 38 ans, me´narche a` 14 ans : « Mes Nana c’e´tait mon petit accessoire de femme dans mon sac a` main, j’e´tais devenue une femme ». E13, me´narche a` 11 ans : « Je me suis dit qu’on devait penser de moi que j’e´tais une femme, [. . .] C’e´tait vraiment d’eˆtre vue de´ja` un peu comme un objet sexuel [. . .] Mais je me sentais pas du tout comme c¸a, j’e´tais un be´be´ ». « Quatorze femmes ont rapporte´ qu’elles connaissaient l’existence des menstruations avant leur me´narche, connaissance allant de la simple information, E4, 18 ans, me´narche a` 9 ans « Je savais que les re`gles existaient on m’en avait de´ja` parle´. . . », a` une ve´ritable pre´paration, E17, 26 ans, me´narche a` 14 ans : « Moi j’ai eu le temps d’eˆtre bien pre´pare´e quand meˆme. J’ai eu aucune surprise ». Trois femmes ont exprime´ qu’elles ignoraient tous des menstruations avant leur me´narche, E7, 52 ans, me´narche a` 9 ans et demi « J’ai pas compris parce que moi j’e´tais pas avertie du tout [. . .] c’e´tait.. c¸a fait peur quand on est petite et qu’on est pas bien avertie ». La source de l’information, avant ou pendant la me´narche, avait e´te´ pour 9 femmes la me`re, pour 3 femmes la sœur, pour 4 femmes le programme scolaire ou les camarades d’e´cole de´ja` menstrue´es, 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche Le ve´cu et les repre´sentations du sang menstruel des femmes ont e´te´ inﬂuence´s par leur ve´cu de la me´narche, leur repre´senta- tion de l’origine du sang menstruel, leur entourage, leur aˆge et leur situation familiale. Deux the´ories se sont distingue´es chez les femmes ayant tente´ de donner une explication scientiﬁque a` leurs saignements. E13, 28 ans : « En descendant il {l’ovule} cre´e une irritation et arrache l’endome`tre, du coup, c’est c¸a qui fait que c¸a saigne ». E22, 24 ans : « Le corps produit du sang pendant trois semaines, puis on l’e´vacue la quatrie`me, c’est les re`gles ». De plus, la diffe´rence entre menstruations et he´morragies de privation n’a pas e´te´ comprise ou explique´e aux femmes, expliquant une totale confusion entre les deux phe´nome`nes. E22, 24 ans : « Le fonctionnement c’est pareil, c¸a oui ». Les repre´sentations de l’entourage, notamment du re´fe´rent fe´minin, semblent avoir inﬂuence´ les repre´sentations personnelles de ces femmes. E7, psychiques du sang menstruel psychiques de l’ame´norrhe´e induite par une contraception par une contraception d une contraception Non rapporte´ Dysme´norrhe´e Me´norragie Trouble de l’humeur Craintes latentes Sentiment de libe´ration Souhaite absolument une ame´norrhe´e the´rapeutique Naturel Absence de grossesse Utilite´. 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche on reste chez soi, on sort un minimum, [..] C¸a posait proble`me pour ma famille, pour mon mari, pour mes enfants.. ». Tableau 2 (Suite ) Entretien La me´narche Repre´sentations psychiques du sang menstruel Ve´cu des menstruations Repre´sentations psychiques de l’ame´norrhe´e induite par une contraception Ve´cu de l’ame´norrhe´e induite par une contraception Souhait inﬂuenc¸ant le choix d’une contraception Aˆge Ve´cu E21 13 ans Angoisse Pas d’information pre´alable. Information lors de la me´narche : scolaire Procre´ation Bonne sante´ Dysme´norrhe´e De´gouˆt Peur d’une accumulation de sang « ailleurs » Non informe´e des possibilite´s d’ame´norrhe´e induite Souhaite une ame´norrhe´e the´rapeutique E22 14 ans Changement de statut social De´phasage physique Angoisse Information et pre´paration a` la me´narche non rapporte´e Information lors de la me´narche : me`re Naturel Puriﬁcation De´toxiﬁant Bien-eˆtre ne´cessaire pour le corps Peur d’induire une ste´rilite´ Apparition de maladies notamment auto- immunes Craintes lie´es aux hormones Refus d’avoir une ame´norrhe´e induite Souhaite une contraception avec he´morragie de privation E23 15ans Changement de statut social De´phasage physique Pas d’information pre´alable. Information lors de la me´narche : voisine Naturel Puriﬁcation De´toxiﬁant Volonte´ divine Dysme´norrhe´e Me´no- me´trorragieSoulagement physique, me´te´orisme moins important Peur d’une qccumulation du sang "ailleurs" Craintes lie´esa` la nocivite´ des hormones Refus d’avoir une ame´norrhe´e induite Souhaite une contraception ave he´morragie de privation 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche Peu de contraintes associe´es Contre-nature Refus d’avoir une ame´norrhe´e induite Souhaite absolument une contraception avec he´morragie de privation Aucune utilite´ sous contraception Sentiment d’injustice par rapport aux hommes Dysme´norrhe´e Contraintes professionnelles Sentiment de liberte´ N’a pas eu l’opportunite´ d’eˆtre en ame´norrhe´e induite Souhaite une ame´norrhe´e the´rapeutique Non rapporte´ Dysme´norrhe´e Contrainte dans les loisirs sportifs Sentiment de liberte´ N’a pas eu l’opportunite´ d’eˆtre en ame´norrhe´e induite Souhaite une ame´norrhe´e the´rapeutique Procre´ation : marqueur de l’ovulation ; absence de grossesse Peu de contraintes associe´es De´calage entre le ressenti et les connaissances scientiﬁques Ame´norrhe´e ne repre´sentant pas d’inte´reˆt pour son quotidien Angoisse Souhaite une contraception avec he´morragie de privation Naturel Dysme´norrhe´e Contrainte dans les loisirs sportifs Non rapporte´ Sentiment de liberation Souhaite absolument une ame´norrhe´e the´rapeutique Naturel Jeunesse Procre´ation De´toxiﬁant Habitude De´calage entre le ressenti et les connaissances scientiﬁquesAngoisse Refus d’avoir une ame´norrhe´e induite Souhaite une contraception avec he´morragie de privation Aurait souhaite´ une ame´norrhe´e the´rapeutique si elle avait e´te´ plus jeune Absence de grossesse Sentiment d’injustice par rapport aux hommes Ste´rilite´ Accumulation de sang Apparition de maladies Refus d’avoir une ame´norrhe´e induite Souhaite absolument une contraception avec he´morragie de privation Naturel Dysme´norrhe´e Me´norragie Contraintes multiples Fe´minite´ (absence de protection hygie´nique) Sentiment de libe´ration Souhaite absolument une ame´norrhe´e the´rapeutique Naturel Appareil reproducteur fonctionnel Utilite´ Accumulation de sang et crainte de la putre´faction Apparition de maladies Refus d’avoir une ame´norrhe´e induite Souhaite absolument une contraception avec he´morragie de privation Non rapporte´ Sentiment d’injustice par rapport aux hommes Dysme´norrhe´e Contraintes multiples Absente´ismes Obligation me´dicale Bien-eˆtre Sentiment de liberation Souhaite absolument une ame´norrhe´e the´rapeutique ge entre le ti et les ssances ﬁquesAngoiss te´ mulation de san tion de malad ite´ (absence d Repre´sentations psychiques du sang menstruel Ve´cu des menstruations Repre´sentations psychiques de l’ame´norrhe´e induite par une contraception Ve´cu de l’ame´norrhe´e induite par une contraception Souhait inﬂuenc¸ant le choix d’une contraception Naturel Asthe´nie Trouble de l’humeur Non-respect du roˆle du corps Pratique Ame´norrhe´e limite´e dans le temps Souhaite une contraception avec he´morragie de privation Naturel Procre´ation Repe`re temporel Contraintes dans la sexualite´ Entourage : peur du cancer du sein Refus d’avoir une ame´norrhe´e induite Souhaite une contraception avec he´morragie de privation Procre´ation Obligation Avant : me´norragie Maintenant : moins abondante, habitude Peu de contraintes Non rapporte´ Refus d’avoir une ame´norrhe´e induite Souhaite une contraception avec he´morragie de privation Aurait souhaite´ une ame´norrhe´e the´rapeutique si elle avait e´te´ plus jeune Procre´ation Sentiment d’injustice par rapport aux hommes Dysme´norrhe´e sur endome´triose Syndrome pre´menstruel Asthe´nie Obligation me´dicale Peur d’une me´nopause artiﬁcielle Craintes lie´es aux hormones Peur d’induire une ste´rilite´ : risque surajoute´ a` celui d’une me´narche tardive Sentiment de libe´ration Souhaite absolument une ame´norrhe´e the´rapeutique Naturel Procre´ation Pas d’utilite´ lorsque le nombre d’enfant de´sire´ est atteint Obligatoire Me´no-me´trorragies Asthe´nie Ane´mie Contraintes vestimentaires et loisirs Obligation me´dicale d’une ame´norrhe´e the´rapeutique Bien-eˆtre Sentiment de libe´ration Souhaite absolument une ame´norrhe´e the´rapeutique Naturel Puriﬁcation De´toxiﬁant Repe`re temporel Ritualise´ par des soins corporels Habitude Contre-nature Malsaine Refus d’avoir une ame´norrhe´e induite Souhaite une contraception avec he´morragie de privation Naturel Puriﬁcation De´toxiﬁant Soulagement physique, me´te´orisme moins important Peur d’une accumulation de sang "ailleurs" Obligation me´dicale mal ve´cue Douleur et me´te´orisme abdominal Souhaite une contraception avec he´morragie de privation Naturel Procre´ation Sentiment d’injustice par rapport aux hommes Dysme´norrhe´e Migraine catame´niale Infection vaginale De´gouˆt Peur d’induire une ste´rilite´ Possible si le nombre d’enfant de´sire´ est atteint Sentiment de libe´ration Souhaite une ame´norrhe´e the´rapeutique Naturel Procre´ation Bonne sante´ Pas d’utilite´ lorsque le nombre d’enfant de´sire´ est atteint De´gouˆt Contraintes multiples Peur d’induire une ste´rilite´ Possible si le nombre d’enfant de´sire´ est atteint Sentiment de libe´ration Souhaite une ame´norrhe´e the´rapeutique Repre´sentations psychiques du sang menstruel Ve´cu des menstruations Repre´sentati psychiques d l’ame´norrhe´ par une cont Naturel Asthe´nie Trouble de l’humeur Non-respect corps Naturel Procre´ation Repe`re temporel Contraintes dans la sexualite´ Entourage : cancer du se Procre´ation Obligation Avant : me´norragie Maintenant : moins abondante, habitude Peu de contraintes Non rapport Procre´ation Sentiment d’injustice par rapport aux hommes Dysme´norrhe´e sur endome´triose Syndrome pre´menstruel Asthe´nie Obligation m Peur d’une m artiﬁcielle Craintes lie´e hormones Peur d’indui ste´rilite´ : ris 52 ans : « C’est du transmis de maman quoi. 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche On ne l’a donne´ mais c’est pas force´ment personnel ». Nous avons observe´ que la repre´sentation du sang menstruel des femmes est e´volutive et en rapport avec l’approche de la me´nopause ou du nombre d’enfant de´sire´ obtenu. E20, 48 ans : « Maintenant c’est bon, c¸a sert plus a` rien [. . .] j’ai eu mes enfants, on peut pas rester jeune toute la vie ». Que ce soit, des menstruations ou des he´morragies de privation sous contraception oestro-progestative, les femmes parlent de leur sang, au sens large, de leur roˆle et de leur ve´cu, positif ou ne´gatif. Dans le groupe des femmes ayant des repre´sentations et un ve´cu positifs, les femmes ont conside´re´ le sang menstruel, comme un e´ve`nement naturel, ayant un pouvoir puriﬁcateur, e´loignant certaines maladies et ayant un roˆle essentiel dans la procre´ation. E17, 26 ans : « Une femme n’est pas sale quand elle a ses re`gles au contraire [. . .] je conside`re que, pour moi, les re`gles c’est de´toxiﬁant ». E23, 40 ans : « C’est Dieu qui de´cide, on est cre´e´e comme c¸a, on fonctionne comme c¸a ». Le ve´cu ne´gatif des menstruations a e´te´ retrouve´ dans les deux groupes de femmes, la diffe´rence re´sidait dans l’intensite´ des contraintes et dans la tole´rance des femmes a` les vivre (Tableau 2). De manie`re non exhaustive, les contraintes e´nonce´es e´taient lie´es a` de nombreux troubles physiques, telle que la gestion des me´no-me´trorragies, E9, 56 ans « fallait penser a` te changer, que t’e´tais re´gulie`rement taˆche´e [..] je me levais d’une chaise de restaurant euh j’e´tais toute salie, c’e´tait la honte », la gestion de la douleur, E11, 27 ans « C’e´tait vraiment l’enfer. 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche Les journe´es sont longues quand on attend que la douleur passe [.] quoi que je fasse, debout, assise, allonge´e, a` droite, a` gauche, peu importe la position que j’essayais de prendre la douleur passait pas », la gestion de l’asthe´nie et des troubles de l’humeur, E11, 27 ans « C’est une pe´riode ou` on est plus fatigue´e, et moi quand je suis plus fatigue´e ben je suis plus susceptible » et la gestion des troubles du transit et de la ﬂore vaginale, E1, 41 ans, « j’avais la diarrhe´e, je perdais beaucoup de sang, apre`s du coup je manquais de fer donc le docteur me donnait du fer, c¸a me cre´ait des grosses constipations, en fait c’e´tait un cycle qui s’arreˆtait jamais ». Les autres contraintes e´nonce´es e´tait dues aux proble´matiques de l’hygie`ne, E1, 41 ans, « J’e´tais dans un lyce´e dans un quartier assez difﬁcile a` Marseille et du coup les toilettes e´taient quasiment inaccessibles et quand j’avais mes re`gles c’e´tait e´videmment une gale`re »; ﬁnancie`res, E11, 27 ans, « C’est quand meˆme des contraintes que ce soit. . .ben alors ﬁnancie`res quand meˆme a` chaque fois », des tenues vestimentaires, E16, 48 ans, « [. . .] pour s’habiller. . .on sait qu’on doit s’habiller en noir, qu’on met pas force´ment du moulant »; de la repre´sentation de la sexualite´ ou de la fe´minite´, E9, 56 ans, « Je me sens beaucoup moins femme quand j’ai une couche au fond de ma culotte [Rires] »; professionnelles, E11, 27 ans, « On est dans une socie´te´ ou` on est oblige´e de gommer cet aspect-la`, on nous demande de ne pas en parler, c’est tabou dans le quotidien, la recherche de la performance. . . »; sportives, E6, 26 ans, « j’avais mes re`gles tous les mois mais c’e´tait tre`s douloureux et c¸a m’embeˆtait parce que je vais a` la piscine trois fois par semaine et c’e´tait pas tre`s pratique »,;du regard des autres, E8, 22 ans, « Pour les protections on est toujours geˆne´e d’en acheter, c’est un peu la honte a` la caisse »; familiales, E16, 48 ans, « Quand je faisais une sortie, il fallait que je m’arreˆte dans un cafe´, consommer rien que pour aller aux toilettes. Pas de mer l’e´te´, pas de plage, j’ai refuse´ meˆme des sorties. 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche on reste chez soi, on sort un minimum, [..] C¸a posait proble`me pour ma famille, pour mon mari, pour mes enfants.. ». 52 ans : « C’est du transmis de maman quoi. On ne l’a donne´ mais c’est pas force´ment personnel ». Nous avons observe´ que la repre´sentation du sang menstruel des femmes est e´volutive et en rapport avec l’approche de la me´nopause ou du nombre d’enfant de´sire´ obtenu. E20, 48 ans : « Maintenant c’est bon, c¸a sert plus a` rien [. . .] j’ai eu mes enfants, on peut pas rester jeune toute la vie ». Que ce soit, des menstruations ou des he´morragies de privation sous contraception oestro-progestative, les femmes parlent de leur sang, au sens large, de leur roˆle et de leur ve´cu, positif ou ne´gatif. Dans le groupe des femmes ayant des repre´sentations et un ve´cu positifs, les femmes ont conside´re´ le sang menstruel, comme un e´ve`nement naturel, ayant un pouvoir puriﬁcateur, e´loignant certaines maladies et ayant un roˆle essentiel dans la procre´ation. E17, 26 ans : « Une femme n’est pas sale quand elle a ses re`gles au contraire [. . .] je conside`re que, pour moi, les re`gles c’est de´toxiﬁant ». E23, 40 ans : « C’est Dieu qui de´cide, on est cre´e´e comme c¸a, on fonctionne comme c¸a ». Le ve´cu ne´gatif des menstruations a e´te´ retrouve´ dans les deux groupes de femmes, la diffe´rence re´sidait dans l’intensite´ des contraintes et dans la tole´rance des femmes a` les vivre (Tableau 2). De manie`re non exhaustive, les contraintes e´nonce´es e´taient lie´es a` de nombreux troubles physiques, telle que la gestion des me´no-me´trorragies, E9, 56 ans « fallait penser a` te changer, que t’e´tais re´gulie`rement taˆche´e [..] je me levais d’une chaise de restaurant euh j’e´tais toute salie, c’e´tait la honte », la gestion de la douleur, E11, 27 ans « C’e´tait vraiment l’enfer. 3.2. Le ve´cu et les repre´sentations du sang menstruel apre`s la me´narche Les journe´es sont longues quand on attend que la douleur passe [.] quoi que je fasse, debout, assise, allonge´e, a` droite, a` gauche, peu importe la position que j’essayais de prendre la douleur passait pas », la gestion de l’asthe´nie et des troubles de l’humeur, E11, 27 ans « C’est une pe´riode ou` on est plus fatigue´e, et moi quand je suis plus fatigue´e ben je suis plus susceptible » et la gestion des troubles du transit et de la ﬂore vaginale, E1, 41 ans, « j’avais la diarrhe´e, je perdais beaucoup de sang, apre`s du coup je manquais de fer donc le docteur me donnait du fer, c¸a me cre´ait des grosses constipations, en fait c’e´tait un cycle qui s’arreˆtait jamais ». Les autres contraintes e´nonce´es e´tait dues aux proble´matiques de l’hygie`ne, E1, 41 ans, « J’e´tais dans un lyce´e dans un quartier assez difﬁcile a` Marseille et du coup les toilettes e´taient quasiment inaccessibles et quand j’avais mes re`gles c’e´tait e´videmment une gale`re »; ﬁnancie`res, E11, 27 ans, « C’est quand meˆme des contraintes que ce soit. . .ben alors ﬁnancie`res quand meˆme a` chaque fois », des tenues vestimentaires, E16, 48 ans, « [. . .] pour s’habiller. . .on sait qu’on doit s’habiller en noir, qu’on met pas force´ment du moulant »; de la repre´sentation de la sexualite´ ou de la fe´minite´, E9, 56 ans, « Je me sens beaucoup moins femme quand j’ai une couche au fond de ma culotte [Rires] »; professionnelles, E11, 27 ans, « On est dans une socie´te´ ou` on est oblige´e de gommer cet aspect-la`, on nous demande de ne pas en parler, c’est tabou dans le quotidien, la recherche de la performance. . . »; sportives, E6, 26 ans, « j’avais mes re`gles tous les mois mais c’e´tait tre`s douloureux et c¸a m’embeˆtait parce que je vais a` la piscine trois fois par semaine et c’e´tait pas tre`s pratique »,;du regard des autres, E8, 22 ans, « Pour les protections on est toujours geˆne´e d’en acheter, c’est un peu la honte a` la caisse »; familiales, E16, 48 ans, « Quand je faisais une sortie, il fallait que je m’arreˆte dans un cafe´, consommer rien que pour aller aux toilettes. Pas de mer l’e´te´, pas de plage, j’ai refuse´ meˆme des sorties. 4.2. Impact de l’expe´rience me´narchale sur le ve´cu et les repre´sentations du sang menstruel Les femmes ayant un ve´cu et/ou des repre´sentations ne´gatives de l’ame´norrhe´e induite ont exprime´ le de´sir de conserver leurs he´morragies de privation. E2, 35 ans : « Justement moi, je fais l’arreˆt volontairement ». Les femmes ayant exprime´ une indiffe´rence ont avoue´, ﬁnalement, avoir une pre´fe´rence en faveur des saignements menstruels. E13, 28 ans : « Non, moi je m’en fous de saigner ou pas saigner [..] Non, en fait c¸a me rassure d’avoir mes re`gles ». Les femmes ayant un ve´cu et/ou des repre´sentations positives de l’ame´norrhe´e induite ont exprime´ le de´sir de ne plus avoir d’he´morragies de privation. E9, 56 ans : « C’est un plus conside´rable. C’est une liberte´. C’est un crite`re de choix absolu ». Malgre´ ce de´sir, une femme a formule´ la crainte d’une sanction biologique a` eˆtre en ame´norrhe´e induite. E1, 41 ans : « Est-ce que je risque pas d’avoir un petit polype, des petits kystes, des hormones qui vont de´clencher des choses comme c¸a et est-ce que c¸a va pas me provoquer des soucis apre`s par la suite. . . ». Pour les femmes ayant e´te´ oblige´es de choisir une contraception hormonale progestative (risque majore´ de phle´bite, he´patite, endome´triose et me´norragies), l’ame´norrhe´e induite a e´te´ accepte´ a posteriori, excepte´ pour celle atteinte d’he´patite. E11, 27 ans : « C’e´tait pas un choix voulu [. . .] maintenant non je l’e´changerais pas » (Tableau 2). Nous n’avons pas constate´ d’effet de la classe socio-professionnelle ou de l’origine ge´ographique des femmes sur le choix d’avoir ou non une he´morragie de privation dans le choix de leur contraception. Nous avons observe´, que malgre´ une culture scientiﬁque, les femmes travaillant dans le me´dical et le parame´dical ont plus exprime´ le de´sir de conserver leurs he´morragies de privation. Nous n’avons pas pu e´tudier les croyances religieuses des femmes du fait des limites e´thiques impose´es a` notre e´tude. D’apre`s les travaux sociologiques re´alise´s en France par le Dr Mardon, le ve´cu de la me´narche se re´percuterait sur les repre´sentations du sang menstruel et l’acceptation des change- ments corporels. Or, la me´narche constituerait une expe´rience ne´gative et angoissante pour la majorite´ des jeunes ﬁlles. 4.2. Impact de l’expe´rience me´narchale sur le ve´cu et les repre´sentations du sang menstruel Les jeunes ﬁlles dont la me´narche a e´te´ pre´coce ou celles qui n’ont pas e´te´ pre´pare´es pre´senteraient des symptoˆmes plus importants de honte et de de´gouˆt, et une image de soi plus ne´gative et elles associeraient, a` l’aˆge adulte, le sang a` la souillure et a` la salete´ [19]. Les jeunes ﬁlles qui conside`reraient le moment de leur premier saignement comme n’e´tant pas opportun pre´senteraient un de´phasage physique, ne´gatif sur l’estime de soi, l’acceptation des pairs et la popularite´ aupre`s du sexe oppose´. En revanche, les jeunes ﬁlles ayant rec¸u une meilleure information auraient une expe´rience me´narchale moins ne´gative et accepteraient plus volontiers leurs changements physiques et les saignements associe´s [20,21]. 4.3. Impact du ve´cu et des repre´sentations du sang menstruel sur les repre´sentations de l’ame´norrhe´e induite 4.3. Impact du ve´cu et des repre´sentations du sang menstruel sur les repre´sentations de l’ame´norrhe´e induite L’abondance des saignements et la douleur contribueraient a` la construction d’une repre´sentation ne´gative des menstruations. Dans une e´tude Franc¸aise de 2004, 25 % des femmes entre 30 et 49 ans souffraient de troubles he´morragiques fonctionnels [21]. En 2016, la pre´valence de la dysme´norrhe´e, en France, variait de 36,4 % a` 62,3 % selon l’aˆge et l’origine ge´ographique. Seulement un tiers des femmes a` l’aˆge adulte ne pre´senteraient aucun symptoˆme douloureux pendant leurs menstruations [22]. Il aurait e´te´ constate´ que les femmes ayant une bonne relation avec leur corps et une repre´sentation positive de leurs menstruations semble- raient trouver moins d’inte´reˆt a` une ame´norrhe´e induite, a` l’inverse des femmes ayant une repre´sentation ne´gative de leurs saignements. 3.4. La pre´sence ou l’absence de sang menstruel comme crite`re de choix d’une contraception 3.4. La pre´sence ou l’absence de sang menstruel comme crite`re de choix d’une contraception 3.3. Le ve´cu et les repre´sentations d’une ame´norrhe´e induite par une contraception hormonale 3.3. Le ve´cu et les repre´sentations d’une ame´norrhe´e induite par une contraception hormonale Le ve´cu et les repre´sentations d’une ame´norrhe´e induite par une contraception hormonale continue sont directement inﬂuen- ce´s par le ve´cu et les repre´sentations du sang menstruel. Les Le biais de se´lection est inhe´rent a` la me´thode qualitative et pour limiter ce biais, l’e´tude a e´te´ pre´sente´e aux femmes comme portant sur la contraception et les saignements menstruels n’ont e´te´ e´voque´s que lors de l’entretien. De plus, ce biais a e´te´ limite´ au maximum en multipliant les lieux de recrutements aﬁn d’e´viter que les re´ponses sur la contraception ou les menstruations soient me´decins-de´pendants. Aﬁn de limiter le biais de confusion, l’aˆge et la classe socio-professionnelle ont e´te´ conside´re´s comme des facteurs de confusion. Aﬁn de limiter le biais de me´morisation le guide d’entretien a e´te´ re´gulie`rement re´e´value´ comme le pre´voit la me´thodologie qualitative. Et aﬁn de limiter le biais d’inter- pre´tation et d’analyse, lie´ a` l’enqueˆteur, nous avons effectue´ une triangulation des donne´es. Une fois retranscrites, certaines informations recueillies n’e´taient pas exploitables et ont donc e´te´ supprime´es. femmes vivant l’e´coulement menstruel de fac¸on positive ont de´crit l’ame´norrhe´e induite comme e´tant « contre nature » et plus a` risque de cancers, de maladies auto-immunes, de ste´rilite´ ou de risque de grossesse non de´sire´e. E22, 24 ans : « La contraception c’est des risques, alors faut pas surajouter les risques en bloquant les re`gles, je pense ». E17, 26 ans : « Avoir ses re`gles, c’est quelque chose de sain. C’est comme manger, boire, aller aux toilettes ». Au contraire, les femmes vivant l’e´coulement menstruel de fac¸on ne´gative, ont de´crit l’ame´norrhe´e induite comme un traitement me´dical les libe´rant de toutes les contraintes associe´es au sang (pre´cise´es au chapitre pre´ce´dent) E9, 56 ans : « J’ai trouve´ c¸a extraordinaire ! [. . .] Quand j’ai e´te´ libe´re´e de c¸a, c’est pfff. . . j’ai presque envie de dire que c’e´tait une renaissance [..] Je re´cupe´rais de la vie quelque part » (Tableau 2). Re´fe´rences [1] Hippocrate, Littre´ E. Des maladies des femmes. In: Œuvres comple`tes, Tome VIII-Livre I. Paris: Baillie`re; 1853. http://remacle.org/bloodwolf/erudits/ Hippocrate/femmes.htm (cite´ 20 mai 2017). [2] Pline l’Ancien, Vinas A. Singularite´s du ﬂux menstrue. In: Histoire naturelle, Livre II-Chapitre XIII. Paris: Dubochet; 1850. http://remacle.org/bloodwolf/ erudits/plineancien (cite´ 15 mars 2017). [3] Carnel M. Le Sang embaume´ des roses : sang et passion dans la poe´sie amoureuse de Pierre de Ronsard. Gene`ve: Librairie Droz; 2004. p. 151–154. https://books.google.fr/books?id=zf6JFE4Bg4oC&pg=PA152&lpg=PA152&dq= la+renaissance%2Bmenstruations&source=bl&ots=CBMj4nvXsL&sig= 0v5aNtzYnfvZ1RLM49DYc8qkHQk&hl=fr&sa=X&ved= 0ahUKEwiUm47O0dHUAhVGSRoKHVV7C0MQ6AEIVzAI#v=onepage&q= la%20renaissance%2Bmenstruations&f=false (cite´ 1 juin 2017).. [4] Collard F. Le poison et le sang dans la culture me´die´vale. Rev Me´die´vales 2011;60:142–3 [http://www.puv-editions.fr/media/ouvr_pdf/516_Le% 20poison%20et%20le%20sang.pdf (cite´ 26 mai 2017)]. [5] Le Naour JY, Valenti C. Du sang et des femmes. Histoire me´dicale de la menstruation a` la Belle E´poque. Rev Clio : Histoire femmes et socie´te´s 2001;14:207–29 [http://clio.revues.org/114 (cite´ 3 mai 2017)]. [6] McClive C. Engendrer le tabou. L’interpre´tation du Le´vitique 15, 18-19 et 20- 18 et de la menstruation sous l’Ancien Re´gime. Rev Ann Demogr historique 2013;125(1):165–210 [https://www.cairn.info/revue-annales-de- demographie-historique-2013-1-page-165.htm (cite´ le 15 avril 2019)]. [ ] ´ h f b i d i l l [7] Laqueur T, De´chaux JH. La fabrique du sexe. Essai sur le corps et le genre en Occident. Rev Ann Sociol 1993;34(3):454–7 [https://www.persee.fr/doc/ rfsoc_0035-2969_1993_num_34_3_4269 (cite´ le 15 avril 2019)]. ( )] [8] Victor H. Choses vues 1830–1846. Paris: Gallimard; 1972. [8] Victor H. Choses vues 1830–1846. Paris: Gallimard; 1972. [9] Olrik H, Le sang impur. Notes sur le concept de prostitue´e-ne´e chez Lombroso Traduit de La Donna delinquente, la prostituta e la donna normale. In: Romantisme. 1981;31. Sangs. p. 167–178. https://doi.org/10.3406/roman. 1981.4479 (cite´ 15 juin 2017). [10] Bobel C, Walden R. A Discussion of Menstrual Activism with Chris Bobel; 2009, https://www.ourbodiesourselves.org/2009/02/ a-discussion-of-menstrual-activism-with-chris-bobel/ (cite´ le 15 avril 2019). [11] Loi n82015–1785 du 29 de´cembre 2015 de ﬁnances pour 2016, Article 10. https://www.legifrance.gouv.fr/afﬁchTexte.do?cidTexte=JORFTEXT00003 1732865&categorieLien=id (cite´ le 18 juin 2019). [12] Direction ge´ne´rale de la concurrence, de la consommation et de la re´pression des fraudes. Se´curite´ des produits d’hygie`ne fe´minine. Rev Re´sultats d’enqueˆte. 2017. https://www.economie.gouv.fr/dgccrf/securite-des- produits-dhygiene-feminine (cite´ 15 avril 2019). [13] Laville E, Corre MF, Pingusson P, Marcel J. Mes courses pour la plane`te. Graines Chang 2018 [http://www.mescoursespourlaplanete.com/Produits/ Santae_et_Beautae_83/Protections_Hygiaeniques_115.html. (cite´ 22 mars 2017)]. [14] Semeraro L, Philippe A. Du point de vue des femmes, quelle est l’inﬂuence des hormones contraceptives sur leur sexualite´ ? E´tude qualitative re´alise´e a` Annecy, entre mars et juillet 2014. [The`se]. Grenoble: universite´ Joseph- Fourier; 2014. p. 1–225. 5. Conclusion [15] Re´gnier-Loilier A, Leridon H. Apre`s la loi Neuwirth, pourquoi tant de grossesses impre´vues ? Rev INED : Population et socie´te´s 2007;439:1–8 [https://www. ined.fr/ﬁchier/s_rubrique/176/publi_pdf1_439.fr.pdf. (cite´ 2 janv 2018)]. ` ´ L’impact du ve´cu et des repre´sentations fantasmatiques du sang menstruel chez les femmes sur le choix et la compliance a` une contraception est peu e´tudie´. Par pudeur, tabou ou manque de temps, la repre´sentation des menstruations est un sujet peu aborde´ par les femmes en consultation me´dicale et bien que le cycle menstruel soit enseigne´ en France aux adolescentes au Colle`ge, les repre´sentations que nous avons rapporte´ dans cette e´tude sont issues de visions ancestrales, socie´tales ou familiales, bien ancre´es dans l’inconscient collectif. Une information pre´coce de qualite´ permettrait probablement un ve´cu moins angoissant de la me´narche chez les adolescentes. Une e´tude sur l’impact d’une consultation me´dicale de´die´e en de´but de puberte´, ou` serait fournie une information simple concernant la me´narche et l’origine cyclique des menstruations, pourrait eˆtre inte´ressante sur le ve´cu a` long terme des symptoˆmes lie´s aux menstruations. Etant donne´ que la prescription d’une contraception inadapte´e est a` risque de grossesse non de´sire´e, les praticiens prescrivant une contraception devraient avoir appre´hende´ au moment de leur prescription les repre´sentations mentales qu’a la femme de ses re`gles, et de l’inte´reˆt me´dical et psychologique de lui proposer ou non une contraception sans he´morragie de privation. [16] Ministe`re des Affaires sociales et de la Sante´. Mise en oeuvre de la politique sur la contraception. Dossier de presse : HAS; 2013. p. 1–47. http:// solidarites-sante.gouv.fr/IMG/pdf/15_05_13_DP_ASS_contraception.pdf. (cite´ 2 janv 2018). [17] Bajos N, Moreau C, Leridon H, Ferrand M. Pourquoi le nombre d’IVG n’a-t-il pas baisse´ en France depuis 30 ans ? Rev INED : Population et socie´te´s 2004;407:1–4 [https://www.ined.fr/ﬁchier/s_rubrique/18727/pop.et.soc. francais.407.fr.pdf. (cite´ 20 nov 2017)]. [18] Bretin H, Kotobi L. Ine´galite´s contraceptives au pays de la pilule. Rev Agone 2016;58(1):123–34. [19] Mardon A. Honte et de´gouˆt dans la fabrication du fe´minin. L’apparition des menstrues. Rev Ethnologie franc¸aise 2011;41:1–9 [https://www.cairn.info/ revue-ethnologie-francaise-2011-1-page-33.htm. (cite´ 27 oct 2018)]. [20] Brooks-Gunn J, Ruble DN. The Experience of Menarche from a Developmental Perspective. In: Girls at Puberty, chapter Psychological Aspects of Puberty [Engl. Ed.]; 1983. p. 155–177. https://link.springer.com/chapter/10.1007/ 978-1-4899-0354-9_8. (cite´ 20 de´c 2018). [21] Fernandez H, Gervaise A, De Tayrac R. Les troubles he´morragiques fonction- nels. Epide´miologie-diagnostic objectif. Extraits et mise a` jour en gyne´cologie me´dicale, CNGOF. 28e`me Journe´e Nationale, 2004 ; Paris. p.1-11. http://www. cngof.asso.fr/d_livres/2004_Gm_005_fernandez.pdf. 4.4. L’ame´norrhe´e induite ou l’he´morragie de privation comme crite`re de choix d’une contraception Dans cette e´tude, 50 % des femmes auraient pour souhait de conserver leur he´morragie de privation et 33 % des femmes auraient pour souhait d’eˆtre en ame´norrhe´e induite. Par ailleurs, 59 % des femmes pre´fe´reraient ne pas avoir de saignement tous les mois [27]. Aux Pays-Bas, 80,5 % des femmes Ne´erlandaises interroge´es auraient pre´fe´re´ avoir eu des mens- truations moins douloureuses, plus courtes voire eˆtre en ame´nor- rhe´e. La repre´sentation de l’ame´norrhe´e induite comme une conse´quence positive plutoˆt que comme un effet secondaire de la contraception hormonale, augmenterait avec l’aˆge. Chez les femmes de plus de 45 ans, l’ame´norrhe´e induite serait mieux accepte´e (62 % contre 25,2 % chez les 15–19 ans) [28]. La culture, souvent appre´hende´e dans la litte´rature par les origines ethniques, inﬂuencerait les repre´sentations de l’ame´norrhe´e et les femmes d’origine caucasiennes seraient plus susceptibles de choisir une ame´norrhe´e induite dans leur contraception que les femmes afro- ame´ricaines (29 % vs 4 %, p = 0,006). Toutefois, quelle que soit leur origine, la majorite´ des femmes accepteraient d’essayer une ame´norrhe´e induite [25]. Aucune donne´e de la litte´rature n’a montre´ que le niveau socioe´conomique pourrait avoir un impact sur le choix d’une ame´norrhe´e induite ou non lors de la prescription d’une contraception. Re´fe´rences https://dumas.ccsd.cnrs.fr/dumas-01096135/ document (cite´ 12 fe´vr 2018). De´claration de liens d’inte´reˆts ou dans les recommandations de l’HAS de 2004, aucune recommandation ne propose de tenir compte du de´sir ou non d’avoir une he´morragie de privation dans le choix d’une contraception par une femme. En 2014, une e´tude qualitative Franc¸aise a observe´ l’importance de l’absence de contrainte ainsi que l’inte´reˆt des be´ne´ﬁces non contraceptifs dans le choix contraceptif des femmes, notamment l’impact de la sante´ sexuelle sur le choix contraceptif [24]. Les re´percussions socioe´conomiques des me´norragies (ane´mie, asthe´nie et absente´isme scolaire ou professionnel) semblent encore trop peu prises en compte en termes de Sante´ Publique. Aux E´tats-Unis, l’inconfort menstruel repre´senterait 40 % des motifs de consultations gyne´cologiques aupre`s des plannings familiaux [25]. Les femmes souffrant de me´tro-me´norragies signaleraient une diminution de leurs activite´s professionnelles de 28 % compare´ aux femmes ne souffrant pas de me´tro-me´norragies ce qui repre´senteraient une perte de 1692 dol- lars par an et par femme [26]. Dans l’e´tude de Andrista et al., 38 % des femmes ame´ricaines entre 18 et 40 ans utiliseraient une contraception a` une autre ﬁn que contraceptive, dont 20 % pour eˆtre en ame´norrhe´e induite. Dans cette e´tude, 50 % des femmes auraient pour souhait de conserver leur he´morragie de privation et 33 % des femmes auraient pour souhait d’eˆtre en ame´norrhe´e induite. Par ailleurs, 59 % des femmes pre´fe´reraient ne pas avoir de saignement tous les mois [27]. Aux Pays-Bas, 80,5 % des femmes Ne´erlandaises interroge´es auraient pre´fe´re´ avoir eu des mens- truations moins douloureuses, plus courtes voire eˆtre en ame´nor- rhe´e. La repre´sentation de l’ame´norrhe´e induite comme une conse´quence positive plutoˆt que comme un effet secondaire de la contraception hormonale, augmenterait avec l’aˆge. Chez les femmes de plus de 45 ans, l’ame´norrhe´e induite serait mieux accepte´e (62 % contre 25,2 % chez les 15–19 ans) [28]. La culture, souvent appre´hende´e dans la litte´rature par les origines ethniques, inﬂuencerait les repre´sentations de l’ame´norrhe´e et les femmes d’origine caucasiennes seraient plus susceptibles de choisir une ame´norrhe´e induite dans leur contraception que les femmes afro- ame´ricaines (29 % vs 4 %, p = 0,006). Toutefois, quelle que soit leur origine, la majorite´ des femmes accepteraient d’essayer une ame´norrhe´e induite [25]. Aucune donne´e de la litte´rature n’a montre´ que le niveau socioe´conomique pourrait avoir un impact sur le choix d’une ame´norrhe´e induite ou non lors de la prescription d’une contraception. Les auteurs de´clarent ne pas avoir de liens d’inte´reˆts. 4.4. L’ame´norrhe´e induite ou l’he´morragie de privation comme crite`re de choix d’une contraception Dans la litte´rature, il existe peu d’e´tudes qualitatives re´centes s’inte´ressant aux repre´sentations psychiques des menstruations par des patientes. L’inte´reˆt de notre travail a donc e´te´ de les mettre en exergue, en espe´rant ainsi ame´liorer la qualite´ du choix contraceptif. Aﬁn d’obtenir le plus de repre´sentations et de ve´cu possibles, il e´tait impe´ratif que la population soit la plus he´te´roge`ne possible, avec un panel de femmes ayant des caracte´ristiques diffe´rentes en terme d’aˆge, de classe socio- e´conomique et d’acce`s aux informations me´dicales sur les menstruations et la contraception. Cet e´chantillon nous a permis de donner un aperc¸u sur le ressenti des femmes vis-a`-vis de leur sang menstruel associe´ ou non a` leur contraception. En 2010, un travail de the`se a e´value´ les recommandations de l’HAS concernant les « strate´gies de choix de me´thodes contra- ceptives chez la femme » par les prescripteurs aupre`s de 92 femmes et aretrouve´ les crite`res de choix suivant pour la prescription d’une contraception : la facilite´ d’utilisation, le degre´ d’efﬁcacite´, le prix, l’absence de douleur lors de la pose ou de l’utilisation, l’absence d’effets secondaires, l’absence de geˆne pour le partenaire, le taux de remboursement par la se´curite´ sociale, la fre´quence d’utilisation et l’absence de contre-indications [23]. Que ce soit dans cette e´tude ou dans les recommandations de l’HAS de 2004, aucune recommandation ne propose de tenir compte du de´sir ou non d’avoir une he´morragie de privation dans le choix d’une contraception par une femme. En 2014, une e´tude qualitative Franc¸aise a observe´ l’importance de l’absence de contrainte ainsi que l’inte´reˆt des be´ne´ﬁces non contraceptifs dans le choix contraceptif des femmes, notamment l’impact de la sante´ sexuelle sur le choix contraceptif [24]. Les re´percussions socioe´conomiques des me´norragies (ane´mie, asthe´nie et absente´isme scolaire ou professionnel) semblent encore trop peu prises en compte en termes de Sante´ Publique. Aux E´tats-Unis, l’inconfort menstruel repre´senterait 40 % des motifs de consultations gyne´cologiques aupre`s des plannings familiaux [25]. Les femmes souffrant de me´tro-me´norragies signaleraient une diminution de leurs activite´s professionnelles de 28 % compare´ aux femmes ne souffrant pas de me´tro-me´norragies ce qui repre´senteraient une perte de 1692 dol- lars par an et par femme [26]. Dans l’e´tude de Andrista et al., 38 % des femmes ame´ricaines entre 18 et 40 ans utiliseraient une contraception a` une autre ﬁn que contraceptive, dont 20 % pour eˆtre en ame´norrhe´e induite. [23] Macchi M. Evaluation de l’application des recommandations de l’HAS de 2004 sur les « strate´gies de choix des me´thodes contraceptives chez la femme » par les prescripteurs. Etude re´trospective sur une population de patientes consultant au centre d’orthoge´nie de l’hoˆpital Robert Ballanger pour une demande d’IVG. Paris VI: universite´ Pierre et Marie Curie; 2010. p. 1–113. http://www.cmge-upmc.org/ IMG/pdf/macchi_these_contraception.pdf. (cite´ 10 fe´vr 2018). [25] Marquillanes S. Regards des me´decins ge´ne´ralistes sur la prescription d’un moyen de contraception pouvant induire une ame´norrhe´e. Lyon-1: universite´ Claude Bernard; 2013. p.1–148. [24] Ducheˆne-Paton AM, Lope`s P. Sexualite´ et choix du mode contraceptif. Rev Sexol Elsevier 2015;24(2):69–81 [http://www.em-consulte.com/en/article/ 973259. (cite´ 10 fe´vr 2018)]. 5. Conclusion [22] Margueritte F, Ringa V, Fritel X. Algies pelviennes chroniques : pre´valence et caracte´ristiques associe´es dans la cohorte Constances. Rev Epidemiol Sante Publique Elsevier 2016;64(2):134. http://www.sciencedirect.com/science/ article/pii/S0398762016001000. (cite´ 10 fe´vr 2018). [23] Macchi M. Evaluation de l’application des recommandations de l’HAS de 2004 sur les « strate´gies de choix des me´thodes contraceptives chez la femme » par les prescripteurs. Etude re´trospective sur une population de patientes consultant au centre d’orthoge´nie de l’hoˆpital Robert Ballanger pour une demande d’IVG. Paris VI: universite´ Pierre et Marie Curie; 2010. p. 1–113. http://www.cmge-upmc.org/ IMG/pdf/macchi_these_contraception.pdf. (cite´ 10 fe´vr 2018). [26] Coˆte´ I, Jacobs P, Cumming D. Work Loss Associated With Increased Menstrual Loss in the United States. Rev Obstet Gynecol [Engl Ed] Elsevier 2002;100(4):683–7 [https://www.sciencedirect.com/science/article/pii/ S002978440202094X. (cite´ 10 fe´vr 2018)]. [27] Andrista LC, Ariasb RD, Nucatolab D, Kaunitz AM, Musselman BL, Reiter S, et al. Women’s and providers’ attitudes toward menstrual suppression with ex- tended use of oral contraceptives. Rev Contracept [Engl Ed] Elsevier 2004;70(5):359–63. [24] Ducheˆne-Paton AM, Lope`s P. Sexualite´ et choix du mode contraceptif. Rev Sexol Elsevier 2015;24(2):69–81 [http://www.em-consulte.com/en/article/ 973259. (cite´ 10 fe´vr 2018)]. [28] Den Tonkelaar I, Oddens BJ. Preferred frequency and characteristics of men- strual bleeding in relation to reproductive status, oral contraceptive use, and hormone replacement therapy use. Rev Contracept [Engl Ed] Elsevier 1999;59(6):357–62. [25] Marquillanes S. Regards des me´decins ge´ne´ralistes sur la prescription d’un moyen de contraception pouvant induire une ame´norrhe´e. Lyon-1: universite´ Claude Bernard; 2013. p.1–148.
https://openalex.org/W2755081174	https://europepmc.org/articles/pmc5622782?pdf=render	English	null	Flavonolignans Inhibit IL1-β-Induced Cross-Talk between Blood Platelets and Leukocytes	Nutrients	2,017	cc-by	9,053	Michal Bijak 1,, Angela Dziedzic 1, Ewelina Synowiec 2, Tomasz Sliwinski 2 and Joanna Saluk-Bijak 1 Michal Bijak 1,, Angela Dziedzic 1, Ewelina Synowiec 2, Tomasz Sliwinski 2 and Joanna Saluk-Bijak 1 Michal Bijak 1,, Angela Dziedzic 1, Ewelina Synowiec 2, Tomasz Sliwinski 2 and Joanna Saluk-Bijak 1 1 Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz Pomorska 141/143, 90-236 Lodz, Poland; angela.dziedzic@outlook.com (A.D.); joanna.saluk@biol.uni.lodz.pl (J.S.-B.) 1 Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland; angela.dziedzic@outlook.com (A.D.); joanna saluk@biol uni lodz pl (J S -B ) j p 2 Laboratory Medical Genetics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland; ewelina.synowiec@biol.uni.lodz.pl (E.S.); tomasz.sliwinski@biol.uni.lodz.pl (T.S.) j p 2 Laboratory Medical Genetics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland; ewelina.synowiec@biol.uni.lodz.pl (E.S.); 2 Laboratory Medical Genetics, Faculty of Biology and Environmental Protection, Univer Pomorska 141/143, 90-236 Lodz, Poland; ewelina.synowiec@biol.uni.lodz.pl (E.S.); t li i ki@bi l i l d l (T S ) Correspondence: michal.bijak@biol.uni.lodz.pl; Tel./Fax: +48-042-635-4336 * Correspondence: michal.bijak@biol.uni.lodz.pl; Tel./Fax: +48-042-635-4336 Received: 15 August 2017; Accepted: 13 September 2017; Published: 15 September 2017 Abstract: Interleukin-1 beta (IL-1β)—the most potent pro-inﬂammatory is responsible for a broad spectrum of immune and inﬂammatory responses, it induces T-cell and B-cell activation and consequently the synthesis of other pro-inﬂammatory cytokines (such as IFN-γ and TNF). IL-1β induces the formation of blood platelet-leukocyte aggregates (PLAs), which suggests that IL-1β signiﬁcantly affects the cross-talk between blood platelets and the immune response system, leading to coronary thrombosis. The aim of our study is to investigate the effect of ﬂavonolignans (silybin, silychristin and silydianin) on the IL-1β-induced interaction between platelets and leukocytes, as well as on the expression and the secretion of pro-inﬂammatory factors. Whole blood samples were pre-incubated with commercially available ﬂavonolignans (silybin, silychristin and silydianin) in a concentration range of 10–100 µM (30 min, 37 ◦C). Next, samples were activated by IL-1β for 1 h. Blood platelet-leukocyte aggregates were detected by using the double-labeled ﬂow cytometry (CD61/CD45). The level of produced cytokines was estimated via the ELISA immunoenzymatic method. IFN-γ and TNF gene expression was evaluated using Real Time PCR with TaqMan arrays. We observed that in a dose-dependent manner, silybin and silychristin inhibit the IL-1β-induced formation of blood platelet-leukocyte aggregates in whole blood samples, as well as the production of pro-inﬂammatory cytokines—IL-2, TNF, INF-α, and INF-γ. Michal Bijak 1,, Angela Dziedzic 1, Ewelina Synowiec 2, Tomasz Sliwinski 2 and Joanna Saluk-Bijak 1 Additionally, these two ﬂavonolignans abolished the IL-1β-induced expression of mRNA for IFN-γ and TNF. Our current results demonstrate that ﬂavonolignans can be novel compounds used in the prevention of cardiovascular diseases with dual-use action as antiplatelet and anti-inﬂammatory agents. Keywords: interleukin 1; anti-inﬂammatory; ﬂavonolignans; silybin; silychristin nutrients nutrients 1. Introduction Interleukin 1 beta (IL-1β) is the most potent pro-inﬂammatory cytokine that is crucial in host-defense responses to infection and injury [1]. IL-1β is expressed by many cells and has multiple functions, including in local inﬂammation. IL-1β is produced by activated macrophages, endothelial cells, B cells, and ﬁbroblasts. This potent pro-inﬂammatory cytokine was initially discovered and classiﬁed as the major endogenous pyrogen. IL-1β mediates the expression of a vast array of genes involved in secondary inﬂammation, which coordinate all aspects of local inﬂammation and also attract and activate the cells of the adaptive immune system at the infection sites [2]. IL-1β is responsible for a broad spectrum of immune and inﬂammatory responses, induces T-cell and B-cell activation, and consequently the synthesis of other pro-inﬂammatory cytokines (such as IFN-γ, IL-6 and Nutrients 2017, 9, 1022; doi:10.3390/nu9091022 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 11 Nutrients 2017, 9, 1022 TNF), and antibody production. This cytokine also induces the expression of itself in newly-arriving monocytes, thus reinforcing the overall process. IL-1β circulating in blood is unregulated under systemic and chronic inﬂammatory conditions and is measurable in pg/mL [3]. The mechanism of IL-1β cell action is based on the binding to type I IL-1 receptor (IL-1RI) and the activation of the intracellular signal pathway. IL-1β ﬁrst binds to the ﬁrst extracellular chain of IL-1RI that recruits the IL-1 receptor accessory protein (IL-1RAcP), which serves as a co-receptor and is necessary for signal transduction. In response to the ligand binding of the receptor, a complex sequence of combinatorial phosphorylation and ubiquitination events results in the activation of nuclear factor κB (NF-κB) signalling and the JNK and p38 mitogen-activated protein kinase pathways. Together, these then induce the expression of canonical IL-1β target genes through transcriptional and post-transcriptional mechanisms [4]. Pro-inﬂammatory cytokines and chemokines can affect all of the coagulation pathways. Therefore, the relationship between the presence of cytokines resulting in inﬂammation and hyper-coagulation state, is particularly relevant in the pathogenesis of thrombosis. Interleukin 1 Receptor 1 and IL-1β have been seen to be increased in cardiovascular diseases [5]. IL-1β is also known to be present in autoimmune conditions and contributes to several chronic diseases, including atherosclerosis [6]. Increased levels of IL-1β are known to play an important role in both acute and chronic inﬂammation, with resulting pathological clotting. 1. Introduction However, there is still little information available about the effects of this interleukin on the properties of blood platelet involved in clot formation. An in vitro study performed using the ﬂow cytometry method indicated that IL-1β signiﬁcantly increases the formation of blood platelet-leukocyte aggregates (PLAs). This suggests that IL-1β signiﬁcantly effects the cross-talk between blood platelets and the immune response system [5]. Flavonolignans are a group of active chemical components of silymarin—an extract obtained from the fruit of the milk thistle—Silybum marianum (L.) Gaernt. [7]. This plant, which is a member of Asteraceae family, has been used for thousands of years as a remedy for a variety of ailments [8]. Flavonolignans are structurally composed of a ﬂavonoid unit (taxifolin) and a phenylpropanoid unit (coniferyl alcohol), linked by an oxeran ring [9,10]. This type of connection is present in the formation of lignans, and gives this group of compounds its name [11]. Silymarin represents 1.5–3% of the dry fruit weight. The main represents of ﬂavonolignans presented in silymarin are silybin, isosilybin, silychristin, isosilychristin silydianin, silimonin [7,12–16], however the highest concentration, approximately 70% of the extract have the silybin, silychristin and silydianin and these compounds are the major bioactive component of extract [17]. In our previous study, we demonstrated that ﬂavonolignans, especially silybin and silychristin, are able to adenosine diphosphate (ADP)-induce blood platelets’ activation through interactions with the P2Y12 receptor [18]. Additionally, silybin and silychristin have an inhibitory effect on platelets cyclooxygenase activity, which blocks arachidonic acid metabolism in these cells [19]. Recent studies demonstrate that the ﬂavonolignans are able to inhibit the NF-κB activation pathway, which is responsible for cell reaction to IL-1β. For this reason, we decided to investigate the effect that ﬂavonolignans (silybin, silychristin and silydianin) have on the IL-1β-induced interaction between platelets and leukocytes, as well as on the expression and secretion of pro-inﬂammatory and prothrombotic factors. 2.1. Reagents Interleukin-1 beta was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Dimethyl sulfoxide (DMSO), Tris and the ﬂavonolignans (silybin, silychristin and silydianin (Figure S1) were all obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Flow cytometry reagents: anti-CD61/FITC, anti-CD61/PE, anti-CD45/PE, isotype controls, BD FACSTM Lysing Solution and CellFix were all obtained from Becton Dickinson (San Diego, CA, USA). All of the other chemicals were of reagent grade or the highest quality available. Nutrients 2017, 9, 1022 3 of 11 2.4. Flow Cytometry Analysis of Platelet-Leukocyte Aggregates First, the blood samples were stained in BD FACS lysing solution. After 1 h of ﬁxation, the samples were stained with speciﬁc antibodies: anti-CD61/FITC, anti-CD45/PE (6 µL of each antibody + 50 µL of sample), and left for 30 min in the dark, at room temperature. Next, 500 µL of 1% Cellﬁx was added to each sample. All of the samples were centrifuged (2500× g, 10 min), and the precipitate obtained was then suspended in 500 µL of 0.9% NaCl. The ﬂuorescence of 10,000 leucocytes (CD45/PE-positive objects) was measured using the CUBE 6 (Pertec, Görlitz, Germany) ﬂow cytometer. Blood platelet-leukocyte aggregates were detected using CD61-FITC and CD45-PE ﬂuorescence (Figure S2). The speciﬁc ﬂuorescence fractions were obtained after the subtraction of nonspeciﬁc ﬂuorescence in the control samples (labelled with proper isotype control). Gates for PE and FITC ﬂuorescents were estimated based on the ﬂuorescence of unstained probes. The percentage values of CD61+/CD45+ positive objects (PLAs) were calculated relative to the total number of leucocytes (CD45 positive cells) present in each sample. All of the data analysis was performed in CyFlow version 1.5.1.2 (Pertec, Görlitz, Germany). 2.3. Samples Preparation The fresh whole blood samples were pre-incubated with ﬂavonolignans (silybin, silychristin and silydianin) in the concentration range of 10–100 µM, at 37 ◦C. All of the compounds tested were initially dissolved in 20% DMSO to a preliminary concentration of 20 mM. Other solutions of the compounds used were also 20% DMSO (prepared in 50 mM Tris-buffered saline [TBS], pH 7.4). The ﬁnal DMSO concentration of all the samples was 0.1%. In the control samples, the same volume of solvent was added (20% DMSO prepared at 50 mM TBS, pH 7.4), with the probes warmed at 37 ◦C [18–20]. After 30 min, to each sample (control or pre-incubated with ﬂavonolignans) IL-1β (10 ng/mL) was added. Treatment with IL-1β was conducted for 1 h at 37 ◦C, and samples were used for appropriate analysis. An additional sample was not activated. 2.2. Blood Samples Blood samples collected from twelve different healthy donors were purchased from the Regional Centre for Transfusion Medicine in Lodz (Poland). All of the samples had been drawn in the morning (between 8 a.m. and 10 a.m.), from fasting donors and immediately transferred to the laboratory. All donors had been checked by a doctor and were found to have had no cardiovascular disorders, allergies, lipid, or carbohydrate metabolism disorders, nor any traces of medication. Blood was collected according to the standard protocol to the CPDA-1 (Citrate Phosphate Dextrose Adenine Solution) containing blood collection bag with double port, 450 mL (KRUUSE, Langeskov, Denmark). Our analysis of the blood samples was performed under the guidelines of the Helsinki Declaration for Human Research, and approved by the Committee on the Ethics of Research in Human Experimentation at the University of Lodz (with Resolution No. 16/KBBN-UŁ/II/2016). 2.6. Isolation of RNA and Reverse Transcription Frozen whole blood samples (−80 ◦C) were lysed using TRI Reagent® (Sigma-Aldrich), after which separation was performed. Then the InviTrap Spin Universal RNA Mini Kit (Stratec Biomedical Systems, Birkenfeld, Germany) was used to purify the RNA-containing aqueous phase. The quantity and purity of RNA were estimated using a Synergy HTX Multi-Mode Microplate Reader equipped with a Take3 Micro-Volume Plate (BioTek Instruments, Inc., Winooski, VT, USA). Total RNA (0.15 µg) was reverse transcribed into cDNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, USA). All of the steps were performed according to the manufacturer’s recommendations. 2.7. Real-Time PCR Expression levels of both studied genes were obtained using the following TaqMan probes: Hs00174128_m1 for the human TNF gene, Hs00989291_m1 for the human INF-γ gene, and Hs99999901_s1 as an endogenous control, which was the human 18S rRNA gene (Life Technologies, Carlsbad, CA, USA). Real-time PCR analyses were performed using a CFX96 real-time PCR system (BioRad Laboratories, Hercules, CA, USA) with a TaqMan Universal Master Mix II, without UNG (Life Technologies). All procedures were performed according to the manufacturers’ protocols. Relative expressions of the studied genes were calculated using the equation 2−∆Ct, where ∆Ct = Cttarget gene −Ct18S rRNA. 2.5. Cytokine Level Analysis After preparation, the samples were centrifuged (2500× g, 15 min) to obtain plasma. The following cytokine levels: Interleukin 2 (IL-2), tumuor necrosis factor (TNF), interferon α (INF-α), interferon γ (INF-γ), transforming growth factor β (TGF-β), were all measured using commercial ELISA kits (Mabtech, Nacka Strand, Sweden) in accordance with the manufacturer’s protocol. All of the measurements were made using MaxiSorp plates (Nunv, Roskilde, Denmark). Absorbance was measured at 450 nm using the SPECTROstar Nano Microplate Reader (BMG Labtech, Ortenberg, Germany). 4 of 11 Nutrients 2017, 9, 1022 2.8. Statistical Analysis All experiments were performed in duplicate, calculated as mean values and expressed as mean ± SD. All-statistical analyses were performed using Stats Direct statistical software Version. 2.7.2 (StatsDirect software, Cheshire, UK). The results obtained were analysed for normality using a Shapiro-Wilk test. Next, the results were analysed for equality of variance using Levene’s test. The signiﬁcance of the differences between the values was analysed using ANOVA, followed by Tukey’s range test for multiple comparisons (for data with normal distribution and equality of variance), and the Kruskal-Wallis test; p < 0.05 was accepted as statistically signiﬁcant. 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) I the e t te e dete i ed the effe t of fla o oli a o IL 1β i du ed ytoki 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) In the next step, we determined the effects of flavonolignans on IL 1β induced cytokine production by blood cells using the ELISA method. For this analysis, we selected 5 cytokines: IL-2, TGF-β, TNF, INF-α, and INF-γ. In all of them, except for TGF-β, after IL-1β treatments of blood samples a statistically significant increase was observed. The highest induction of production by IL- 1β was observed for TNF (about 5 times—from 355 pg/mL to 1632 pg/mL), and INF-γ (about 7 times—from 255 pg/mL to 1701 pg/mL). Subsequently, we evaluated the inhibitory effect of flavonolignans on the IL-1β-induced production of cytokines by blood cells. In all samples in which the blood had been treated with silybin and silychristin, a reduction of cytokine concentration was observed (Table 1). In samples treated with 100 µM silychristin and 100 µM silybin, the levels of produced cytokines were reduced by about 90%. In samples treated with silydianin, we observed some inhibitory tendencies, however, none were statistically significant. In the next step, we determined the effects of flavonolignans on IL-1β-induced cytokine production by blood cells using the ELISA method. For this analysis, we selected 5 cytokines: IL-2, TGF-β, TNF, INF-α, and INF-γ. In all of them, except for TGF-β, after IL-1β treatments of blood samples a statistically significant increase was observed. The highest induction of production by IL- 1β was observed for TNF (about 5 times—from 355 pg/mL to 1632 pg/mL), and INF-γ (about 7 times—from 255 pg/mL to 1701 pg/mL). Subsequently, we evaluated the inhibitory effect of flavonolignans on the IL-1β-induced production of cytokines by blood cells. In all samples in which the blood had been treated with silybin and silychristin, a reduction of cytokine concentration was observed (Table 1). In samples treated with 100 µM silychristin and 100 µM silybin, the levels of produced cytokines were reduced by about 90%. In samples treated with silydianin, we observed some inhibitory tendencies, however, none were statistically significant. In the next step, we determined the effects of ﬂavonolignans on IL-1β-induced cytokine production by blood cells using the ELISA method. For this analysis, we selected 5 cytokines: IL-2, TGF-β, TNF, INF-α, and INF-γ. In all of them, except for TGF-β, after IL-1β treatments of blood samples a statistically signiﬁcant increase was observed. 3.1. Flavonolignans Effect on IL-1β-Induced Formation of Blood Platelet-Leukocyte Aggregates Results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 2. The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; p < 0.01, ** p < 0.001. 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) In the next step, we determined the effects of flavonolignans on IL-1β-induced cytokine production by blood cells using the ELISA method. For this analysis, we selected 5 cytokines: IL-2, TGF-β, TNF, INF-α, and INF-γ. In all of them, except for TGF-β, after IL-1β treatments of blood Figure 2. The effect ﬂavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label ﬂow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) In the next step, we determined the effects of ﬂavonolignans on IL-1β-induced cytoki oduction by blood cells using the ELISA method. For this analysis, we selected 5 cytokin 2 TGF β TNF INF α and INF γ In all of them except for TGF β after IL 1β treatments Nutrients 2017, 9, 1022 N t i t 2017 9 1022 , , Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. Results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. Results of double-label ﬂow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Nutrients 2017, 9, 1022 5 of 11 Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. 3.1. Flavonolignans Effect on IL-1β-Induced Formation of Blood Platelet-Leukocyte Aggregates Results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. Results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. Results of double-label ﬂow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. Results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 2. The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 2. The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Figure 2. The effect ﬂavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label ﬂow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Figure 2. 3.1. Flavonolignans Effect on IL-1β-Induced Formation of Blood Platelet-Leukocyte Aggregates The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Figure 2. The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Figure 2. The effect ﬂavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label ﬂow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Figure 2. The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Figure 2. The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. Figure 2. The effect ﬂavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label ﬂow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. 3.2. 3.1. Flavonolignans Effect on IL-1β-Induced Formation of Blood Platelet-Leukocyte Aggregates To determine the level of interaction between blood platelets and leucocytes we applied double-label ﬂow cytometry as an investigative method. Based on ﬂow cytometry measurements, our results clearly indicate that IL-1β is statistically signiﬁcant (p < 0.001) in the induction (about three times–6.9% vs. 22.8%) of the formation of platelets-leukocytes aggregates (Figure 1). Next, we observed that, dose dependent, two of the three tested ﬂavonolignans–silychristin and silybin–inhibit the IL-1β-induced formation of blood platelet-leukocyte aggregates in the whole blood samples (Figure 2). In the highest used concentration (100 µM), it was observed that both silychristin and silybin are able to reduce the formation of platelet-leukocyte aggregate formation in IL-1β-induced samples to similar values, as observed in the control samples (without IL-1β)—22.8% vs. 7.5% and 7.6%, respectively. 5 of 11 5 of 11 5 of 11 rients 2017, 9, 1022 5 of Nutrients 2017, 9, 1022 5 of 11 Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. Results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Figure 2. The effect flavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the formation of blood platelet-leukocyte aggregates. The results of double-label flow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12; * p < 0.01, ** p < 0.001. 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) In the next step, we determined the effects of flavonolignans on IL-1β-induced cytokine production by blood cells using the ELISA method. For this analysis, we selected 5 cytokines: IL-2, TGF-β, TNF, INF-α, and INF-γ. In all of them, except for TGF-β, after IL-1β treatments of blood l t ti ti ll i ifi t i b d Th hi h t i d ti f d ti b IL Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. Results of double-label ﬂow cytometry measurements are expressed as the amount of CD61+/CD45+ objects in the whole CD45+ population (presented as %), n = 12, p < 0.001. Nutrients 2017, 9, 1022 5 of 11 Figure 1. The effect of IL-1β (10 ng/mL) on the formation of blood platelet-leukocyte aggregates. 3.1. Flavonolignans Effect on IL-1β-Induced Formation of Blood Platelet-Leukocyte Aggregates Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) I th t t d t i d th ff t f fl li IL 1β i d d t ki 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) I the e t te e dete i ed the effe t of fla o oli a o IL 1β i du ed ytoki 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) The highest induction of production by IL-1β was observed for TNF (about 5 times—from 355 pg/mL to 1632 pg/mL), and INF-γ (about 7 times—from 255 pg/mL to 1701 pg/mL). Subsequently, we evaluated the inhibitory effect of ﬂavonolignans on the IL-1β-induced production of cytokines by blood cells. In all samples in which the blood had been treated with silybin and silychristin, a reduction of cytokine concentration was observed (Table 1). In samples treated with 100 µM silychristin and 100 µM silybin, the levels of produced cytokines were reduced by about 90%. In samples treated with silydianin, we observed some inhibitory tendencies, however, none were statistically signiﬁcant. 6 of 11 6 of 11 Nutrients 2017, 9, 1022 Table 1. The effect of ﬂavonolignans; silychristin, silybin and silydianin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the production of pro-inﬂammatory cytokines (IL-2, TNF, INF-α, INF-γ, TGF-β). The levels of pro-inﬂammatory cytokines were estimated in plasma obtained from whole blood samples treated with IL-1β and ﬂavonolignans and presented as a mean of concentration ± SD, n = 12; * p < 0.05, p < 0.01, * p < 0.001. 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes I o de to e aluate the e ha i of ily h i ti a d ilybi a ti i fla 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes I d t l t th h i f il h i ti d il bi ti i f 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF γ and TNF Genes In order to e aluate the mechanism of silychristin and silybin anti infl In order to evaluate the mechanism of silychristin and silybin anti-inﬂammatory effects, we performed a gene expression analysis at the mRNA level using the Real-Time PCR method. We decided to evaluate changes in mRNA expression for INF-γ and TNF genes, for which expression was induced by IL-1β (about 25 [0.0101 vs. 0.2473] and 11 [0.0462 vs. 0.508] times, respectively). In our measurements, we found that, in a dose-dependent manner, both silychristin and silybin inhibit the IL-1β-induced expression of INF-γ and TNF genes at the mRNA level (Figures 3 and 4). Both compounds have similar inhibitory effects, with the strongest observed at the highest concentration (100 µM), in which gene expressions were reduced to a level observed in the control samples without IL-1β treatment. In order to evaluate the mechanism of silychristin and silybin anti-inflammatory effects, we performed a gene expression analysis at the mRNA level using the Real-Time PCR method. We decided to evaluate changes in mRNA expression for INF-γ and TNF genes, for which expression was induced by IL-1β (about 25 [0.0101 vs. 0.2473] and 11 [0.0462 vs. 0.508] times, respectively). In our measurements, we found that, in a dose-dependent manner, both silychristin and silybin inhibit the IL-1β-induced expression of INF-γ and TNF genes at the mRNA level (Figures 3 and 4). Both compounds have similar inhibitory effects, with the strongest observed at the highest concentration (100 µM), in which gene expressions were reduced to a level observed in the control samples without IL-1β treatment. In order to evaluate the mechanism of silychristin and silybin anti inflammatory effects, we performed a gene expression analysis at the mRNA level using the Real-Time PCR method. 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes I o de to e aluate the e ha i of ily h i ti a d ilybi a ti i fla g g y y µ on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0.001. Figure 3. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < Figure 3. The effect of ﬂavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0.001. g g y y µ on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0.001. 0.001. Figure 4 The effect of flavonolignans; silychristin and silybin in concentrations of 10 50 and 100 µM Figure 4 The effect of ﬂavonolignans; silychristin and silybin in concentrations of 10 50 and 100 µM on Figure 4. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2-∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; p < 0 001 Figure 4. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2-∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; p < 0.001. Figure 4. The effect of ﬂavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes I o de to e aluate the e ha i of ily h i ti a d ilybi a ti i fla We decided to evaluate changes in mRNA expression for INF-γ and TNF genes, for which expression was induced by IL-1β (about 25 [0.0101 vs. 0.2473] and 11 [0.0462 vs. 0.508] times, respectively). In our measurements, we found that, in a dose-dependent manner, both silychristin and silybin inhibit the IL-1β-induced expression of INF-γ and TNF genes at the mRNA level (Figures 3 and 4). Both compounds have similar inhibitory effects, with the strongest observed at the highest concentration (100 µM), in which gene expressions were reduced to a level observed in the control samples without IL-1β treatment. Figure 3. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0 001 Figure 3. The effect of ﬂavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0.001. Figure 3. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0.001. stin and silybin in concentrations of 10 50 Figure 3. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0 001 Figure 3. The effect of ﬂavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of INF-γ gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene-18S rRNA) ± SD, n = 12; p < 0.001. 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) I the e t te e dete i ed the effe t of fla o oli a o IL 1β i du ed ytoki 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) 3.2. Flavonolignans Effect on IL-1β-Induced Cytokine Production (IL-2, TGF-β, TNF, INF-α and INF-γ) Cytokine Control (without IL1-β) Control (with IL1-β) IL1-β + Silychristin (µM) IL1-β + Silybin (µM) IL1-β + Silydianin (µM) 10 50 100 10 50 100 10 50 100 INF-γ (pg/mL) 255 ± 90 1701 ± 411 * 1278 ± 339 555 ± 175 * 262 ± 82 * 1339 ± 337 574 ± 149 * 283 ± 87 * 1677 ± 398 1665 ± 483 1542 ± 405 TNF (pg/mL) 355 ± 110 1632 ± 473 * 1216 ± 396 478 ± 160 * 338 ± 119 * 1286 ± 408 * 466 ± 115 * 352 ± 115 * 1614 ± 453 1610 ± 423 1494 ± 360 INF-α (pg/mL) 13.3 ± 6.4 20.8 ± 8.0 ** 16.8 ± 6.1 15.0 ± 4.9 * 13.3 ± 4.0 *** 16.3 ± 4.4 * 14.8 ± 4.5 * 12.6 ± 3.5 * 20.1 ± 5.1 18.8 ± 4.4 17.9 ± 3.9 IL-2 (pg/mL) 116 ± 28 189 ± 49 168 ± 45 129 ± 19 116 ± 24 * 164 ± 38 135 ± 21* 112 ± 17 * 191 ± 46 175 ± 31 172 ± 30 TGF-β (pM) 78.3 ± 23.3 86.8 ± 27.9 84.2 ± 24.1 83.6 ± 28.1 83.6 ± 27.9 86.8 ± 31.8 82.8 ± 19.2 82.3 ± 27.7 84.1 ± 24.5 87.2 ± 23.5 85.3 ± 25.1 Nutrients 2017, 9, 1022 Nutrients 2017, 9, x FOR Nutrients 2017, 9, x FOR 7 of 11 7 of 11 7 of 11 7 of 11 7 of 11 7 of 11 4. Discussion Thrombosis and inﬂammation are closely related pathophysiological processes with multicellular activation involving blood platelets and leukocytes. Coronary artery disease, including Acute Coronary Syndromes (ACS), which refers to group of clinical symptoms is compatible with acute myocardial ischaemia, associated with coronary artery thrombosis is one of the most common causes of death in the world. It is now believed that elevated levels of inﬂammatory factors in the blood promote the development of cardiovascular events, and that chronic inﬂammation plays a key role in the pathogenesis of atherosclerosis and acute coronary syndromes. Much intensive study by various leading scientiﬁc centres around the world conﬁrms that the balance between pro-and anti-inﬂammatory processes inﬂuence the risk of developing ACS [21]. In blood samples obtained from persons with acute coronary syndrome episodes, elevated concentrations of chemokines (IL-8, MCP-1, eotaxin, MIP-1α, and IP-10) and cytokines (IL-1, IL-6, IL-7, IL-12, IL-17, IFN-α, and granulocyte-macrophage colony-stimulating factor) regulating both innate and adaptive immunity have been observed [22]. Numerous studies form cardiovascular disease have shown that the platelet-leukocyte interaction (so-called crosstalk) was increased [23]. Thrombosis and inﬂammation involve complex platelet-leukocyte interaction, the details of which have not been fully elucidated. Under conditions that mimic a physiological state, the platelet-leukocyte cross-talk involves multiple mediators and mechanisms [24], and is a common feature of atherothrombosis and inﬂammatory immune reactions. In the last few years, it has been suggested that platelet-leukocyte interactions contribute to cardiovascular disease [25]. The creation of PLAs involves the recruitment leukocytes to the atherosclerotic plaques and stimulates them to release collagenases such as MMP-8, MMP-9, and proteinase 2, which affect the reduction of the atherosclerotic plaque stability by degrading the collagen of the extracellular matrix [26]. As a result of pathological platelet activation, there is an increased immune response and an increasing number of platelet-leukocyte complexes formed at the site of the atherosclerotic plaque, which can lead to its rupture [27]. An increased number of platelet-leukocyte aggregates circulating in blood have been observed in patients with ACS [28]. Additionally, in patients who died following an ACS episode, platelet-neutrophil interactions occurring at the site of ruptured plaques have been observed [29]. Flow-Cytometric analysis of platelet aggregation showed the signiﬁcant effect of IL-1β on the formation of PLAs, showed the signiﬁcant effect of IL-1β on the formation of PLAs, which suggests that IL-1β signiﬁcantly affects pro-inﬂammatory and prothrombotic cross-talk between platelets and leukocytes. 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes 3.3. Silychristin and Silybin Effect on mRNA Expression for INF-γ and TNF Genes I o de to e aluate the e ha i of ily h i ti a d ilybi a ti i fla The results are expressed as a mean of 2−∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; p < 0.001. Figure 4. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2-∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; Figure 4. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2-∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; p < 0.001. Figure 4. The effect of ﬂavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; p < 0.001. Figure 4. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2-∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; Figure 4. The effect of flavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2-∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; p < 0.001. Figure 4. The effect of ﬂavonolignans; silychristin and silybin in concentrations of 10, 50, and 100 µM on the IL-1β (10 ng/mL) induced the expression of TNF gene (measured at the mRNA level). The results are expressed as a mean of 2−∆ct (according to the reference gene—18S rRNA) ± SD, n = 12; p < 0.001. Nutrients 2017, 9, 1022 8 of 11 4. Discussion In the current study, we have also observed the very strong ability of IL-1β to induce the formation of blood platelet-leukocyte aggregates (Figure 1). However, this effect was abolished with the application of two ﬂavonolignans: silychristin and silybin. Depending on the dose, these two inhibit the formation of blood platelet-leukocyte aggregates induced by IL-1β (Figure 2). Leukocytes enhance the inﬂammatory process within the atherosclerotic plaque’s formation. Pro-inﬂammatory cytokines, such as TNF and INF-γ, as inducers of endothelial cell activation and expression of adhesive particles, play a key role in the recruitment of leukocytes, particularly monocytes, from the blood stream [30]. TNF and IFN-γ are particularly toxic to endothelial cells, and activate monocytes, macrophages, and phospholipase A2, which intensiﬁes the synthesis of pro-inﬂammatory eicosanoids in the atherosclerotic plaque [31]. Additionally, these inﬂammatory mediators exacerbate the expression of MMPs in macrophages, as well as in endothelial and smooth muscle cells. MMP activity, regulated by inﬂammatory mediators, is responsible for the disintegration of interstitial collagen, leading to thinning and ﬁbrous cup (FC) depletion, causing a susceptibility to atherosclerotic plaque’s fracture [32]. INF-γ also inhibits the ability of smooth muscle cells to synthesize the new interstitial collagen ﬁbres required for the repair of the FC extracellular matrix [33]. In our study, we observed that of all the tested cytokines (IL-2, TNF, INF-α, INF-γ, TGF-β), IL-1β most induces the production of TNF and INF-γ (Table 1). Additionally, in this study, we have observed that the two tested ﬂavonolignans: silybin and silychristin, reduce the IL-1β-induced Nutrients 2017, 9, 1022 9 of 11 production of cytokines. In the highest concentration of the tested compounds (100 µM), we observed the almost complete abolition of the pro-inﬂammatory action of IL-1β. Contrary to numerous in vitro and in vivo studies on ﬂavonolignans, including investigations of hepatoprotective activity [34], their anti-inﬂammatory properties and the therapeutic effects have been less thoroughly described. However, some information is available [35]. The biochemical mechanisms include the modulation of a variety of cell-signalling pathways, resulting in the reduction of pro-inﬂammatory mediators. Both silymarin [36] and pure silybin [37] suppress NF-κB, which plays a crucial role in regulating immune response and inﬂammation through the regulation of the expression of various genes involved in these processes [38]. A non-activated NF-κB is maintained in the cytoplasm by the inhibitory protein 1-κBα (IκBα). 4. Discussion The activation of NF-κB occurs via the phosphorylation of IκBα, leading to its proteasome-mediated degradation, release from IκBα complex, and the translocation to the nucleus. NF-κB pathway plays an essential role in activating genes encoding pro-inﬂammatory cytokines (TNF, IFN, IL-1β, IL-2, IL-6, and granulocyte macrophage colony-stimulating factors), chemokines (IL-8, macrophage inﬂammatory protein 1α, macrophage chemotactic protein 1), enzymes that generate mediators of inﬂammation (5-lipoxygenase), immune receptors (interleukin-2 receptors), and also adhesion molecules (E-selectin, intercellular adhesion molecule 1) [39]. The study presented by Trappoliete et al. [40], shows that silybin is able to inhibit the IκBα phosphorylation, which suppresses the IL-1β-induced activation of the NF-κB pathway in hepatic stellate cells (HSC). Silybin was also able to suppress the antigen-stimulated calcium uptake and the activation of NF-κB, resulting in the signiﬁcant reduction of TNF and IL-6 production [41]. In the present study, in samples treated with silybin and silychristin, we observed a reduction of the levels of cytokines secreted into plasma, as well as the inhibition of IL-1β-induced expression of TNF and IFN-γ, which conﬁrms the anti-inﬂammatory effect of these two ﬂavonolignans. Additionally, in the last few years, novel forms of ﬂavonolignans administration have been developed that posses a very high bioavailability (with plasma concentrations ranging from 60–70 µM) [42]. This corresponds with the concentrations of ﬂavonolignans that have a biological effect in our study (10–100 µM). 5. Conclusions In summary, our results indicate that ﬂavonolignans may be used in the prevention of cardiovascular disease with dual action as antiplatelet and anti-inﬂammatory agents. Further studies using a larger sample size and additional studies, demonstrating NF-κB activity is necessary before the ﬁnal statement about the role of the inhibitory effect of these two ﬂavonolignans on the NF-κB pathway. Supplementary Materials: The following are available online at www.mdpi.com/2072-6643/9/9/1022/s1. Figure S1: Chemical structures of ﬂavonolignans used in this study, Figure S2: Flow cytometry dot plot presented blood platelet-leukocyte aggregates. Acknowledgments: This study was supported by grants: B1611000001144.02, B161100000004601 and grant 506/1136 from the University of Lodz. Author Contributions: M.B. (corresponding author) and J.S.-B. conceived and designed the study; M.B. prepared all samples and collected and analyzed the data; M.B., A.D., E.S. performed research analysis; All co-authors wrote the paper, have read and approved the ﬁnal manuscript s of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W4301858536	https://darchive.mblwhoilibrary.org/bitstream/1912/9614/1/1-s2.0-S0304380017304969-main.pdf	English	null	A high-resolution modeling study on diel and seasonal vertical migrations of high-latitude copepods	RePEc: Research Papers in Economics	2,018	cc-by	19,391	Kanchana Bandara a,b,∗, Øystein Varpe b,c, Rubao Ji d, Ketil Eiane a a Faculty of Biosciences and Aquaculture, Nord University, 8049, Bodø, Norway b The University Centre in Svalbard, 9171, Longyearbyen, Norway c Akvaplan-niva, Fram Centre, 9296, Tromsø, Norway p y d Woods Hole Oceanographic Institution, Redﬁeld 2-14, Woods Hole, MA 02543, USA a r t i c l e i n f o Article history: Received 20 September 2017 Received in revised form 12 December 2017 Accepted 12 December 2017 Available online 26 December 2017 Keywords: Vertical migration Seasonality Phenology Optimization model Genetic algorithm Habitat choice Article history: Received 20 September 2017 Received in revised form 12 December 2017 Accepted 12 December 2017 Available online 26 December 2017 Despite diel and seasonal vertical migrations (DVM and SVM) of high-latitude zooplankton have been studied since the late-19th century, questions still remain about the inﬂuence of environmental season- ality on vertical migration, and the combined inﬂuence of DVM and SVM on zooplankton ﬁtness. Toward addressing these, we developed a model for simulating DVM and SVM of high-latitude herbivorous cope- pods in high spatio-temporal resolution. In the model, a unique timing and amplitude of DVM and SVM and its ontogenetic trajectory were deﬁned as a vertical strategy. Growth, survival and reproductive performances of numerous vertical strategies hardwired to copepods spawned in different times of the year were assessed by a ﬁtness estimate, which was heuristically maximized by a Genetic Algorithm to derive the optimal vertical strategy for a given model environment. The modelled food concentration, temperature and visual predation risk had a signiﬁcant inﬂuence on the observed vertical strategies. Under low visual predation risk, DVM was less pronounced, and SVM and reproduction occurred ear- lier in the season, where capital breeding played a signiﬁcant role. Reproduction was delayed by higher visual predation risk, and copepods that spawned later in the season used the higher food concentrations and temperatures to attain higher growth, which was efﬁciently traded off for survival through DVM. Consequently, the timing of SVM did not change much from that predicted under lower visual preda- tion risk, but the body and reserve sizes of overwintering stages and the importance of capital breeding diminished. Altogether, these ﬁndings emphasize the signiﬁcance of DVM in environments with elevated visual predation risk and shows its contrasting inﬂuence on the phenology of reproduction and SVM, and moreover highlights the importance of conducting ﬁeld and modeling work to study these migratory strategies in concert. Keywords: Vertical migration Seasonality Phenology Optimization model Genetic algorithm Habitat choice Keywords: Vertical migration Seasonality Phenology Optimization model Genetic algorithm Habitat choice © 2017 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ∗Corresponding author at: Faculty of Biosciences and Aquaculture, Nord Univer- sity, 8049, Bodø, Norway. E-mail addresses: kanchana.bandara@nord.no, kanchana bandara@live.com (K. Bandara). Contents lists available at ScienceDirect Contents lists available at ScienceDirect https://doi.org/10.1016/j.ecolmodel.2017.12.010 0304-3800/© 2017 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A high-resolution modeling study on diel and seasonal vertical migrations of high-latitude copepods Kanchana Bandara a,b,∗, Øystein Varpe b,c, Rubao Ji d, Ketil Eiane a https://doi.org/10.1016/j.ecolmodel.2017.12.010 0304-3800/© 2017 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Ecological Modelling 368 (2018) 357–376 Ecological Modelling 368 (2018) 357–376 g g ( ) Contents lists available at ScienceDirect Ecological Modelling journa l h om epa ge: www.elsevier.com/locate/ecolmodel Table 2 Table 2 Evolvable (soft) parameters optimized in the model. The ﬁrst six are proxies that deﬁne the vertical strategy. Vertical strategies of copepods spawned in different times of the year (tB) are optimized using the GA. Term Deﬁnition Range Interval Unit Light sensitivity parameter 0–Imaxa 1 mol m−2 s−1 ˇ Size-speciﬁcity of light sensitivity parameter 0–10 1 dim.less Growth allocation parameter 0–1 0.01 dim.less ı Seasonal descent parameter 0–1 0.01 dim.less Overwintering depth 1–500 10 m ε Seasonal ascent parameter 0–1 0.01 dim.less tB Time of birthb 1–8760 1 h a The upper limit of changes with the maximum surface irradiance of the model environment, i.e. Imax = 1500 mol m−2 s−1 for Environment-L, 1300 mol m−2 s−1 for Environment-M and 1100 mol m−2 s−1 for Environment-H (cf. Fig. 1). b Time of being spawned. g Mechanistic models offer an alternative means of studying zoo- plankton vertical strategies in higher resolution. Models related to DVM usually encompass the highest spatial (≤1 m), temporal (≤1 h) and biological (=individual) resolution (e.g. Fiksen and Giske, 1995; Eiane and Parisi, 2001; Liu et al., 2003; Burrows and Tarling, 2004; Hansen and Visser, 2016). Models related to SVM and diapause (i.e. hibrnation in deeper waters, e.g. Hirche, 1996) encompass the same biological resolution, but are usually coarse in spatio- temporal resolution. Here, the time intervals range from 1 h to 1d and vertical spatial elements are usually resolved to either abso- lute depth units (e.g. 1 m bins) or segregated habitats (e.g. Fiksen and Carlotti, 1998; Miller et al., 1998; Hind et al., 2000; Ji, 2011; Ji et al., 2012; Sainmont et al., 2015; Banas et al., 2016). The choice of a coarser spatio-temporal resolution of these models reﬂects the broader space and time scales at which the SVM and diapause occurs. This contrasting spatio-temporal scale makes it difﬁcult to harbor lifetime dynamics of DVM to be simulated in SVM mod- els without signiﬁcantly increasing computer time. Consequently, most models that simulate SVM tend to either fully (e.g. Hind et al., 2000) or partly (i.e. of younger developmental stages, e.g. Fiksen and Carlotti, 1998) disregard DVM. However, the validity of such simpliﬁcations are questionable, given the geographically and tax- onomically widespread nature of zooplankton DVM behavior and its ontogenetic patterns (Huntley and Brooks, 1982; Huang et al., 1993; Osgood and Frost, 1994; Hays, 1995). Table 2 It is thus interesting to investigate whether the extra biological information resulting from modeling DVM and SVM in concert is a worthy trade-off for the ele- vated computer time. If so, such models may lead to improvements of the current understanding about how environmental seasonal- a The upper limit of changes with the maximum surface irradiance of the model environment, i.e. Imax = 1500 mol m−2 s−1 for Environment-L, 1300 mol m−2 s−1 for Environment-M and 1100 mol m−2 s−1 for Environment-H (cf. Fig. 1). b Time of being spawned. ity shapes up vertical strategies, and the means of which the latter inﬂuences life histories of high latitude zooplankton. In this study, we present a model of zooplankton vertical strate- gies. The model operates in a high-latitude setting and simulates both DVM and SVM of a herbivorous copepod with an annual life cycle in high spatial (vertical) and temporal resolution. Using this model, we aim to investigate the inﬂuence of environmental vari- ables on vertical strategies, and how vertical strategies affect ﬁtness and phenology in seasonal environments. We further discuss how short-term behavior (DVM) inﬂuences and interacts in the longer- term and shape-up different life history components of copepod strategies. 1. Introduction Ohman, 1990; Loose and Dawidowicz, 1994). The long-term sea- sonal vertical migration (SVM) has a periodicity of up to one year, and reﬂects adaptations to seasonal extremities of food availabil- ity (Head and Harris, 1985; Hind et al., 2000; Bandara et al., 2016), temperature (Hirche, 1991; Astthorsson and Gislason, 2003) and predation risk (Kaartvedt, 1996; Bagøien et al., 2000; Varpe and Fiksen, 2010). In either case, since both DVM and SVM can alter feeding, growth, survival and reproduction, and ultimately affect ﬁtness (Aidley, 1981; Alerstam et al., 2003; Cresswell et al., 2011; Litchman et al., 2013), these migratory strategies are termed verti- cal strategies (Bandara et al., 2016). Vertical migration is a common behavior of many zooplankton taxa. Based on the periodicity, vertical migrations of high-latitude zooplankton are classiﬁed into diel and seasonal components, which have been studied since the late-19th century (reviewed in Russell, 1927; Cushing, 1951; Banse, 1964). The short-term diel vertical migration (DVM) has a periodicity of up to 24 h, and is understood as a strategy that trades off growth potential to reduce the mortality risk imposed by visual predators (Lampert, 1989; Empirical knowledge on zooplankton vertical strategies largely comes from studying the dynamic vertical positioning of popu- lations in a water column, and are often rather coarse in spatial (vertical) and temporal resolution (Pearre, 1979). This can under- mine the key concept that such migrations are individual responses K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 358 Table 1 (1991) to certain cues or stimuli and not a property of the population (Zink, 2002), and may complicate the understanding of the rela- tionships between vertical strategies and environmental variables (see Table 1 for examples). Moreover, since diel and seasonal ver- tical migrations occur on different spatial and temporal scales, studying these migrations together in the ﬁeld in adequate res- olution remains a major challenge. Although novel optical and acoustic methods of in-situ observation offer a solution to some of these problems (e.g. Basedow et al., 2010; Sainmont et al., 2014b; Bozman et al., 2017; Darnis et al., 2017), long-term deployment and accurately resolving the identity of the migrants remain as key challenges. to certain cues or stimuli and not a property of the population (Zink, 2002), and may complicate the understanding of the rela- tionships between vertical strategies and environmental variables (see Table 1 for examples). Moreover, since diel and seasonal ver- tical migrations occur on different spatial and temporal scales, studying these migrations together in the ﬁeld in adequate res- olution remains a major challenge. Although novel optical and acoustic methods of in-situ observation offer a solution to some of these problems (e.g. Basedow et al., 2010; Sainmont et al., 2014b; Bozman et al., 2017; Darnis et al., 2017), long-term deployment and accurately resolving the identity of the migrants remain as key challenges. Table 1 Table 1 Some endogenous and exogenous cues that are believed to proximately or ultimately regulate diel and seasonal vertical migrations of marine and freshwater zooplankton. Literature do not come from an exhaustive review and only serve as examples. Cue DVM SVM Temperature McLaren (1963), Enright (1977) Hirche (1991), Heath and Jónasdóttir (1999), Astthorsson and Gislason (2003) Light (absolute or relative irradiance from sun, moon, stars, or aurora borealis, photoperiod, spectral quality, polarization etc.) Clarke (1933), Gliwicz (1986), Frank and Widder (1997), Berge et al. (2009), Båtnes et al. (2015), Cohen et al. (2015), Bianchi and Mislan (2016), Bozman et al. (2017) Sømme (1934), Ussing (1938), Miller et al. (1991) Dissolved oxygen Devol (1981), Bianchi et al. (2013) – Water depth, transparency and UV radiation Rhode et al. (2001), Williamson et al. (2011), Ekvall et al. (2015) Dupont and Aksnes (2012) Tides, currents and advective transport Hardy (1935), Wroblewski (1982), Kimmerer and McKinnon (1987) Berge et al. (2012), Irigoien (2004) Food availability Hardy and Gunther (1935), Huntley and Brooks (1982), George (1983), Johnsen and Jakobsen (1987) Herman (1983), Hind et al. (2000), Head and Harris (1985), Bandara et al. (2016) Visual and tactile predation Zaret and Suffern (1976), Iwasa (1982), Ohman (1990), Bollens et al. (1992), Loose and Dawidowicz (1994) Kaartvedt (1996), Kaartvedt (2000), Dale et al. (1999), Bagøien et al. (2000), Varpe and Fiksen (2010) Body size, ontogeny and pigmentation Zaret and Kerfoot (1975), Uye et al. (1990), Hays et al. (1994), Dale and Kaartvedt (2000) Østvedt (1955), Hind et al. (2000) Nutritional state and lipid reserves Fiksen and Carlotti (1998), Sekino and Yamamura (1999) Visser and Jónasdóttir (1999), Thorisson (2006) Endogenous rhythms and internal biological clocks Cohen and Forward (2009), van Haren and Compton (2013) Carlisle and Pitman (1961), Miller et al. (1991), Hirche (1996) Table 1 Some endogenous and exogenous cues that are believed to proximately or ultimately regulate diel and seasonal vertical migrations of marine and freshwater zooplankton. Literature do not come from an exhaustive review and only serve as examples. Some endogenous and exogenous cues that are believed to proximately or ultimately re Literature do not come from an exhaustive review and only serve as examples. Hirche (1991), Heath and Jónasdóttir (1999), Astthorsson and Gislason (2003) Sømme (1934), Ussing (1938), Miller et al. 2. Materials and methods Although the model is not strictly individual-based, it is described following the Overview, Design concepts and Details (ODD) protocol (Grimm et al., 2006, 2010) to improve reproducibil- ity. K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 359 K. Bandara et al. / Ecological Modelling 368 (2018) 357 376 359 Fig. 1. The modelled dynamics of irradiance incident on the sea surface (hourly estimates; a, d, g), temperature (b, e, h) and food availability (c, f, i, expressed as Chlorophyll-a biomass) in the three model environments. See Appendix A1 in Supplementary material for a detailed comparison. Fig. 1. The modelled dynamics of irradiance incident on the sea surface (hourly estimates; a, d, g), temperature (b, e, h) and food availability (c, f, i, expressed as Chlorophyll-a biomass) in the three model environments. See Appendix A1 in Supplementary material for a detailed comparison. Fig. 1. The modelled dynamics of irradiance incident on the sea surface (hourly estimates; a, d, g), temperature (b, e, h) and food ava biomass) in the three model environments. See Appendix A1 in Supplementary material for a detailed comparison. 2.1. Purpose iological activity (Hirche, 1996). Overwintering stages ascend to near-surface waters as the primary production commences in the following year, molt into adults and start to reproduce (Conover, 1988; Varpe, 2012). The life cycle of the two species is usually completed within one year in most sub-Arctic and Arctic locations (Falk-Petersen et al., 2009; Daase et al., 2013), within which reside the model environments of this study. The purpose of the model is to investigate the bottom-up and top-down inﬂuences of environmental variability (i.e. irradi- ance, temperature, food-availability and predation risk) on vertical strategies of a high-latitude herbivorous copepod, and to under- stand the inﬂuences of vertical strategies on its ﬁtness and phenology. y The model runs in three 500-m deep artiﬁcial seasonal envi- ronments that represent three high-latitude locations along the southern and southeastern coast of Norway (60–70◦N). These envi- ronments do not point to speciﬁc geographic locations, but the modelled environmental dynamics were adopted from ﬁeld mea- surements from the above region (Appendix A1 in Supplementary material). The baseline model simulation (hereafter, the basic run) runs in Environment-L, representing the lower end (ca. 60◦N) of the selected geographical range. Here, the modelled irradiance, tem- perature and food availability are highly seasonal and vertically structured (Fig. 1a–c), but are assumed constant between years. The irradiance incident on the sea surface follows the global clear-sky horizontal irradiance model of Robledo and Soler (2000), and peaks at ca. 1500 mol m−2 s−1 (Fig. 1a, Appendix A1 in Supplementary material). The sea surface temperature reaches a maximum of 18 ◦C in the summer (e.g. Bagøien et al., 2000), and distributes evenly in the surface mixed layer (Fig. 1b). Below this, the temperature decreases with depth and converges to a minimum of 4 ◦C at ca. 100 m (e.g. Ingvaldsen and Loeng, 2009). The pelagic productive season extends ca. 180 days, with a chlorophyll-a peak at 8 mg m−3 in mid-April (Fig. 1c: Sakshaug et al., 2009; Daase et al., 2013). We manipulated the environmental parameters of Environment-L to formulate two additional artiﬁcial environments: Environment- M (ca. 65◦N, Fig. 1d–f) and Environment-H (ca. 70◦N, Fig. 1g–i), 2.2. Entities, state variables and scales The model consists of three entities: vertical strategies, model organism and the model environment. Vertical strategies deﬁne the timing, amplitude and the ontogenetic trajectories of DVM and SVM, and are described using six evolvable (soft) parameters (Table 2). These are hardwired to the model organism, i.e. copepods spawned in different times of the year. The model organism is a hypothetical herbivorous semelparous female copepod (hereafter, the copepod) with an annual life cycle that resembles Calanus ﬁnmarchicus and C. glacialis in terms of body size, behavior and life history strategies (Conover, 1988). These two species often dominate the copepod biomass in the North Atlantic and most Eurasian sub-Arctic and Arctic seas and shelves (Falk- Petersen et al., 2009). Their life cycle consists of an embryonic stage (egg), six naupliar stages (NI–NVI), ﬁve copepodite stages (CI–CV) and an adult. Eggs that are released in near-surface waters in the spring usually develop into CIV or CV stages toward the end of the productive season. As further development is typically constrained by the duration of the productive season and seasonal peaks of visual predation risk, CIVs and CVs descend into deeper waters and remain in a state of diapause/dormancy with minimal phys- K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 2. The model overview. Vertical strategies that deﬁne the timing and amplitude of DVM and SVM are hardwired to copepods born in different times of the year. Growth val and reproduction of these copepods are simulated in a seasonal environment to derive a ﬁtness estimate that is heuristically maximized by the GA to derive the mal vertical strategy, time of birth and several associated life history traits emerging from the model. Dashed line represents the indirect dependency of the ﬁtness mate on growth (Section 2.6.4). K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 360 / g g ( ) Fig. 2. The model overview. Vertical strategies that deﬁne the timing and amplitude of DVM and SVM are hardwired to copepods born in different times of the year. Growth, survival and reproduction of these copepods are simulated in a seasonal environment to derive a ﬁtness estimate that is heuristically maximized by the GA to derive the optimal vertical strategy, time of birth and several associated life history traits emerging from the model. Dashed line represents the indirect dependency of the ﬁtness estimate on growth (Section 2.6.4). formances. 2.4.1. Basic principles The high spatial and temporal resolution implemented in the model allow both DVM and SVM to be simulated over the entire annual life cycle of the copepod. Carlotti and Wolf (1998) have implemented a similar construct, but the SVM of their model was constrained by ﬁxing the timing of ascent and descent to match the ﬁeld observations of the region of interest. In contrast, the timing and the amplitude of DVM and SVM of our model are ﬂexible and allowed to evolve according to the environmental conditions. To 2.2. Entities, state variables and scales The ﬁtness is heuristically maximized using a Genetic Algorithm (GA, Holland, 1975) to estimate the optimal vertical strategy and optimal time of birth for a given set of environmental conditions (Fig. 2). representing the mid-point and the higher end of the selected geo- graphical range (Appendix A1 in Supplementary material). Copepods are characterized by six states: vertical location (depth), structural body mass, energetic reserve, reproductive out- put (fecundity), survivorship and developmental stage. The model has a temporal coverage of an annual cycle and a unidimensional (vertical) spatial coverage of 500 m. The time and space consist of 1 h and 1 m discreet intervals. 2.4.6. Observations For a given model environment, the model produces heuristic estimates of the optimal vertical strategy and optimal time of birth, along with a range of associated life history traits (Fig. 2, Table 3). 2.4.2. Emergence The behavioral strategies and life history traits emerging from the model are presented in Fig. 2 and described in Table 3. 2.4.3. Adaptation and sensing Copepods are sensitive to their internal states (i.e. structural body mass, mass of the energetic reserve and developmental stage) and external stimuli (i.e. irradiance, temperature, food con- centration and depth). Altogether, these determine the size- or stage-speciﬁc patterns of growth, metabolism, reproduction and vertical behavior (Section 2.6). For ambient food concentrations (F: g C ml−1) above a spe- ciﬁc saturation concentration (f), growth is food-independent, and occurs at a maximum rate (GT: g C ind−1 h−1) dependent only on the ambient temperature (T) as; (GT)i,t,z = G ′ max t,z · Wi,t (1) (1) Here, i represents individual, t time and z is depth, where G’ max (g C mg dry mass h−1) is the maximum temperature-dependent mass-speciﬁc growth rate, assuming a Carbon: dry body mass (W, mg) ratio of 0.40 (Huntley and Boyd, 1984), deﬁned as; Here, i represents individual, t time and z is depth, where G’ max (g C mg dry mass h−1) is the maximum temperature-dependent mass-speciﬁc growth rate, assuming a Carbon: dry body mass (W, mg) ratio of 0.40 (Huntley and Boyd, 1984), deﬁned as; Table 3 E Table 3 Emergent properties of the model. The timing and amplitude and of DVM and SVM altogether forms the vertical strategy of a copepod. Trait/attribute Units Description Time of birth Day of the year Time of being spawned Surface time h Uniﬁed estimate representing the timing of DVM, i.e. the stage-speciﬁc mean no. of hours per day occupied in waters with highest growth potential (usually the surface waters) Amplitude of diel vertical migration m The vertical range corresponding to the above Time of seasonal descent and ascent Day of the year Separate estimates representing the timing of SVM (ascent and descent) Amplitude of seasonal vertical migration m Overwintering depth Body mass at seasonal descent g C Structural and energetic reserve mass at the onset of diapause Onset of egg production Day of the year – Fecundity No. of eggs No. of eggs produced during the lifetime Breeding mode index dim.less Proportion of capital breeding eggs (0 = pure income breeding, 1 = pure capital breeding) Food limitation index dim.less Stage-speciﬁc total no. hours with food-limited growth (Eq. (3)) as a fraction of stage duration (0 = no food limitation, 1 = total food limitation) Development time d From egg to a given stage Longevity d Duration of the life cycle, from birth to death achieve this level of ﬂexibility, we used multiple evolvable prox- ies to represent vertical migration (Table 2). This resulted in a complex seven-dimensional optimization problem that can be efﬁ- ciently solved using heuristic techniques (Zanakis and Evans, 1981). As evolutionary algorithms provide an efﬁcient means of solving multi-dimensional optimization problems (Deb, 2001; Eiben and Smith, 2003), we used a GA as the optimization platform of this model. Further, to increase the precision of the evolvable param- eters and that of the behavioral strategies and life history traits ensued (Fig. 2), we used a GA variant with ﬂoating point represen- tation (i.e. a Real-Coded Genetic Algorithm, Davis, 1989; Lucasius and Kateman, 1989; Herrera et al., 1998). achieve this level of ﬂexibility, we used multiple evolvable prox- ies to represent vertical migration (Table 2). This resulted in a complex seven-dimensional optimization problem that can be efﬁ- ciently solved using heuristic techniques (Zanakis and Evans, 1981). As evolutionary algorithms provide an efﬁcient means of solving multi-dimensional optimization problems (Deb, 2001; Eiben and Smith, 2003), we used a GA as the optimization platform of this model. Table 3 E Further, to increase the precision of the evolvable param- eters and that of the behavioral strategies and life history traits ensued (Fig. 2), we used a GA variant with ﬂoating point represen- tation (i.e. a Real-Coded Genetic Algorithm, Davis, 1989; Lucasius and Kateman, 1989; Herrera et al., 1998). 2.5. Initialization The model initializes with seeding of N (=106) eggs at random times of the year to random depths (<50 m) of the water column. Each seed represents an embryonic stage of a copepod with a spe- ciﬁc vertical strategy, which is determined by randomly assigning values to the evolvable proxies. The ranges (bounds) and resolu- tions of these proxies are listed in Table 2. The strategy-oriented construct of this model contrasts classic individual-based models of zooplankton life history and behavior in two main ways: ﬁrst, trading off of biological resolution (strategies vs. individuals) to accommodate higher spatio-temporal resolution, and second, the lack of population-level responses such as density dependence. As a result, modelled vertical strategies do not inter- act with each other and show no quantitative feedbacks with the model environment (e.g. impact of grazing on food concentration and duration of the productive season). 2.4.4. Objectives The model uses a ﬁtness estimate that evaluates the expected reproduction and survival performances rendered by different ver- tical strategies (Section 2.6.4). G′max t,z = 0.903 · exp (0.110 · Tt,z) (2) (2) 2.6.1. Growth and development 2.6.1. Growth and development We modelled somatic growth in Carbon units (g C) accord- ing to the growth model of Huntley and Boyd (1984) (Eqs. (1)–(8) below), using a Chlorophyll-a/C ratio of 0.030 (Båmstedt et al., 1991; Sakshaug et al., 2009). This growth sub-model was used due to its simplicity and general applicability, which are shown by its utility to model several different copepod taxa with varying body sizes representing a wide range of geographical locations (e.g. Robinson, 1994; Fiksen and Giske, 1995; Roman et al., 2000). Deﬁ- nitions and units of all the terms described henceforth are listed in Table 4. 2.3. Process overview and scheduling At each timestep, the model follows vertical strategies hard- wired to copepods born in different times of the year and simulates their growth, survival and reproduction. State variables are updated simultaneously. Vertical strategies are evaluated using a ﬁtness function based on the expected survival and reproductive per- K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 361 Table 3 E Table 4 ﬁ Table 4 Deﬁnitions, values and units of the terms used in the model. Term Deﬁnition Value/formula Units a Assimilation coefﬁcient 0.70b – bt,z Clearance coefﬁcient Eq. (4)a ml mg dry mass h−1 E Egg development parameter 717e,f – fi,t,z Saturation food concentration Eq. (8)a g C ml−1 Ft,z Ambient food concentration Section 2.2 g C ml−1 (G´ımax)t,z Maximum mass-speciﬁc growth rate Eq. (2)a g C mg dry mass h−1 (GF)i,t,z Food-limited growth rate Eq. (3)a g C ind−1 h−1 (GT )i,t,z Non food-limited growth rate Eq. (1)a g C ind−1 h−1 Hi,t,z Survivorship Eq. (15) – i Individual – – I´ıt,z Remapped It,z 0.9 ≥ I´ı ≥ 0.1 – It ,0 Irradiance incident on sea surface Appendix A1 in Supplementary materialc mol m−2 s−1 It,z Downwelling irradiance at depth z Eq. (9) mol m−2 s−1 j Developmental stage 0–12 (Egg–Adult) – Ki,t Scalar for visual predation risk 1 > K > 0 – kt,z Respiratory coefﬁcient Eq. (5)a g C mg dry mass h−1 (Mn)t,z Non-visual predation risk Section 2.6.2.1 – (Ms)i,t,z Starvation risk Eq. (12) – mt,z Exponent (respiration) Eq. (6)a – (Mv)i,t,z Visual predation risk Eq. (10) – N No. of initial seeds 1,000,000 – nt,z Exponent (clearance) Eq. (7)a – Ri Fecundity Eq. (13) no. of eggs t Time 1–8760 h Tt,z Ambient temperature Section 2.2 ◦C Ui,t Cruising velocity Eq. (11) m h−1 (Wc)i,t Structural mass – g C WE Unit egg mass 0.55d g C Wi,t Dry body mass (assuming 40% C) – mg (Wq)i,t Catabolized structural mass (proportion to the maximum lifetime structural mass) 0 ≥ Wq ≥ 0.5 – (WR)i,t,z Matter allocated for egg production – g C (Ws)i,t Storage (energetic reserve) mass – g C Wx Stage-speciﬁc critical molting mass Table 5 g C z Depth 0–500 m ˚ Termination condition of the RCGA Section 2.6.4 – Light attenuation coefﬁcient 0.06g m−1 ω Parameter for weighing ﬁtness 0 or 1 – ˝i Fitness Eq. (14) – a Huntley and Boyd (1984) (2)). The growth of early developmental stages (NI–CIII) is solely allocated to the building up of structural mass (Wc, g C, Fig. 3a, b and Table 5). where two terms of the right-hand side of the equation refer to the assimilation and respiratory rates respectively. 2.4.5. Prediction and stochasticity 2.4.5. Prediction and stochasticity If the ambient food concentration drops below the saturation concentration, the growth occurs at a rate limited by food avail- ability (GF) as; The vertical search pattern of copepod behavior is based on a semi-stochastic predictive algorithm (Section 2.6.2.2 and Appendix A2 in Supplementary material). Stochasticity plays a central role in the model initialization (Section 2.5) and selection, recombination and mutation operators of the GA (Section 2.6.4). (GF)i,t,z = a · bt,z · Wnt,z i,t · Ft,z − k · Wmt,z i,t (3) (3) K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 362 Table 4 ﬁ The assimila- tion coefﬁcient (a) is assumed to be constant (Table 4), but Huntley and Boyd (1984) found that the coefﬁcients of clearance (b) and respiration (k), and the exponents (n and m) vary with ambient temperature as; where two terms of the right-hand side of the equation refer to the assimilation and respiratory rates respectively. The assimila- tion coefﬁcient (a) is assumed to be constant (Table 4), but Huntley and Boyd (1984) found that the coefﬁcients of clearance (b) and respiration (k), and the exponents (n and m) vary with ambient temperature as; The embryonic development follows a Bˇelehrádek temperature function (Campbell et al., 2001; Ji et al., 2012). The post-embryonic development (from stage j to j + 1) occurs only if Wc exceeds a stage-speciﬁc critical molting mass (Wx, g C, Table 5). However, for simplicity, we did not model the dependence of molting process on the physiological state (Nival et al., 1988) and the limitation of growth by the exoskeleton (Mauchline, 1998). bt,z = 1.777 · exp (0.234 · Tt,z) (4) kt,z = 0.375 · exp (0.0546 · Tt,z) (5) nt,z = 0.671 · exp (0.0199 · Tt,z) (6) mt,z = 0.858 · exp (−0.008 · Tt,z) (7) (7) 2.6.2.1. Predation risk. Visual (v) and non-visual (n) predators induce mortality, which is estimated as a probability following Eiane and Parisi (2001) as; At the point where F reaches f, Eqs. (1) and (3) balance out, and the f becomes; fi,t,z = G ′ max t,z · Wi,t + kt,z · W mt,z i,t a · bt,z · W nt,z i,t (8) It,z = It,0 · exp − · z (9) (9) (8) (8) where Iz and I0 are irradiance at depth z and surface at a given time, and (=0.06 m−1) is the attenuation coefﬁcient for downwards directed irradiance in the water column. We remapped irradiance (I) between 0.1–0.9 (I′) so that visual predation risk is not nulliﬁed even at the lowest levels of irradiance, and the copepod has some chance of survival even at highest levels of irradiance. This growth sub-model is not applicable to the ﬁrst two nau- plii stages, which do not feed (Fig. 3a, Marshall and Orr, 1972; Mauchline, 1998). For simplicity, we assumed the growth of NI and NII stages to occur at a temperature-dependent rate (Eqs. (1) and K. Bandara et al. Table 4 ﬁ / Ecological Modelling 368 (2018) 357–376 3 Fig. 3. Simpliﬁed physiological pathways modelled in this study. Some life stages are grouped together due to their similarities in energy allocation patterns (a–f). Starvati (highlighted in red) triggers catabolic pathways marked in red. T and F are Boolean values true and false. is the growth allocation parameter (Table 2). A comparat summary is given in Table 5. K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 363 Fig. 3. Simpliﬁed physiological pathways modelled in this study. Some life stages are grouped together due to their similarities in energy allocation patterns (a–f). Starvation (highlighted in red) triggers catabolic pathways marked in red. T and F are Boolean values true and false. is the growth allocation parameter (Table 2). A comparative summary is given in Table 5. modelled in this study. Some life stages are grouped together due to their similarities in energy allocation patterns (a–f). Starvation hways marked in red. T and F are Boolean values true and false. is the growth allocation parameter (Table 2). A comparative 2.6.2.2. Diel vertical migration. The copepod may perform DVM to trade off growth potential to minimize the visual predation risk. We used the photoreactive behavior as a proxy to estimate the tim- ing and amplitude of DVM (e.g. Kerfoot, 1970; Carlotti and Wolf, 1998). Here, , an evolvable light sensitivity parameter (Table 2) was used to deﬁne an irradiance threshold above which induces a negative phototatic response in the vertical swimming behavior (Båtnes et al., 2015; Cohen et al., 2015). At any given time, the cope- pod occupies a depth with an irradiance level (It,z) below . From a series of possible depth bins that satisfy the It,z < condition, we assumed that the copepod searches and occupies the depth that maximizes its growth potential. For simplicity, we further assumed that internal state-dependent factors, such as hunger and satia- tion have a negligible inﬂuence on the modelled DVM. The vertical search pattern was predicted using a biased random walk algorithm (Codling, 2003, Appendix A2 in Supplementary material), assum- ing that the copepod is neutrally buoyant and vertically moves in the water column at a maximum velocity (hereafter cruising veloc- 2.6.2.2. Diel vertical migration. The copepod may perform DVM to trade off growth potential to minimize the visual predation risk. Table 5 a Feeding ceases during diapause. b Does not allocate surplus growth to develop the energetic reserve, but inherit the reserves from its developmental progression. c Reduces during diapause. Feeding ceases during diapause. b Does not allocate surplus growth to develop the energetic reserve, but inherit the reserves from its developmental progression. c Reduces during diapause Fig. 4. Relationships of (a) visual predation risk scalar, (b) cruising velocity, (c) l sensitivity parameter and (d) the total body mass of the copepod with its struct mass (Wc). The cruising velocity (U) model was ﬁtted using laboratory and ﬁ estimates of Calanus spp. from Hardy and Bainbridge (1954), Greene and Lan (1985) and Heywood (1996) (points in panel b). The different linear models fo that scale the light sensitivity parameter () are optimized in the model (Tabl The lower and upper border of the shaded polygon (panel d) represent the t body mass for growth allocation parameter () = 0 and 1 respectively. ity, U). We used several stage-speciﬁc cruising velocity estimates of Calanus spp. available in the literature (Fig. 4b), and related those to body mass as; Ui,t = 8.0116 · (Wc)0.4531 i,t (11) Ui,t = 8.0116 · (Wc)0.4531 i,t (11) We considered the size- or stage-speciﬁc variability of DVM as a response to size-dependent visual predation risk (Zaret and Kerfoot, 1975; Uye et al., 1990; Hays et al., 1994; Eiane and Ohman, 2004) and modelled it by scaling the light sensitivity parameter () with the body mass (Wc). As data on the light sensitivity of younger developmental stages (NI–CIII) of Calanus spp. is rare, we could not derive a general relationship between Wc and . To address this, we deﬁned an evolvable parameter ˇ that describes the size speci- ﬁcity of , which, at its maximum (ˇ = 10) downscales of the adult female to 10% of that of the egg/NI (Fig. 4c). Higher trajectories than ˇ = 10 were not used, as it was shown in the trial runs that the model always converges on ˇ < 10 even at highest levels of visual predation risk. 2.6.2.3. Energy storage. CIV and CV stages can allocate a speciﬁc fraction from surplus growth (evolvable growth allocation param- eter: , Table 2) to build up an energy reserve (Fig. 3c) that possesses a maximum size of 70% of the total body mass (Fig. 4d, Fiksen and Carlotti, 1998). 2.6.2.4. Seasonal vertical migration. Table 5 Table 5 Developmental stages, their maximum structural body masses (Wx) and stage-speciﬁc variability in several biological processes modelled in this study (cf. Fig. 3). Dashes indicate non-applicability. Stage Wx (gC) Feeding Structural growth Energetic Reserve Respiration Swimming Egg production Egg 0.55 – – – – – – NI 0.55 – x – x x – NII 0.68 – x – x x – NIII 0.91 x x – x x – NIV 1.84 x x – x x – NV 2.72 x x – x x – NVI 3.92 x x – x x – CI 6.01 x x – x x – CII 9.84 x x – x x – CIII 17.58 x x – x x – CIV 36.42 xa xd x xc xd – CV 110.03 xa xd x xc xd – Adult 332.27 x – xb x x x Wx values resemble those published for C. ﬁnmarchicus and C. glacialis by Båmstedt et al. (1991); and Campbell et al. (2001). a Feeding ceases during diapause. b Does not allocate surplus growth to develop the energetic reserve, but inherit the reserves from its developmental progression. c Reduces during diapause. Table 5 Developmental stages, their maximum structural body masses (Wx) and stage-speciﬁc variability in several biological processes indicate non-applicability. Table 5 Developmental stages, their maximum structural body masses (Wx) and stage-speciﬁc variability in several biological processes modelled in this study (cf. Fig. 3). Dashes indicate non-applicability Developmental stages, their maximum structural body masses (Wx) and stage-speciﬁc variability in several biological processes modelled in this study (cf. Fig. 3). Dashes indicate non-applicability. Stage Wx (gC) Feeding Structural growth Energetic Reserve Respiration Swimming Egg production Egg 0.55 – – – – – – NI 0.55 – x – x x – NII 0.68 – x – x x – NIII 0.91 x x – x x – NIV 1.84 x x – x x – NV 2.72 x x – x x – NVI 3.92 x x – x x – CI 6.01 x x – x x – CII 9.84 x x – x x – CIII 17.58 x x – x x – CIV 36.42 xa xd x xc xd – CV 110.03 xa xd x xc xd – Adult 332.27 x – xb x x x Wx values resemble those published for C. ﬁnmarchicus and C. glacialis by Båmstedt et al. (1991); and Campbell et al. (2001). Table 4 ﬁ We used the photoreactive behavior as a proxy to estimate the tim- ing and amplitude of DVM (e.g. Kerfoot, 1970; Carlotti and Wolf, 1998). Here, , an evolvable light sensitivity parameter (Table 2) was used to deﬁne an irradiance threshold above which induces a negative phototatic response in the vertical swimming behavior (Båtnes et al., 2015; Cohen et al., 2015). At any given time, the cope- pod occupies a depth with an irradiance level (It,z) below . From a series of possible depth bins that satisfy the It,z < condition, we assumed that the copepod searches and occupies the depth that maximizes its growth potential. For simplicity, we further assumed that internal state-dependent factors, such as hunger and satia- tion have a negligible inﬂuence on the modelled DVM. The vertical search pattern was predicted using a biased random walk algorithm (Codling, 2003, Appendix A2 in Supplementary material), assum- ing that the copepod is neutrally buoyant and vertically moves in the water column at a maximum velocity (hereafter cruising veloc- The detection efﬁciency of visually orientating planktivores increases with the size of their prey (Brooks and Dodson, 1965; Batty et al., 1990). For simplicity, we modelled the size-dependent visual predation risk using a linear model, assuming that the largest developmental stage is ca. 10 times more vulnerable to visual predators compared to the smallest developmental stage (Fig. 4a, Table 5, De Robertis, 2002). This was implemented using the scalar K (1 > K > 0) as; (Mv)i,t,z = I ′ t,z · Ki,t (10) (10) (Mv)i,t,z = I ′ t,z · Ki,t The initial value of K (i.e. K value at the embryonic stage, range = 1 × 10−4–1.5 × 10−2) was decided so that it produces hourly estimates of visual predator-induced mortality. We assumed the mortality risk caused by non-visual predators (non-visual predation risk, Mn) to be 1% of the maximum visual predation risk and constant over time and depth (Eiane and Parisi, 2001). K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 364 Table 5 Similar to most high-latitude marine zooplankton, which descend to depths during the unpro- ductive part of the year (reviewed in Conover, 1988; Hagen and Auel, 2001; Falk-Petersen et al., 2009), the copepod may perform SVM. We used the state of the energetic reserve as a proxy of timing of the SVM (cf. Visser and Jónasdóttir, 1999). Here, the cope- pod descends to a speciﬁc depth (evolvable overwintering depth , Table 2) when the stores account for a speciﬁc fraction of the total body mass (evolvable seasonal descent parameter: ı, Table 2). Upon reaching the overwintering depth, the copepod remains stag- nant at a diapause state (Hirche, 1996) with its metabolic rate reduced by 90% from that under normal conditions (Fig. 3d, Table 5, Pasternak et al., 1994; Varpe et al., 2007). The overwintering period terminates when a speciﬁc fraction (evolvable seasonal ascent parameter: ε, Table 2) of the energetic reserve is exhausted. After the overwintering period, surplus gains are not allocated to develop further energetic reserves, but may be used for structural growth and reproduction (Fig. 3e and f, Table 5). Fig. 4. Relationships of (a) visual predation risk scalar, (b) cruising velocity, (c) light sensitivity parameter and (d) the total body mass of the copepod with its structural mass (Wc). The cruising velocity (U) model was ﬁtted using laboratory and ﬁeld estimates of Calanus spp. from Hardy and Bainbridge (1954), Greene and Landry (1985) and Heywood (1996) (points in panel b). The different linear models for ˇ, that scale the light sensitivity parameter () are optimized in the model (Table 2). The lower and upper border of the shaded polygon (panel d) represent the total body mass for growth allocation parameter () = 0 and 1 respectively. K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 365 / g g ( ) Fig. 5. (a–c) Mechanism of weighing ﬁtness. Fitness of a copepod is multiplied by a binary weight ω = 0 if its egg production season (tD − tR) does not overlap the time of birth (tB, May 1 in this example, denoted by a black dot) and vice versa. (d) Simpliﬁed workﬂow of initialization and optimization steps of the model. The initial set of strategies enter the optimization loop after going through the ﬁrst life cycle simulation (LS1). Table 5 The GA optimizes seven evolvable (soft) parameters (Table 2) by repeatedly applying selection, recombination and mutation operators until a termination condition (ϕ) is satisﬁed. T and F are Boolean true and false conditions. No. of strategies (i.e. size of the GA-population, N or 2N) at each operation is indicated to the right. Fig. 5. (a–c) Mechanism of weighing ﬁtness. Fitness of a copepod is multiplied by a binary weight ω = 0 if its egg production season (tD − tR) does not overlap the time of birth (tB, May 1 in this example, denoted by a black dot) and vice versa. (d) Simpliﬁed workﬂow of initialization and optimization steps of the model. The initial set of strategies enter the optimization loop after going through the ﬁrst life cycle simulation (LS1). The GA optimizes seven evolvable (soft) parameters (Table 2) by repeatedly applying selection, recombination and mutation operators until a termination condition (ϕ) is satisﬁed. T and F are Boolean true and false conditions. No. of strategies (i.e. size of the GA-population, N or 2N) at each operation is indicated to the right. sat’s rule (Chossat, 1843), which posits that starving animals may catabolize about half of their body weight before death. Irrespective of the age of this generalized rule, it has been used as a constraint in starvation studies of many vertebrate and invertebrate taxa (e.g. Threlkeld, 1976; Spencer, 1997; Costello, 1998; Loos et al., 2010). 2.6.2.5. Metabolism. The basal metabolic cost relates with the body mass and ambient temperature, expressed as k·Wm in Eq. (3) (terms as deﬁned above and in Table 4). The metabolic cost of zooplankton vertical movements can account for 0–300% of the basal metabolic demand (Vlymen, 1970; Foulds and Roff, 1976; Morris et al., 1985; Dawidowicz and Loose, 1992). For simplicity, we assumed the cost of vertical movement to be 150% of the basal metabolic cost (mid- point of the above range). This additional cost is subtracted from the growth Eqs. (1) or (3). The energy reserve is used to balance the metabolic demands that cannot be sustained under low ambient food concentrations (Fig. 3c–f). 2.6.4. Fitness function and optimization 2.6.4. Fitness function and optimization 2.6.4. Fitness function and optimization 2.6.4. Fitness function and optimization for a particular environment, we can assume that those predicted optima should persist from one generation to the next if the envi- ronment remains constant. If a copepod’s spawning period lasts from time tR to tD (time of death) we assumed that it produces a series of offspring with the same vertical strategy, but born at different times of the year (ranging from tR to tD). However, only the offspring with a time of birth matching that of the mother can represent the entire evolvable (soft) parameter set of the mother, and guarantee its persistence from one generation to another (Fig. 5a–c). Therefore, we adjusted the ﬁtness using a binary weight (ω) by setting ω = 0 if the copepod’s spawning season does not overlap its time of birth (Fig. 5a and b) and vice versa (Fig. 5c). To evaluate the performance of a vertical strategy, we derived a ﬁtness estimate (˝) as a function of survivorship and fecundity as; ˝i = tX tB Hi,t,z · Ri,t,z · ω (14) (14) Here, ω is a weight that adjusts ﬁtness (see below) and H is the survivorship, i.e. the probability of survival from birth (tB) to a given time horizon (tX) estimated as a function of visual, non-visual and starvation risks (Mv, Mn and Ms) as; Here, ω is a weight that adjusts ﬁtness (see below) and H is the survivorship, i.e. the probability of survival from birth (tB) to a given time horizon (tX) estimated as a function of visual, non-visual and starvation risks (Mv, Mn and Ms) as; Hi,t,z = tX tB 1 − (Mv)i,t,z + (Mn)t,z + (Ms)i,t (15) We used a Real-Coded Genetic Algorithm (RCGA) to derive heuristic estimates of optimal vertical strategy and time of birth that maximizes ﬁtness in a given model environment (Fig. 5d). In the RCGA, six proxies of vertical strategies and the time of birth of the copepod that those are hardwired to (Table 2) are con- sidered as genes on a single chromosome. The RCGA begins by selecting a mating pool of N chromosomes (=parents, i.e. N verti- cal strategies seeded in different times of the year) from the initial (15) The term ˝ technically resembles the net reproductive rate (e.g. Stearns, 1992), and is used in some optimization models (e.g. 2.6.3. Reproduction We assumed that somatic growth ceases after the ﬁnal molt, and all adults become sexually mature at a constant structural body mass (Fig. 3f, Table 5). Energetic input to egg production may be sourced from food intake (income breeding) or allocating a spe- ciﬁc amount of matter (C) equivalent to the maximum growth rate (GT: Eqs. (1) and (2)) from the remaining energetic reserve (capital breeding, cf. Varpe et al., 2009). The fecundity (R) from the time of sexual maturity (tR = time of ﬁnal molt) to a given time horizon (tX) is estimated using the matter allocated to egg production (WR) and the unit egg mass (WE = 0.55 g C) as; 2.6.2.6. Starvation risk. When energy reserves are depleted, the metabolic demands that cannot be balanced by food intake are met by catabolizing structural body mass (Fig. 3b–f). This elevates the mortality risk due to starvation (starvation risk, Ms), which is deﬁned as a probability that increases as a linear function of catab- olized structural mass as; (Ms)i,t = 2 · Wq i,t (12) (Ms)i,t = 2 · Wq i,t (12) Here, Wq is the catabolized structural mass expressed as a pro- portion of the maximum structural mass prior to structural catabolization. Wq can reach a maximum of 0.5, during which Ms peaks following Eq. (12), and the copepod dies according the Chos- Ri = tX tR (WR)i,t,z WE (13) Ri = tX tR (WR)i,t,z WE (13) K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 366 366 K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 Fig. 6. Predicted optimal vertical strategy and associated growth and reproductive performances of the copepod in the basic run at Environment-L (cf. Fig. 1a–c). The surfac ime (a) is the stage-speciﬁc mean no. of hours per day that the copepod occupies food-rich surface waters, and amplitude (b) is its vertical range. Panel d compares predicte development times (excluding overwintering duration) to those estimated for C. ﬁnmarchicus and C. glacialis following Bˇelehrádek functions parameterized by Campbell et a 2001) and Ji et al. (2012). Wc and Ws refer to structural body mass and size of the energetic reserve respectively. Fig. 6. Predicted optimal vertical strategy and associated growth and reproductive performances of the copepod in the basic run at Environment-L (cf. Fig. 1a–c). The surface time (a) is the stage-speciﬁc mean no. 2.6.3. Reproduction of hours per day that the copepod occupies food-rich surface waters, and amplitude (b) is its vertical range. Panel d compares predicted development times (excluding overwintering duration) to those estimated for C. ﬁnmarchicus and C. glacialis following Bˇelehrádek functions parameterized by Campbell et al. (2001) and Ji et al. (2012). Wc and Ws refer to structural body mass and size of the energetic reserve respectively. Fig. 6. Predicted optimal vertical strategy and associated growth and reproductive performances of the copepod in the basic run at Environment-L (cf. Fig. 1a–c). The surface time (a) is the stage-speciﬁc mean no. of hours per day that the copepod occupies food-rich surface waters, and amplitude (b) is its vertical range. Panel d compares predicted development times (excluding overwintering duration) to those estimated for C. ﬁnmarchicus and C. glacialis following Bˇelehrádek functions parameterized by Campbell et al. (2001) and Ji et al. (2012). Wc and Ws refer to structural body mass and size of the energetic reserve respectively. Fig. 6. Predicted optimal vertical strategy and associated growth and reproductive performances of the copepod in the basic run at Environment-L (cf. Fig. 1a–c). The surface time (a) is the stage-speciﬁc mean no. of hours per day that the copepod occupies food-rich surface waters, and amplitude (b) is its vertical range. Panel d compares predicted development times (excluding overwintering duration) to those estimated for C. ﬁnmarchicus and C. glacialis following Bˇelehrádek functions parameterized by Campbell et al. (2001) and Ji et al. (2012). Wc and Ws refer to structural body mass and size of the energetic reserve respectively. 2.6.4. Fitness function and optimization Kiørboe and Hirst, 2008) but may not bare the same interpreta- tion given the strategy-oriented construct of this model. When the model predicts an optimal vertical strategy and time of birth 367 K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 Fig. 7. Graphical summary of the sensitivity analysis. Model parameters and environmental variables tested for sensitivity are presented on the vertical dimension, and the model-predicted optima of time of birth and vertical strategy, and the associated ﬁtness on the horizontal dimension. +/−: 25% increase/decrease in the parameter value, E/D: 15-d earlier/delayed and S/L: 15-d shorter/longer scenarios regarding timing and duration of the productive season (see Appendix A3 in Supplementary material). Fig. 7. Graphical summary of the sensitivity analysis. Model parameters and environmental variables tested for sensitivity are presented on the vertical dimension, and the model-predicted optima of time of birth and vertical strategy, and the associated ﬁtness on the horizontal dimension. +/−: 25% increase/decrease in the parameter value, E/D: 15-d earlier/delayed and S/L: 15-d shorter/longer scenarios regarding timing and duration of the productive season (see Appendix A3 in Supplementary material). 2.7. Programming, execution and analysis of the model seeds using a binary (two-way) deterministic tournament selection (Goldberg and Deb, 1991; Miller and Goldberg, 1995). Genes of two randomly selected parents from the mating pool are recombined through blend crossover following the BLX- method (Eshelman and Schaffer, 1993), which produces two offspring (recombinants). Genes of the recombinants are mutated at a probability of 0.02 by random replacement (uniform mutation: Eiben and Smith, 2003; Haupt and Haupt, 2004). The population of strategies resulting from these operations comprises of N parents, whose ﬁtness is known and N offspring, whose ﬁtness is not yet known. Parents with unique gene combinations are selected to construct a library (hereafter, the reference library), which is updated at each itera- tion. Each offspring is compared with those in the reference library to assess their ﬁtness. Fitness of the offspring with similar gene combination to those in the library are assigned in-situ, while the rest goes through the life cycle simulation to determine ﬁtness (LS- 2 in Fig. 5d). Once the ﬁtness of all 2N individuals are known, N survivors are selected following a round-robin (all-play-all) tour- nament of size 10 (Harik et al., 1997; Eiben and Smith, 2003). This process is repeated for a minimum of 100 iterations, and terminated when the mean ﬁtness of the population shows no improvement for 25 consecutive iterations ( in Fig. 5d, Eiben and Smith, 2003). seeds using a binary (two-way) deterministic tournament selection (Goldberg and Deb, 1991; Miller and Goldberg, 1995). Genes of two randomly selected parents from the mating pool are recombined through blend crossover following the BLX- method (Eshelman and Schaffer, 1993), which produces two offspring (recombinants). Genes of the recombinants are mutated at a probability of 0.02 by random replacement (uniform mutation: Eiben and Smith, 2003; Haupt and Haupt, 2004). The population of strategies resulting from these operations comprises of N parents, whose ﬁtness is known and N offspring, whose ﬁtness is not yet known. Parents with unique gene combinations are selected to construct a library (hereafter, the reference library), which is updated at each itera- tion. Each offspring is compared with those in the reference library to assess their ﬁtness. Fitness of the offspring with similar gene combination to those in the library are assigned in-situ, while the rest goes through the life cycle simulation to determine ﬁtness (LS- 2 in Fig. 5d). 2.7. Programming, execution and analysis of the model Once the ﬁtness of all 2N individuals are known, N survivors are selected following a round-robin (all-play-all) tour- nament of size 10 (Harik et al., 1997; Eiben and Smith, 2003). This process is repeated for a minimum of 100 iterations, and terminated when the mean ﬁtness of the population shows no improvement for 25 consecutive iterations ( in Fig. 5d, Eiben and Smith, 2003). We used R version 3.3.1 (R Core Team, 2016) and R Studio inte- grated development environment (IDE) version 1.0.136 (RStudio Team, 2016) along with the high-performance computing packages Rcpp (Eddelbuettel et al., 2011) and bigmemory (Kane et al., 2013) to construct, simulate and analyze the model. A basic run was performed in the Environment-L using default values for model parameters (Table 4). In order to test the inﬂuence of model parameters and environmental variables on model- predicted vertical strategies and ﬁtness, we performed a sensitivity analysis following (Jørgensen and Bendoricchio, 2001). Here, we calculated a sensitivity score (Sx) as; Sx = (XBR − XM) /XBR (PBR − PM) /PBR (16) Sx = (XBR − XM) /XBR (PBR − PM) /PBR (16) where X is the predicted model output of the basic run (XBR) and the modiﬁed run (XM) for a given change (±25%) of input param- eter value between the basic run (PBR) and the modiﬁed run (PM). We tested the sensitivity of vertical strategies and ﬁtness for 13 dif- ferent input parameters (Appendix A3 in Supplementary material). For the convenience of interpretation of these results, we presented the sensitivity scores under three categories: no-sensitivity (Sx = 0), low sensitivity (0 < Sx ≤ 3) and high sensitivity (Sx > 3). Finally, we K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 368 studied the changes of heuristically optimized vertical strategies by performing model simulations in the three model environments at different levels of visual predation risk, while maintaining the rest of the parameter values at its default levels. Table 6 Predicted variability of emergent behavioral strategies and life history traits (Fig. 2, Table 3) induced by model parameters and environmental variables analyzed for sensitivity (Fig. 7, Appendix A3 in Supplementary materia Only the input parameters with highest inﬂuence on ﬁtness (sensitivity index >3) are tabulated. 3.1. The basic run In the basic run, the life cycle emerging from the model began as an egg spawned at 20 m depth in late-April. The ﬁrst two nauplii stages did not perform DVM, but DVM and the associated metabolic cost (swimming cost) increased ontogenetically from NIII onwards (Fig. 6a, b, e and f). The somatic growth of all developmental stages beyond NV occurred under food limitation (Fig. 6c), and because of reduced growth rates, their predicted development times were higher than those estimated from Bˇelehrádek temperature func- tions (Fig. 6d). As the energetic reserve reached 65% of the total body mass (Ws ≈ 196 g C, Fig. 6g), the developmental stage CV descended to an overwintering depth of 150 m in mid-June, ca. 2 months before the pelagic primary production had terminated (Fig. 6e). It remained there for ca. 290 days, and ascended into near- surface waters again in early-April of the following year, ca. 10 days after the pelagic primary production had commenced (Fig. 1c), with fully depleted energetic reserves. Although the energetic cost of seasonal migration was quite high, the cost of ascent was ca. ¼ of that of the descent due to the loss of body mass during overwin- tering (Fig. 6f and g). The copepod developed to an adult female in mid-April, and thenceforth produced eggs (ca. 3000) via income breeding until mid-June and then died (Fig. 6h). 2.7. Programming, execution and analysis of the model Parameter Variation Time of birth (day) Surface timea (h) DVM amplitudea (m) Onset of seasonal descent (day) Onset of seasonal ascent (day) Overwintering depth (m) Development timeb (d) Food limitation indexa Body mass at seasonal descent (g C) Onset of egg production (day) Fecundity Breeding mode index Longevity (d) Absolute ﬁtness Wc Ws Basic run 0% 120 20.5 5 166 92 150 68.1 0.33 120.4 195.7 114 2972 0.000 414 4.10 Visual predation risk (K) −25% 118 22.7 3 163 91 150 65.2 0.38 127.0 203.2 111 3834 0.000 430 21.2 +25% 123 19.8 7 168 90 170 69.4 0.29 116.0 185.6 114 1446 0.000 389 0.90 Food concentration (F) −25% 105 22.9 3 183 85 150 98.4 0.25 221.1 342.7 105 415 0.018 384 1.00 +25% 122 19.8 6 157 99 150 59.5 0.63 120.8 196.3 123 3957 0.000 424 8.70 Temperature −25% 103 23.0 2 181 82 150 99.1 0.21 229.7 346.3 103 654 0.022 387 1.20 +25% 127 19.7 7 160 107 150 47.0 0.68 124.4 202.5 121 5285 0.000 412 16.1 W = structural mass W = mass of energetic reserve As GAs produce heuristic estimates of the maximum ﬁtness, there is no guarantee that it would converge on the global max- imum given a potentially diverse ﬁtness landscape (Record et al., 2010). Therefore, we replicated each model run 10 times with dif- ferent starting values assigned to the soft parameters (Table 2) to check if the algorithm converges on the same set of solutions. As the optimized parameter values showed little variability between replicate runs (<5%), we used the mean of the replicates for each parameter for analyses. 3.2. Sensitivity analysis The model-predicted ﬁtness was highly sensitive to visual pre- dation risk, food concentration and temperature (Fig. 7). A 25% change in the visual predation risk (K = 7.5 × 10−3 and 1.25 × 10−2) inﬂuenced the DVM, which intensiﬁed at the higher-end of K and vice versa (Table 6). Although the overwintering depth deepened by ca. 13% under higher visual predation risk, it did not change under lower visual predation risk. Furthermore, higher visual predation risk lowered the fecundity and longevity, and vice versa (Table 6). A 25% change in food concentration (F = 10 and 6 mg m−3 Chl.-a) notably inﬂuenced the DVM, timing of SVM and time of birth, but not the overwintering depth (Fig. 7). Under low food concentra- tion, the DVM was less pronounced and the seasonal descent was delayed ca. 15 d compared to the basic run, possibly because of foraging later into the feeding season due to lower growth poten- tial sustained under increased food limitation (Table 6). However, the copepod overwintered as a signiﬁcantly large CV with elevated energetic reserves, and made ca. 7-d earlier spring ascent (late March), followed by spawning that preceded the pelagic bloom by ca. 2 days (cf. Fig. 1c). Here, ca. 2% of the total egg production was sourced from capital breeding (Table 6). Early seasonal ascent, capi- tal breeding and early spawning thus appear as strategies employed to avoid seasonal peak in visual predation risk (cf. Fig. 1a, Eqs. (9) and (10)) when foraging efforts are elevated to cope with lower K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 369 of time of birth (tB), vertical strategy and associated life history traits of the copepod in the three model environments u dation risk is scaled by varying the parameter K in a range of 1–150 × 10−4. The ﬁtness is cube-root transformed for the c of the year, where day 1 = 1 January. Wc and Ws are structural and energy reserve masses, and tR is time of ﬁrst reprod Fig. 8. Predicted optima of time of birth (tB), vertical strategy and associated life history traits of the copepod in the three model environments under variable levels of visual predation risk. Visual predation risk is scaled by varying the parameter K in a range of 1–150 × 10−4. The ﬁtness is cube-root transformed for the convenience of visualization. 3.2. Sensitivity analysis 7, Table 6), and highlight the equally important roles played by food availability and temperature in growth and development. growth potential. However, this came with a cost of decreased fecundity (80%) and longevity (7%) (Fig. 7, Table 6). Increased food concentration had the opposing effects on the predicted behavioral strategies and life history traits described above (Fig. 7, Table 6). stant across the three model environments, with the exception that it occurred the earliest in mid-April at Environment-H under the lowest visual predation risk (K = 10−4, Fig. 8d). At lower levels of visual predation risk, the copepod overwintered as relatively large CVs with elevated energetic reserves at relatively shallow depths (Fig. 8b and c). However, as the visual predation risk increased, the copepod overwintered as smaller CVs or CIVs with relatively less energetic reserves at greater depths. Although the predicted longevity decreased by ca. 80 d along the modelled visual predation risk gradient (Fig. 8g), longevity at Environment-H under K = 10−4 was lower (365 d) compared to those predicted under higher visual predation risk levels (384–430 d). g y ( g , ) The inﬂuence of 4.5 ◦C change in temperature (T = 22.5 and 13.5 ◦C) on copepod’s behavior and life history followed the same general trends described for food concentration (Fig. 7, Table 6), and highlight the equally important roles played by food availability and temperature in growth and development. 3.2. Sensitivity analysis Time is presented as day of the year, where day 1 = 1 January. Wc and Ws are structural and energy reserve masses, and tR is time of ﬁrst reproduction. Fig. 8. Predicted optima of time of birth (tB), vertical strategy and associated life history traits of the copepod in the three model environments under variable levels of visual predation risk. Visual predation risk is scaled by varying the parameter K in a range of 1–150 × 10−4. The ﬁtness is cube-root transformed for the convenience of visualization. Time is presented as day of the year, where day 1 = 1 January. Wc and Ws are structural and energy reserve masses, and tR is time of ﬁrst reproduction. Fig. 8. Predicted optima of time of birth (tB), vertical strategy and associated life history traits of the copepod in the three model environments under variable levels of visual predation risk. Visual predation risk is scaled by varying the parameter K in a range of 1–150 × 10−4. The ﬁtness is cube-root transformed for the convenience of visualization. Time is presented as day of the year, where day 1 = 1 January. Wc and Ws are structural and energy reserve masses, and tR is time of ﬁrst reproduction. 0 K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 g. 9. Predicted stage-speciﬁc surface time (a), DVM amplitude (b), food limitation index (c) and the development times (egg to a given stage excluding the overwinterin riod, d) of the copepod in the three model environments under variable levels of visual predation risk. K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 370 Fig. 9. Predicted stage-speciﬁc surface time (a), DVM amplitude (b), food limitation index (c) and the development times (egg to a given stage excluding the overwintering period, d) of the copepod in the three model environments under variable levels of visual predation risk. growth potential. However, this came with a cost of decreased fecundity (80%) and longevity (7%) (Fig. 7, Table 6). Increased food concentration had the opposing effects on the predicted behavioral strategies and life history traits described above (Fig. 7, Table 6). The inﬂuence of 4.5 ◦C change in temperature (T = 22.5 and 13.5 ◦C) on copepod’s behavior and life history followed the same general trends described for food concentration (Fig. 3.3. Latitudinal environmental variability and visual predation risk The predicted timing of SVM showed signiﬁcant variability across the three model environments, but was less affected by visual predation risk (Fig. 8a). Both the descent and ascent occurred earliest at the lowest latitude environment, but happened later in the season at higher-latitude environments, with a shift of about a month. Although this reﬂects the delayed occurrence of the pelagic bloom along the modelled latitudinal gradient (Fig. 1c, f Model-predicted optimal time of birth, body mass (Wc and Ws) at seasonal descent, overwintering depth and longevity changed with visual predation risk (K), but showed less variability along the modelled latitudinal environmental gradient (Fig. 8b–d and g). The predicted optimal time of birth changed from late-April to mid-May with increasing visual predation risk, and was con- K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 371 1987). However, the largely exogenous-regulated DVM behavior emerging from this model does not render a complete view on the subject matter, as DVM can also be sensitive to internal (physio- logical) states of zooplankton (e.g. hunger and satiation, Hays et al., 2001; Pearre, 2003). and i, Appendix A1 in Supplementary material), the seasonal ascent at Environment-H under lowest visual predation risk (K = 10−4) occurred ca. 25 days before the pelagic primary production had commenced (Figs. 1i and 8a). The predicted onset of spawning, fecundity and breeding mode index (Fig. 8d–f), along with the predicted timing and amplitude of DVM, food limitation index and development time (Fig. 9) varied with both the visual predation risk and latitudinal environmental gradient. In all model environments, the spawning started earli- est under the lowest visual predation risk (K = 10−4, Fig. 8d). Here, spawning commenced ca. 5–7 days earlier than the onset of spring primary production in lower latitude environments (Fig. 1c and f), and ca. 2.5–5% of the total egg production were sourced from capital breeding (Fig. 8f). Spawning at Environment-H commenced ca. 25 d prior to the pelagic bloom, but lasted only for ca. 10 days (Figs. 1i, 8d and g). Consequently, the expected fecundity was the lowest (ca. 145 eggs, Fig. 8e) and all eggs were sourced from cap- ital breeding (Fig. 8f). The onset of spawning shifted later into the season at higher latitude model environments at higher lev- els of visual predation risk, and occurred after the commencement of the pelagic primary production (Figs. 1c, f, i and 8d). 3.3. Latitudinal environmental variability and visual predation risk Here, all eggs were produced via income breeding (Fig. 8f). At lower levels of visual predation risk (10−4 ≤ K ≤ 5 × 10−3), the predicted DVM pattern was similar across the three model environments, where developmental stages until early copepodites did not perform DVM (Fig. 9a and b). Although the model predicted the younger developmental stages (NIII onwards) to perform DVM under ele- vated visual predation risk at Environment-L, this effect gradually waned in higher-latitude model environments. The food limitation index strongly followed the DVM pattern, where developmental stages that performed DVM suffered from increased food limitation (Fig. 9c). Food limitation signiﬁcantly reduced the growth rates (cf. Eqs. (1)–(3), see also Appendix A4 in Supplementary material), and consequently, the development times increased along the modelled environmental gradient (Fig. 9d). Further, in each model environ- ment, lowest development times were predicted under the lowest level of visual predation risk. The effect of temperature on growth potential further explains the diminished inﬂuence of visual predation risk on the tim- ing and amplitude of DVM predicted at higher latitude model environments (Fig. 9). Albeit similar food concentrations, the mod- elled temperatures decreased from lower- to higher-latitude model environments, reﬂecting a decreasing gradient of growth potential (Fig. 1, Appendix A4 in Supplementary material). Consequently, the model-predicted optimal DVM strategy for higher-latitude envi- ronments was to elevate the growth potential by spending more time foraging in near-surface waters (Fig. 9a and b). This effect was most pronounced among younger developmental stages (NIII–CI), whose DVM reduced from environment-L to -M, and completely ceased at environment-H. DVM of younger developmental stages (NIII onwards) are most commonly reported from lower latitudes for Calanus spp. (e.g. Huntley and Brooks, 1982; Uye et al., 1990; Huang et al., 1993; Osgood and Frost, 1994; Zakardjian et al., 1999) and Metridia spp. (e.g. Hays, 1995). While some ﬁeld studies failed to detect notable DVM in high-latitudes (e.g. Blachowiak-Samolyk et al., 2006; Basedow et al., 2010), others reported ontogenetic increase of DVM (CI onwards, e.g. Dale and Kaartvedt, 2000; Daase et al., 2008) in Calanus spp. However, empirical data with high spa- tial, temporal and biological (i.e. developmental stage) resolution is needed to test the ontogenetic and latitudinal patterns of DVM predicted by our model. The predicted DVM amplitudes spanned across the productive part of the water column (i.e. upper 30 m, cf. Figs. 3.3. Latitudinal environmental variability and visual predation risk 1, 6 and 9), and showed a positive relationship with the vertical extent of food availability (Fig. 7). Moreover, strategies that involve higher- amplitude DVM lead to increased food-limitation, where younger developmental stages with no energetic reserves tend to suffer from starvation risk due to low temperatures and food concentra- tions that prevail in deeper parts of the model environments (Fig. 1). Therefore, low-amplitude DVM appears to be a strategy that efﬁ- ciently trades off growth potential for survival, by balancing both the visual predation and starvation risks (Kerfoot, 1970; Fiksen and Giske, 1995; De Robertis, 2002). Although higher-amplitude DVM can be predicted either by not modeling starvation risk or impos- ing starvation tolerance (e.g. Andersen and Nival, 1991; Carlotti and Wolf, 1998; Zakardjian et al., 1999; Tarling et al., 2000), we did not follow these approaches because our model sufﬁciently represents the relative importance of DVM across the modelled environmental gradients. 4.1. Inﬂuence of environmental variables on vertical strategies 4.1. Inﬂuence of environmental variables on vertical strategies 4.1.1. Diel vertical migration 4.1.1. Diel vertical migration g In this model, visual predation risk had the highest inﬂuence on the DVM, which diminished under low visual predation risk (Figs. 7 and 9, Table 6) and completely ceased when visual pre- dation was removed from the model (K = 0, data not presented). Conversely, under high visual predation risk, also younger devel- opmental stages reduced the time spent in food-rich surface waters by performing low-amplitude (shallow) DVM (Fig. 9a and b). Food concentration and temperature also inﬂuenced the DVM (Fig. 7, Table 6). Lower food concentrations or temperatures produced low- amplitude DVM, possibly due the low growth potential attained in cold, food-limited conditions (Fig. 9, Eqs. (1)–(8), Appendix A4 in Supplementary material). Under these conditions, it appears that modelled copepods do not possess sufﬁcient growth poten- tial to trade off for survival and perform high-amplitude (deep) DVM, a conclusion also drawn in empirical work (e.g. Huntley and Brooks, 1982; Loose and Dawidowicz, 1994). Reduced or absence of DVM under low food concentrations and temperatures are reported from several other modeling studies on copepods and euphausi- ids (e.g. Andersen and Nival, 1991; Fiksen and Giske, 1995; Fiksen and Carlotti, 1998; Tarling et al., 2000) and from empirical work on marine copepods and freshwater cladocerans (e.g. Hardy and Gunther, 1935; Huntley and Brooks, 1982; Johnsen and Jakobsen, 4.1.2. Seasonal vertical migration Table 7 However, direct comparisons between vertical migratory patterns predicted by a strategy-oriented model and ﬁeld estimates should be done with caution, as an environment-speciﬁc optimal vertical strategy predicted by the model contrasts the diversity of vertical behavior exhibited by individuals of a zooplankton population. conditions (e.g. Reid et al., 1998; Eiane and Parisi, 2001; Ji, 2011). However, direct comparisons between vertical migratory patterns predicted by a strategy-oriented model and ﬁeld estimates should be done with caution, as an environment-speciﬁc optimal vertical strategy predicted by the model contrasts the diversity of vertical behavior exhibited by individuals of a zooplankton population. Visual predation risk and depth of thermal stratiﬁcation (summer–autumn) were the only environmental variables that inﬂuenced the model-predicted overwintering depth (Figs. 7 and 8b, Table 6). The overwintering depth deepened at higher levels of visual predation risk and deeper thermal stratiﬁcation depths, and agrees with Hirche (1991), Kaartvedt (1996), Dale et al. (1999), Bagøien et al. (2000) and Astthorsson and Gislason (2003) that Calanus spp. prefer colder water masses with low predator abundance for overwintering. However, the overwintering depths predicted by or model underestimate those of ﬁeld observations, which can extend well below 1000 m (e.g. Østvedt, 1955). Apart from the shallow bottom depths modelled, this discrepancy largely reﬂects how overwintering habitat selection of Calanus spp. is inﬂu- enced by the buoyancy-effect of stored lipid reserves (Visser and Jónasdóttir, 1999), convective mixing of surface waters (Irigoien, 2004) and vertical distribution of water masses and predator pop- ulations, such as mesopelagic ﬁsh, predatory ctenophores and krill (Hirche, 1991; Kaartvedt 1996; Bagøien et al., 2000; Bandara et al., 2016). Compared to the phenology of reproduction, the elevated visual predation risk had little inﬂuence on the timing of seasonal migra- tion (Fig. 8a). The SVM strategy predicted by the model was to descend to overwintering depth approximately at the same period of the year (mid–late summer), but with ca. 1⁄3 lesser the body mass (both structural and energetic reserve mass) compared to that under the lowest visual predation risk (Fig. 8a and c). As higher visual predation risk tends to intensify DVM in this model (Fig. 9a and b), it appears that trading off growth potential for survival makes an earlier seasonal descent unfavorable, as food- limitation and slower growth rates (Fig. 9c and d) demands more time to acquire sufﬁcient energy reserves to overwinter, despite the smaller body mass of the overwintering stage. 4.1.2. Seasonal vertical migration Food availability and temperature had the most notable inﬂu- ence on the model-predicted timing of SVM (Fig. 7, Table 6). The predicted shift in timing of seasonal descent and ascent coincided with those of the pelagic algal bloom and thermal stratiﬁcation along the modelled latitudinal gradient (Figs. 1 and 8a). This agrees with the argument that food availability is the ultimate factor inﬂu- encing the timing of seasonal vertical migration of Calanus spp. (Herman, 1983; Head and Harris, 1985; Hind et al., 2000). How- ever, ﬁeld estimates of timing of SVM of Calanus spp. from low to high latitudes do not point to a simple south–north gradient as predicted in our model (Table 7, see also Melle et al., 2014). This discrepancy of model predictions and ﬁeld estimates underlies the differences between location-speciﬁc variability in hydrogra- phy, algal bloom dynamics and species composition (e.g. Hirche, 1991; Daase et al., 2013), diversity of generation lengths and breed- ing strategies (e.g. Conover, 1988; Falk-Petersen et al., 2009) and climate-driven and other stochastic oscillations of environmental K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 372 Table 7 Further, as the model-predicted body mass of the overwintering stage reaches a lower threshold at K > 10−4 (Fig. 8c), it is likely that modelled copepods overwintered with minimum reserves to last the over- wintering duration, and therefore makes an earlier descent (with lesser energy reserves) nearly impossible. In the contrary, occu- pying near-surface waters later into the season and descend to 4.2. Inﬂuence of vertical strategies on ﬁtness and phenology Table 7 (1)–(8), Appendix A4 in Supplementary material), which is then efﬁciently traded off for survival through DVM to counter the risk of increasing visual predation risk (Fig. 9). Conversely, due to lower temperatures, copepods born earlier in the season must ele- vate the time spent foraging in near-surface waters to attain higher growth rates, and become more vulnerable to visual predation risk. However, it should be noted that our model does not consider the ability of Calanus spp. to use the darker and seasonally ice-covered period of the year to attain growth with minimal inﬂuence from visual predators by feeding on alternative food sources, such as ice algae and microzooplankton (Conover and Siferd, 1993; Søreide et al., 2010). to those spawned earlier (Fig. 8h). This seems counterintuitive as zooplankton are more vulnerable to visual predation risk later in the season due to the higher irradiance levels that persist in late- spring and summer. In this model, the early feeding season (i.e. until the time of peak pelagic bloom) is characterized by higher food concentrations and lower temperatures (Fig. 1). Although the food concentration decreases by ca. 20% by late spring or early-summer, the ambient temperature increases by ca. 2–4 times. Further, even at the onset of the productive season, the visual predation risk had reached ca. 70% of its maximum in all model environments (Fig. 1a, d and g). Therefore, it is likely that copepods born relatively later in the season use the higher temperatures to attain a higher growth (Eqs. (1)–(8), Appendix A4 in Supplementary material), which is then efﬁciently traded off for survival through DVM to counter the risk of increasing visual predation risk (Fig. 9). Conversely, due to lower temperatures, copepods born earlier in the season must ele- vate the time spent foraging in near-surface waters to attain higher growth rates, and become more vulnerable to visual predation risk. However, it should be noted that our model does not consider the ability of Calanus spp. to use the darker and seasonally ice-covered period of the year to attain growth with minimal inﬂuence from visual predators by feeding on alternative food sources, such as ice algae and microzooplankton (Conover and Siferd, 1993; Søreide et al., 2010). conditions (e.g. Reid et al., 1998; Eiane and Parisi, 2001; Ji, 2011). Table 7 Table 7 Timing of seasonal ascent and descent of Calanus spp. estimated by several high-latitude ﬁeld investigations. These estimates are based on observation of zooplankton populations oftentimes containing various combinations of C. helgolandicus, C. ﬁnmarchicus, C. glacialis and C. hyperboreus. Geographical location is approximate. Data for North-Atlantic are available in Melle et al. (2014). Table 7 Timing of seasonal ascent and descent of Calanus spp. estimated by several high-latitude ﬁeld investigations. These estimates are based on observation of zooplankton populations oftentimes containing various combinations of C. helgolandicus, C. ﬁnmarchicus, C. glacialis and C. hyperboreus. Geographical location is approximate. Data for N th Atl ti il bl i M ll t l (2014) Study Lat. Lon. Onset of descent Onset of ascent Hirche (1984) 58◦N 11◦E mid-October mid-May Bagøien et al. (2000) 59◦N 10◦E July–August March Heath (1999) 61◦N 4◦W – May Gislason and Astthorsson (2000) 62◦N 20◦W – April 64◦N 28◦W – April Østvedt (1955) 66◦N 2◦E July April Kosobokova (1999) 66◦N 35◦E mid-July mid-May Astthorsson and Gislason (2003) 68◦N 13◦E – May Madsen et al. (2001) 69◦N 54◦W – mid-April Madsen et al. (2008) 69◦N 54◦W late-September early-April Hirche (1997) 71◦N 4◦E July–August April 74◦N 1◦E July–August April Unstad and Tande (1991) 75◦N 30◦E – May Arashkevich et al. (2002) 76◦N 33◦E July May Hirche and Kosobokova (2011) 77◦N 25◦E – March Hirche and Kosobokova (2003) 78◦N 82.5◦E – mid-May Bandara et al. (2016) 78◦N 16◦E July–August mid-February Dawson (1978) 84◦N 112◦W August June to those spawned earlier (Fig. 8h). This seems counterintuitive as zooplankton are more vulnerable to visual predation risk later in the season due to the higher irradiance levels that persist in late- spring and summer. In this model, the early feeding season (i.e. until the time of peak pelagic bloom) is characterized by higher food concentrations and lower temperatures (Fig. 1). Although the food concentration decreases by ca. 20% by late spring or early-summer, the ambient temperature increases by ca. 2–4 times. Further, even at the onset of the productive season, the visual predation risk had reached ca. 70% of its maximum in all model environments (Fig. 1a, d and g). Therefore, it is likely that copepods born relatively later in the season use the higher temperatures to attain a higher growth (Eqs. Acknowledgements SVM was essential for the wintertime survival of the modelled copepods, given its food source is only available during the primary production season (spring–autumn, Fig. 1). Different combinations of proxies (Table 2) yielded non-seasonally migrating strategies, in which the copepods developed to adults and reproduced within the same productive season. Although this strategy had the potential to produce more than one generation per year (especially in the rela- tively lower-latitude environment-L, Fig. 1a–c), we did not peruse this further, as our focus was on an annual life cycle (see the ﬁtness weighing process, Eq. (14), Fig. 5a–c). This project was funded by VISTA (project no. 6165), a basic research program in collaboration between The Norwegian Academy of Science and Letters and Statoil. ØV received fund- ing from the Fulbright Arctic Initiative and thanks the Woods Hole Oceanographic Institution for hosting during the Fulbright exchange. Authors are also thankful to the two anonymous review- ers for critically reading the earlier draft of the manuscript and suggesting substantial improvements. g g p , q ( ), g ) The body mass and the size of energetic reserve at seasonal descent together with the timing of seasonal ascent had a pro- found inﬂuence on the predicted timing of reproduction, breeding strategy and fecundity (Fig. 8). Overwintering as large CVs with elevated energetic reserves at lower visual predation risk enabled the copepod to allocate the post-overwintering surplus energetic reserves to capital breeding in the following year (Fig. 8c and f, and see also Sainmont et al., 2014a; Ejsmond et al., 2015; Halvorsen, 2015). As capital breeding emerged in environments with lower temperatures and food concentrations (Fig. 7, Table 6), it appears as a strategy that allows the new generation to feed from the very start of the feeding season, while avoiding the seasonal peak in visual predation risk later in the year (Fig. 1, see also Varpe et al., 2009). The proportional increase of capital breeding eggs from relatively lower-latitude environment-L to higher-latitude environment-H reﬂects the decreasing temperature gradient that occur at overwintering depths of these environments (Figs. 1, 8b and f). Overwintering in colder water masses reduces the metabolic costs and conserves the energetic reserve, which ultimately boosts the fecundity through capital breeding (Hirche, 1991; Hirche, 1996; Astthorsson and Gislason, 2003). The pure capital breeding strat- egy predicted at environment-H under the lowest visual predation risk more resembles the spawning strategy of C. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ecolmodel.2017.12. 010. 4.2.1. Diel vertical migration In the model, high-amplitude DVM caused increased food lim- itation that led to slow growth and development and reduced fecundity (Figs. 7 and 9, Table 6). This ultimately resulted in lower ﬁtness relative to that predicted for low-amplitude DVM. It is there- fore apparent that decisions to fully or partly disregard DVM in models focusing SVM and other seasonal strategies should be made with caution, as our ﬁndings indicate that DVM can have a notable negative inﬂuence on growth and development of younger devel- opmental stages, especially at lower latitudes. At higher levels of visual predation risk, the model predicted up to a one month delay in the onset of spawning (Fig. 8d), and high- lights the inﬂuence of predation risk on the reproductive phenology (Magnhagen, 1991; Stibor, 1992; Varpe et al., 2007). These late- spawned copepods appeared to possess higher ﬁtness compared K. Bandara et al. / Ecological Modelling 368 (2018) 357–376 373 Acknowledgements hyperboreus than C. ﬁnmarchicus and C. glacialis (Conover, 1988; Falk-Petersen et al., 2009). The sensitivity of the model-predicted breeding strategy to visual predation risk indicates an extensive pre-breeding cost of capital breeding imposed by the size-dependent visual preda- tion risk and acquisition and carriage of energy reserves (Jönsson, 1997; Varpe et al., 2009). Moreover, excess energy storage (i.e. more than to overwinter) and capital breeding do not emerge as dom- inant strategies in this model as the environmental parameters are modelled in a perfectly predictable manner, without any year- to-year variability. However, capital breeding and energy storage may possess a much larger adaptive signiﬁcance in nature, where spatio-temporal environmental heterogeneity and unpredictabil- ity are more pronounced compared to our model (e.g. Jönsson, 1997; Fischer et al., 2009). 4.3. Concluding remarks overwintering depths with elevated energetic reserves is also unfa- vorable as the visual predation risk in this model is not nulliﬁed even at the deepest parts of the water column (Eq. (10)). The lesser inﬂuence of visual predation risk on the timing of SVM does not align with Kaartvedt (2000) and Varpe and Fiksen (2010) who view predation by planktivorous ﬁsh as a key driver of generation lengths and timing of seasonal descent in C. ﬁnmarchicus in the Norwegian Sea. However, the consequences of DVM on the timing of seasonal descent presented here may diminish if there is an energetic beneﬁt of DVM (e.g. McLaren, 1963; Enright, 1977), if copepods are capable of utilizing alternative food sources (e.g. Runge and Ingram, 1991; Hirche and Kwasniewski, 1997) or if there is a strong size selection against larger developmental stages by visual predators than the linear relationship applied in our model (cf. Fig. 4a with Brooks and Dodson, 1965; Batty et al., 1990; Langbehn and Varpe, 2017). 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https://openalex.org/W4210543137	https://ijpds.org/article/download/1712/3318	English	null	Identifying researcher learning needs to develop online training for UK researchers working with administrative data: CENTRIC training	International journal of population data science	2,022	cc-by	7,641	International Journal of Population Data Science (2022) 7:1:2 International Journal of Population Data Science (2022) 7:1:2 ∗Corresponding Author: Email Address: LuggFV@cardiff.ac.uk (Fiona Lugg-Widger) Results 107 researchers responded to the online survey and four data providers participated in the focus groups. We identified five main themes, relating to research training needs for UK researchers working with administrative data: communication; timelines; changes & amendments; future-proofing applications; and, the availability of training and support. Data providers either provided additional evidence on these learning needs or ways to address identified challenges. Six modules were developed addressing these training needs. Quotes from the survey and focus groups are used anonymously in the online training modules. Methods A mixed-methods design informed curriculum content, including surveys with researchers, focus group discussions with data providers and workshops with members of the public. Researchers were identified from relevant administrative data networks and invited to participate in an online survey identifying training needs. Data providers were approached with a request to input to a face-to-face or online meeting with two members of the research team about their experiences of working with researchers. Data were analysed within the broad framework of the interview schedule, free text responses in the survey were analysed thematically. Abstract Submission History Submitted: 22/10/2021 Accepted: 13/12/2021 Published: 02/02/2022 1Centre for Trials Research, Cardiff University, Cardiff, CF14 4YS 2DECIPHer - Centre for Development, Evaluation, Complexity and Implementation in Public Health Improvement, 1-3 Museum Place, Cardiff. CF10 3BD Submission History Submitted: 22/10/2021 Accepted: 13/12/2021 Published: 02/02/2022 1Centre for Trials Research, Cardiff University, Cardiff, CF14 4YS 2DECIPHer - Centre for Development, Evaluation, Complexity and Implementation in Public Health Improvement, 1-3 Museum Place, Cardiff. CF10 3BD Submission History Submitted: 22/10/2021 Accepted: 13/12/2021 Published: 02/02/2022 1Centre for Trials Research, Cardiff University, Cardiff, CF14 4YS 2DECIPHer - Centre for Development, Evaluation, Complexity and Implementation in Public Health Improvement, 1-3 Museum Place, Cardiff. CF10 3BD https://doi.org/10.23889/ijpds.v7i1.1712 February 2, 2022 © The Authors. Open Access under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/deed.en) Background g The use of administrative data in health and social science research continues to expand, with increased availability of data and interest from funders. Researchers, however, continue to experience delays in access, storage and sharing of administrative data. Training opportunities are limited and typically specific to individual data providers or focussed on the analytical aspects of working with administrative data. The CENTRIC study was funded by the Information Commissioners Office, with the aim of developing a broader training curriculum for researchers working with administrative data in the UK. Identifying researcher learning needs to develop online training for UK researchers working with administrative data: CENTRIC training Identifying researcher learning needs to develop online training for UK researchers working with administrative data: CENTRIC training Fiona Lugg-Widger1*, Kim Munnery1, Julia Townson1, Rob Trubey1, and Michael Robling1,2 International Journal of Population Data Science Journal Website: www.ijpds.org Conclusion Conclusion The CENTRIC online training curriculum was launched in September 2020 and is available, free of charge for UK researchers. CENTRIC specifically addresses commonly identified training needs of researchers working with administrative data. Keywords administrative data; routine data; training; data access ∗Corresponding Author: Submission History Submitted: 22/10/2021 Accepted: 13/12/2021 Published: 02/02/2022 1Centre for Trials Research, Cardiff University, Cardiff, CF14 4YS 2DECIPHer - Centre for Development, Evaluation, Complexity and Implementation in Public Health Improvement, 1-3 Museum Place, Cardiff. CF10 3BD https://doi.org/10.23889/ijpds.v7i1.1712 Methods This was a mixed-methods study designed to understand learners’ needs from the perspective of both researchers and data providers, in order to co-produce a training package to address specific learning outcomes linked to the ICO strategic objectives [20]. Ethical approval for the study was provided by Cardiff University School of Medicine Research Ethics committee (Ref 19/51). The terms routine data and administrative data were used interchangeably during this study, with administrative data being used in the final training curriculum. [ ] The UK Clinical Research Collaboration (UKCRC), National Institute for Health Research (NIHR) and Health Research Authority (HRA) have historically attempted to bridge the gap between researchers and data providers, facilitating discussions and change informed by those experiencing the challenges [13–15] However, applications to access and process data shared by UK data providers are usually undertaken by researchers working alone or in small teams. This is evidenced for example, by the large number of data security protection toolkits listed for users of NHS Digital, often within the same university [16]. This fragmentation means that despite some central coordination by university research governance departments, much onus remains on individual teams in gaining access to and processing data. Problems accessing, storing and sharing data continue to be experienced in silos [11]. Data providers work with a large number of data recipients. Indeed, the breadth of research topics and projects that request administrative data means each application presents its own nuanced challenges making guidance and support challenging to address by each data provider. Introduction The Information Commissioner’s Office (ICO) funded the CENTRIC project in 2019 to address this gap. The aim was to provide a broader training course that would better support researchers in understanding and navigating the process of gaining access to routine public sector data, and to help them process it in a regulatory compliant manner [19]. Importantly, the content of the UK-focused CENTRIC training course was to be informed through consultation with researchers, data providers and members of the public. Aligning with the ICO strategy, this funded work addressed three of their strategic goals: (1) increasing public trust and confidence in how data are used and made available; (2) improving standards of information rights practice through clear, inspiring and targeted engagement and influence; and (3) staying relevant, providing excellent public service and keeping abreast of evolving technology [20]. The use of administrative data for health and social care research continues to expand [1–3]. In health research, the UK Health Data Research (HDRUK) Alliance is leading the way in bringing together data, expertise and infrastructures enabling health research in the UK [4]. NHS DigiTrials is one of the initiatives born out of HDRUK, which aims to streamline trials applying for data through NHS Digital in England and offering support to clinical trials such as recruiting potential participants [5]. The use of administrative data for social science research is also expanding. In England, administrative data research (ADRUK) and Data First have facilitated linkages between the Department for Education and Ministry of Justice, enabling criminal justice, education and social care data to be linked for the first time [6]. Funding initiatives encouraging the use of administrative data are also increasing [7]. [ ] This paper sets out the methods applied to identify the key areas of training needs as identified by researchers and data providers. Methods of co-production with members of the public are to be published separately. Despite the increased availability of data and interest from funding bodies, there remain challenges for data access, storage and sharing, and difficulties in conducting cross- national research [2, 8–11]. In addition, accessing data from multiple data providers remains a challenge with different applications, requirements and governance across providers [9]. Processes and legal frameworks also change over time, as is expected, with post-GDPR changes having most recently moved the goal posts for researchers applying for data [12]. Keywords d i i https://doi.org/10.23889/ijpds.v7i1.1712 February 2, 2022 © The Authors. Open Access under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/deed.en) Researcher survey We developed a survey aimed at UK researchers who work with administrative data to identify training needs and training preferences. The aim was to use the learning from these surveys, along with feedback from data providers, to develop an online curriculum with two face-to-face workshops. The survey comprised four sections: (1) respondent characteristics; (2) a training needs assessment; (3) information about personal learning style; and (4) researcher experience of public involvement. These domains were selected to enable the team to understand who was completing the survey (i.e., their level of experience in routine data), the key areas a curriculum should cover to identify the desirability of different learning styles to ultimately be included for both the online course and face-to-face workshops and to explore current experiences of public involvement in routine data research aligning with the ICO strategic goals to increase trust and confidence. To date, there have been limited training opportunities for health and social care researchers to learn about the challenges and opportunities involved in preparing, applying for and working with administrative data. Some data providers require researchers to undertake some form of training prior to working with administrative data. Mandated training invariably relates to safe researcher training (data protection, information security, confidentiality) and use of data safe havens specific to one data provider. Other established training (delivered by academic organisations) for researchers is heavily focused upon technical challenges such as data cleaning, standardisation and models of data linkage (i.e., probabilistic, deterministic linkage techniques) [17, 18]. i The survey was piloted in June 2019, with eight researchers, five of whom participated in a de-brief interview. These individuals were accessed via a local routine data network of academics. The pilot ensured the flow of questions was acceptable, including checking the logic (i.e., skips) and user feedback helped to identify questions that could be worded better and topics felt to be missing. The time taken 2 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 to complete the pilot surveys were recorded, to inform future users of an expected completion time. both inform the scope and content of the training but also used as evidence in the training (i.e., quotes) to emphasise or explain a point. The main survey was hosted by Online Surveys (https://www.onlinesurveys.ac.uk) and respondents provided their consent before starting the survey. Analysis (Survey and focus group) The different language [Data Provider] uses and learning it. Understanding what they mean in the questions on their application form. They do provide support, but it would be good to get a very good first draft prepared before needing the help. (RS44) We analysed survey free text responses and the focus group transcripts using framework analysis [22] based on the structure of the questions. This was performed by KM and FLW respectively. Other themes that emerged outside of the framework were identified and coded as such. “We are essentially people who are from different backgrounds looking at things from different perspectives, and trying to communicate in ways that we can both understand.” (DP1) “We are essentially people who are from different backgrounds looking at things from different perspectives, and trying to communicate in ways that we can both understand.” (DP1) Data provider characteristics Twelve individuals working within data access roles from four data providers contributed to the focus groups. These data providers represented health, education and social care data from England, Scotland and Wales (NHS Digital, Department for Education, electronic Data Research and Innovation Service and the Secure Anonymised Information Linkage Databank). Theme 1: communication The first and most prominent theme from both the survey and focus groups was communication challenges. Two issues were apparent through the survey and focus group: a difference in conceptual understanding (i.e., understanding of some of the requirements involved in data applications); and terminological differences (the same term being consistently used differently or being understood differently). Both researchers and data providers mentioned speaking different languages and at times, at crossed purposes. This challenge, perceived by both parties, resulted in delays on both sides while meetings and subsequent drafts of data request applications were (re-)reviewed to ensure mutual understanding. Researcher survey We used a non- probability convenience sampling approach with the intention to include researchers from a range of disciplinary backgrounds and across different public sectors but not with the aim of producing generalisable findings. We expected that we may attract in excess of n=100 respondents. Recruitment for the survey was via social media, e-mail distribution lists (e.g. AllStats, UK Trial Manager Network and UK CRC) and via local contacts in this field. The survey was open for 10 weeks. Survey questions were made up of both open and closed questions. Quantitative data were analysed descriptively. Participant responses in the free text boxes were analysed thematically. Codes were recorded using NVivo (version 12) and themes identified by authors (FLW, KM). All data for the survey are stored within Online Surveys secure data centres operated by Amazon Web Services and Cardiff University servers. Themes There were five high level themes that appeared in both the focus groups with data providers (DP) and the survey with researchers (RS). These are illustrated by a sample of quotes in text and in Supplementary Appendix 1. Quantitative survey data supporting these themes are also presented. Focus Groups with UK data providers We held focus groups with key UK data providers between July – September 2019, both face-to-face and by videoconference. We approached key staff at five data providers, who were involved in data access requests, from England, Wales and Scotland. We targeted those data providers who were considered national administrative data providers for researchers covering the health and social care sectors, informed by the literature [21]. Staff within each data provider were invited to take part in these focus groups, provided with an information sheet and those interested then contacted the study team and a mutual date and time was identified. Two researchers with experience in conducting focus groups (FLW with KM or MR) facilitated the sessions, which each followed the same topic guide. Topic guides were focused on the context of developing a training curriculum for UK researchers, and covered: a description of the data providing organisation and their data access process; challenges and successes of data request applications; available training; the role of the public; monitoring and evaluation of their data provision (audits and at the service level). These areas aligned to both the outputs of the researcher survey and the ICO strategic goals. Consent was recorded via consent forms and confirmed verbally at the start of each session. All focus groups were recorded and transcribed verbatim. Transcripts were uploaded into NVivo (version 12). Survey respondent characteristics A total of 107 responses were received. Not all questions were mandatory, so denominators are provided for each question. 88/99 (89%) respondents were based in a university and the remaining 11/99 (11%) were based at an NHS or Government agency. A breakdown by country of employer is available in Table 1. 95% (82/87) had worked with health administrative data but other data were represented also (Table 2). Curriculum development Data from the survey and the focus groups were reviewed and discussed by the study team. The data gathered were used to 3 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Table 1: Country of employer1 N (%) England 65 (60.1%) Northern Ireland <5 (<5.0%) Scotland 13 (12.1%) Wales 16 (14.9%) UK wide2 <5 (<5.0%) Missing 9 (8.4%) 1Respondents provided the name of current main employing organisation which has been aggregated to country. 2Organisations which worked across more than one nation. Table 2: Data types respondents reported accessing Type of data N (%)2 Health (incl. registries) 82 (95.3) Office for National Statistics (ONS) 34 (39.5) Education 13 (15.1) Social Care 11 (12.8) Criminal Justice 8 (9.3) Third / Charitable Sector 5 (5.8) Work and Pensions 2 (2.3) Other1 4 (4.7) 1Other included transport, housing, environment and census data (Scotland). 2Multiple responses were recorded per respondent. England Northern Ireland Scotland Wales UK wide2 Missing Table 2: Data types respondents reported accessing Type of data N (%)2 Health (incl. registries) 82 (95.3) Office for National Statistics (ONS) 34 (39.5) Education 13 (15.1) Social Care 11 (12.8) Criminal Justice 8 (9.3) Third / Charitable Sector 5 (5.8) Work and Pensions 2 (2.3) Other1 4 (4.7) 1Other included transport housing environment and census data (Scotland) Table 2: Data types respondents reported accessing Type of data Health (incl. registries) Office for National Statistics (ONS) Education Social Care Criminal Justice Third / Charitable Sector Work and Pensions Other1 1Other included transport, housing, environment and census data (Scotland). 2Multiple responses were recorded per respondent. your own research project, it’s an impact on other projects as well that we’re trying to, trying to process.” (DP3) We explored with data providers how best to address this challenge. From their perspective, the key challenge was researchers being able to simplify their language for non-expert readership, to ensure a shared understanding of the project and methods. Researchers regularly referred to a lack of transparency from data providers on such aspects as timelines and the likely time required to approve applications. From a data provider perspective, they had to manage what they felt were often unrealistic expectations on the speed of approval. Theme 2: timelines Although linked to communication, timelines appeared as a separate theme for both researchers and data providers including expectations of the process and related timelines for data access requests. Exploring this from both sides of the application process, it is clear that delays are incurred, and a source of frustration, for both parties. We explored with data providers how best to identify realistic timelines, and how they felt that researchers could potentially mitigate delays in data request applications that impacted time-sensitive aspects of their research. All four data providers emphasised the importance of researchers engaging with their application team as early as possible in the project. They all felt that this would address expectations on timescales, and identify any obvious stop-go criteria that would need to be addressed before proceeding with data request approval. “It is also challenging when you hear nothing / no updates for a period of time and have no information as to whether the application is progressing as it should or it’s been forgotten about (we have experienced both).” (RS96) Theme 3 – changes and amendments “An application is submitted, and we try and speak to the researcher to refine the form, and discuss the governance panel, and for one reason or another, they become quite incommunicado. So the forms can’t be progressed if we can’t have that engagement with the researcher. . . And not having that two-way engagement all the time slows down the process...it’s not always as easy to. . . hold their place in the queue, because while we’re waiting for them we’re processing other cases . . . because the delay, the impact on Another challenge related to timelines were the changing requirements of a research project over the course of the project. In part, this reflects the changing nature of research as new information / research comes to light, but does also highlight the need for the research team and data providers to engage at an earlier stage to agree datasets and associated costs. “we do come across projects where they started off and they want three data sets, and by the 4 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 time they come to permissions they’re looking for an additional six or seven data sets linked into that study. Um that can cause some interesting conversations if they’ve gone for funding based on the original.” (DP4) they’ll do a trial linkage so we’re covering consent here but also just also in terms of data quality and linkage variables knowing what your collecting, is collected in the right way and the right format, is it linkable, have you got errors all these little things. . . it will help you out in the long term, rather than failing really hard at the end of the process.” (DP1) they’ll do a trial linkage so we’re covering consent here but also just also in terms of data quality and linkage variables knowing what your collecting, is collected in the right way and the right format, is it linkable, have you got errors all these little things. . . it will help you out in the long term, rather than failing really hard at the end of the process.” (DP1) Data providers felt that significant changes could often have been avoided by discussing the data flows and datasets earlier in the study process. Theme 4 – future-proofing the application A significant challenge, and an area in which the needs of researchers and data providers could often diverge, related to the length of time that the research data could be held, for archiving and/or re-use in further future research. Consent considerations for both of these scenarios, as well as associated costs require up front consideration. Consent wording will need to be approved at the data request application, and costs agreed at grant application stage. Researchers felt that this is not always feasible or realistic. The survey asked researchers “What key challenges do you face in planning a study using routine data?”: The focus groups with data providers explored training that they make available, or recommend to those applying for data. Much of their training was in the form of guidance documents available on their website or sent to applicants alongside the application form or phone calls with applicants. Two data providers noted challenges relating to their guidance not being read when applications were being submitted and one data provider highlighted the problem of researchers often relying on informal training, or seeking support from colleagues who had previously applied for data under different governance arrangements. Lack of clarity about what is required on consent forms to link trial data to routine data in future (especially non-health data) (RS89) Lack of clarity about what is required on consent forms to link trial data to routine data in future (especially non-health data) (RS89) Lack of clarity about what is required on consent forms to link trial data to routine data in future (especially non-health data) (RS89) How long you are allowed to keep data, in order to plan whether several applications for extension will be needed over the course of a project or not. (RS5) Costs over time change (e.g. costs for archiving is now a consideration whereas before it did not need to be costed) (RS107) Costs over time change (e.g. costs for archiving is now a consideration whereas before it did not need to be costed) (RS107) “So in my experience I feel as though a lot of the experience in academic research is handed down. So we get new researchers coming through, PhD students etc, and they speak to people who’ve done research before, which obviously makes sense. And they might be told, ‘we use this consent form’ . . . Theme 3 – changes and amendments Designing data flows to maximise regulatory compliance requires input from all parties (data providers, regulators and the sponsor). A data flow set-up for one project may not be appropriate for all future study designs, and as technology continues to advance, different methods of data transfer and storage may become a preferred option for data providers. One challenge (for both researchers and data providers) is when regulations change rendering earlier decisions incompatible with current regulations. The impact of General Data Protection Regulation (GDPR) on historic trials (i.e. set-up and consented years before the DPA changes were in development) remains a challenge today. Data providers and researchers continue to navigate this hurdle. Considerations related to section 251 support to enable a legal flow of data is a useful option for those working with health-related data, and awareness of this as an option seems important to enable research projects to continue their work. “As a department we don’t necessarily want to feel backed in or forced into a route. We want to work with, what’s the most secure way and most efficient way of achieving your project’s um desired outcome.” (DP3) Theme 5 - training for researchers This quote also raises co-production of data sharing approaches and the importance of early discussions with those involved in the release of data. When asked about formal routine data training they had completed, one third of researchers (26/77) indicated they had completed no formal training related to working with routine data. 43% (n = 33) stated training such as the Medical Research Council (MRC) Research, GDPR and confidentiality training [23] and the ONS Safe Researcher Training [24] – which are mandatory for those accessing data from some data providers. Data provider specific training (including workshops and webinars) accounted for 13% of the training listed, in- house training was mentioned by 17% of researchers and 13% stated having received training on the statistical methods applied to routine data. Informal support researchers received primarily came from colleagues (43%), Data Providers (16%) and networking/conferences (19%). Multiple answers were possible for both of these questions. Theme 4 – future-proofing the application and then they’ll use that consent form virtually word for word with just the particulars of their project passed on. And actually they’re not getting the current guidance. . . Somebody who’s done research for 40 years will say what you do is you get the data, This highlights the ongoing challenge for researchers to put things in place at the start of a project that will still be relevant years down the line. Time between study set-up and linkage may well be one aspect that can be managed in future projects by conducting more than one linkage as the below data provider describes: “I would always say to research projects: don’t leave it so long . . . people that tend to do this better will not collect stuff for 5 years and then try and link it. They’ll collect for 6 months and 5 • Process of applying (flow diagram) • Interdependencies on getting an application approved (ethics, security, etc) • Opportunity for applicants to see data before requesting (dummy data) • Required preparation before applying • How to write a good application (using the right language) • Safe haven (what it is, how it can be used) • Disclosure control • How to have a successful two-way conversation with data providers • GDPR concepts / ICO requirements • Understanding the legislative framework • Privacy notices • Timelines / project management • Data management (recording what you’re doing, decisions made) and you store it here and so on. Whereas actually I think it’s worth people considering whether they actually need to have the data there, whether that’s the optimum way forward, whether it’s the most cost-effective way forward. But also as part of that decision they should be talking to the data providers, because there’ll be an awful lot that people don’t know. People won’t necessarily know about [accessing data via a safe haven] for example. And their mentors in the profession won’t necessarily know that either, despite our best efforts to try and get the information out there.” (DP2) communication and timelines both contribute to the problem of the other (i.e. poor communication leads to increased timelines and unrealistic expectations of timelines is due to poor communication). In a similar vein, changes to the study design or datasets can impact timelines of the study approvals and could be avoided with better communication, earlier in the study. Other ongoing work Many challenges highlighted here have been anecdotally reported in relation to specific projects or data providers [9, 11, 25]. Through this work, we have identified the broader current issues relating to working with administrative data, as well as developing a more detailed understanding and context around known issues by working with both researchers and data providers. Funding has been directed to address some of these ongoing limitations of administrative data with a report being issued by Department of Health and Social Care to review the current state of the use of health data for research and analysis (Goldacre Review[26]). The Health Data Research UK (HDR UK) was established in 2018 as the national institute for data science in health. Through the UK HDR Alliance (launched in February 2019) and funding Theme 4 – future-proofing the application The experiences and challenges of researchers attempting to future proof their applications are echoed by the data providers. Indeed, changing regulations have impacted both parties and the required changes to processes and data application requests. The availability of training for researchers for working with administrative data primarily focusses on the secure access and processing of such data or technical methodological approaches to analysing these data. Informal support from colleagues was the most relied upon form of training, however, data providers highlighted the risk in this approach (i.e. providing out-dated information) and the potential negative impact on data requests. Data providers reported providing support to the research community via local research networks, roadshows, conference workshops and workshops hosted at the organisation. Future plans included webinars, animations and videos to be made available on their website. Data providers were specifically asked what they would want to see in a training curriculum to address the challenges they experience with applicants, and also made suggestions throughout the focus groups. These were thematically coded and Box 1 presents all suggestions identified from the discussions. The CENTRIC training curriculum Six modules were agreed upon to cover the content of training (Figure 1). Across the six modules developed for this training curriculum, all of the five mentioned themes and challenges are addressed and included. The training includes tips, checklists and examples throughout the curriculum to improve communication between researchers and data providers, highlighting common pitfalls and quotes from the focus groups. Modules 1 and 2 provide an overview of how accessing administrative data fits into the study lifecycle and notes key points in which delays can occur and ways to avoid this. To reduce the need for amendments, Modules 3 and 4 provide considerations to data storage and applicable regulatory considerations that may result in changes to aspects of study design (e.g., data flows). Module 5 provides detailed information on the application process which aims to familiarise the learner with this process and set realistic expectations related to timelines. Other relevant training courses are linked to throughout the course, where these fall pre-pandemic, and experiences of data access reflect a pre- pandemic view. Access to data has changed, in particular for COVID-19 related projects receiving expedited approvals causing delays for other (non-COVID-19 related) research. Processes in place to deliver COVID-19 research rely on the suspension of Regulation 3(4) of the Health Service Control of Patient Information Regulations 2002 which is only a temporary situation during pandemic times [30]. Calls for these changes to be implemented to the post-pandemic business-as- usual procedures have been supported by over 350 researchers in the UK [31, 32]. This may impact some elements of the CENTRIC curriculum and highlights a key challenge in this ever-evolving area of research – the need for regular review to ensure sustainability and ongoing relevance. from UK Research and Innovation and medical charities such as the Wellcome Trust and the British Heart Foundation, HDR UK Hubs and HDR Gateway, HDR UK brings together health data assets with specialist expertise across academia, industry and healthcare [4, 27]. HDR UK have recently published, via HDR UK Futures, a range of bitesize videos across a range of curriculums including analytical skills, using the HDR UK Gateway, health data access and PPIE for health data research [28]. The Trials Methodology Research Partnership (TMRP), funded by NIHR and MRC, are also progressing the methodological challenges through the Health Informatics working group and routine data topic group [29]. Both national networks are addressing broad health-related and trial-related challenges respectively. At a more local level, data providers are reviewing their application forms, processes and communication with researchers. Summary of findings Five main themes identified areas for improvement from which we elicited specific learning needs for UK researchers working with administrative data. These were: Communication; Timelines; Changes & amendments; Future-proofing applications; and, (lack of) available training and support. Although presented as two separate themes due to the wealth of data, 6 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Figure 1: CENTRIC training module details Conclusion 7. UKRI ESRC: https://www.adruk.org/news-publications/ funding-opportunities/ The CENTRIC online training curriculum has been developed using a co-production model informed by the public, researchers and data providers and is available, free of charge, for UK researchers. The training addresses the most commonly identified challenges and training needs, as described in this paper, for both researchers and data providers. We hope that this training course will facilitate improved access and management of administrative data in the UK and help reduce barriers that we have identified between researchers and data providers. 8. Jones KH, Heys SM, Daniels H, Ford DV. Exploring barriers and solutions in advancing cross-centre population data science. Int J Popul Data Sci. 2019 Aug 5;4(1):1109. https://doi.org/10.23889/ijpds.v4i1.1109. PMID: 34095536; PMCID: PMC8142621. 9. Lugg-Widger FV, Angel L, Cannings-John R, Hood K Hughes K, Moody G and Robling R (2018) Challenges in accessing routinely collected data from multiple providers in the UK for primary studies: Managing the morass. IJPDS Special issue: Cross-Centre Working, 3:3:2, https://doi.org/10.23889/ijpds.v3i3.432 Strengths and limitations Aligned with the ICO strategic goals [20] we have developed a public-informed training programme through a process of co-production which explores public views and public understanding, tailored to the needs of researchers. We sampled researchers from many disciplines and levels of experience in using administrative data as well as four of the largest data providers in the UK. To note, this did not include a data provider in Northern Ireland (NI) and unique features of the data access arrangements and customers for NI data may not be well represented in our current curriculum. We will seek to address such potential gaps in future iterations of the training. Through co-production with members of the public we have ensured the “working with the public” module in particular is relevant and addresses areas of importance felt by the public. This training was developed 7 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Statement on conflicts of interest 11. Macnair, A., Love, S.B., Murray, M.L. et al. Accessing routinely collected health data to improve clinical trials: recent experience of access. Trials 22, 340 (2021). https://doi.org/10.1186/s13063-021-05295-5 The authors declare that they have no competing interests. Acknowledgments We would like to thank all those who took part in the surveys, workshops and focus groups as part of this study. The Centre for Trials Research receives funding from Health and Care Research Wales and Cancer Research UK. The study was funded by the Information Commissioner’s Office. 10. National Cancer Research Institute. The researchers’ experience when attempting to access health data for research. Available at: https://www.ncri.org. uk/accessing-health-data-for-research/ Accessed 22 Oct 2021. References outside the remit of the curriculum: for example, statistical and data management skills. Public input and engagement is covered in Module 6, reflecting the importance as well as the practical challenges of public input and engagement in administrative data research. This final module was co- produced by members of the public and the development will be reported separately. The online course went live in September 2020. Face-to-face training days were planned for the summer of 2020, however, this was moved to online webinars (Getting a grip! Regulatory requirements when using administrative data; Involving the public in studies using administrative data – the How and the Why.) held in October 2020 due to the COVID-19 pandemic. 1. Hemingway, H., Lyons, R., Li, Q., Buchan, I., Ainsworth, J., Pell, J. and Morris, A. (2020) “A national initiative in data science for health: an evaluation of the UK Farr Institute”, International Journal of Population Data Science, 5(1). https://doi.org/10.23889/ijpds.v5i1.1128 2. Lee, S., Xu, Y., D’Souza, A. G., Martin, E. A., Doktorchik, C., Zhang, Z. and Quan, H. (2020) “Unlocking the Potential of Electronic Health Records for Health Research”, International Journal of Population Data Science, 5(1). https://doi.org/10.23889/ijpds.v5i1.1123 To assess impact, in the short-term, we evaluated the training curriculum in two ways. Firstly, within the online training course, learners were invited to provide module specific feedback via a brief survey. Secondly, we encouraged reflection and feedback from participants who took part in our online webinars, to assess whether the content of the online course (and the webinars themselves) matched the needs of the learners. For the longer-term, we are engaging with data providers, regulators and funders for feedback and endorsement. We will also continue to monitor the feedback being provided by learners in the course modules, to ensure that the content continues to meet researcher and data provider needs. 3. Mc Cord KA, Al-Shahi Salman R, Treweek S, Gardner H, Strech D, Whiteley W, et al. Routinely collected data for randomized trials: promises, barriers, and implications. Trials. 2018;19(1):29. https://doi.org/10.1186/s13063- 017-2394-5 4. UK Health Data Research Alliance. Available at https://ukhealthdata.org/. Accessed 22 Oct 2021. 5. NHS Digitrials. Available at https://digital. nhs.uk/services/nhs-digitrials. Accessed 22 Oct 2021. 6. ADRUK website: https://www.adruk.org/our-work/ browse-all-projects/data-first-harnessing-the-potential- of-linked-administrative-data-for-the-justice-system-169/ Ethics Statement 12. Russell, A.E., Ford, T., McIntosh, A., Jones, P.B., Shenow, S., Russell, G. and McManus, S., 2021. Researcher access to mental health data: results from an online consultation. Ethical approval for the study was provided by Cardiff University School of Medicine Research Ethics committee (Ref 19/51) 8 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 13. Cross, L., Carson, L. E., Jewell, A., Heslin, M., Osborn, D., Downs, J. and Stewart, R. (2020) “Guidance for researchers wanting to link NHS data using non- consent approaches: a thematic analysis of feedback from the Health Research Authority Confidentiality Advisory Group: A thematic analysis of feedback from the Health Research Authority Confidentiality Advisory Group”, International Journal of Population Data Science, 5(1). https://doi.org/10.23889/ijpds.v5i1.1355 24. Office for National Statistics. Accessing secure research data as an accredited researcher. Available at: https://www.ons.gov.uk/aboutus/whatwedo/statistics/ requestingstatistics/approvedresearcherscheme Accessed 29 Nov 2021. 25. Taylor JA, Crowe S, Espuny Pujol F, et al The road to hell is paved with good intentions: the experience of applying for national data for linkage and suggestions for improvementBMJ Open 2021;11:e047575. https://doi.org/10.1136/bmjopen-2020-047575 14. NHS Digital Event Review: 25 November 2020 Using health and social care datasets in research. Available at: https://digital.nhs.uk/services/research-advisory- group/using-health-and-social-care-datasets-in-research Accessed 22 Oct 2021. 26. GOV.UK. New review into use of health data for research and analysis. Available at: https://www. gov.uk/government/news/new-review-into-use-of- health-data-for-research-and-analysis Accessed 22 Oct 2021. 15. Applebe, D., Parker, C.,and Hartley, S. Making Requests to NHS Digital Easier. Available at: https://www. nihr.ac.uk/documents/explore-nihr/Efficient%20studies/ Liverpool%202017%20Final%20Report- Making%20Access%20to%20NHS%20Digital%20Easier.pdf Accessed 22 Oct 2021 27. HDRUK. Our funders. Available at: https://www. hdruk.ac.uk/about-us/funders/ Accessed 29 Nov 2021. 28. Health Data Research UK. Continued Professional Development. Available at: https://www.hdruk.ac.uk/ careers-in-health-data-science/continued-professional- development/power-up-your-health-data-science- knowledge/ Accessed 22 Oct 2021 16. Data Security and Protection Toolkit. Organisation Search. Available at: https://www.dsptoolkit. nhs.uk/OrganisationSearch Accessed 22 Oct 2021. 17. University of York. Analysing Patient-Level Data using Hospital Episode Statistics (HES). Available at: https://www.york.ac.uk/che/courses/patient-data/ Accessed 22 Oct 2021. 29. Trials Methodology Research Partnership. TMRP Working Group Funding Awards https://www. methodologyhubs.mrc.ac.uk/about/tmrp-working- group-funding-awards/ 18. University College London. Introduction to Hospital Episode Statistics. Available at: https://www.ucl. ac.uk/child-health/events/2021/oct/introduction- hospital-episode-statistics Accessed 22 Oct 2021. 30. Department of Health and Social Care. Coronavirus (COVID-19): notice under regulation 3(4) of the Health Service (Control of Patient Information) Regulations 2002. Available at: https://www.gov.uk/ government/publications/coronavirus-covid-19- notification-of-data-controllers-to-share-information/ coronavirus-covid-19-notice-under-regulation-34-of-the- health-service-control-of-patient-information-regulations- 2002-general–2 Accessed 29 Nov 2021. 19. Information Commissioner’s Office. Cardiff University. Available at: https://ico.org.uk/about-the-ico/what-we- do/grants-programme/cardiff-university/ Accessed 22 Oct 2021. 20. Information Commissioner’s Office. Ethics Statement Our mission, vision, strategic goals and values. Available at: https://ico.org.uk/about-the-ico/our-information/ mission-and-vision/ Accessed 22 Oct 2021. 31. Cavallaro, F., Robling, M., Lugg-Widger, F., Cannings- John, R., Aldridge, R., Gilbert, R., Harron, K. Open letter Open letter to the ICO, CMOs and UK data providers: Reducing barriers to data access for research in the public interest-lessons from covid-19 (with signatories). Available at: https:// eprints.ncl.ac.uk/file_store/production/267907/ 3FF575A5-4795-40EA-B0EB-447290C11B3E.pdf Accessed 22 Oct 2021. 21. Lensen, S., Macnair, A., Love, S.B. et al. Access to routinely collected health data for clinical trials – review of successful data requests to UK registries. Trials 21, 398 (2020). https://doi.org/10.1186/s13063-020-04329-8 22. Gale, N.K., Heath, G., Cameron, E. et al. Using the framework method for the analysis of qualitative data in multi-disciplinary health research. BMC Med Res Methodol 13, 117 (2013). https://doi.org/10.1186/1471- 2288-13-117 32. Cavallaro, F., Lugg-Widger, F., Cannings-John, R., Harron, K. 2020. Open letter to the ICO, CMOs and UK data providers: Reducing barriers to data access for research in the public interest-lessons from covid-19. British Medical Journal. 23. MRC. e-Learning. Available at: https://byglearning. com/mrcrsc-lms/course/index.php?categoryid=1 Accessed 29 Nov 2021. 9 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Changes and amendments “It could be, we see this commonly with section 251, people go and get section 251 support, and then we’re actually not happy with the methodology, because it involves us flowing more data than is actually needed to. Or sometimes receiving a huge amount of data from a third party, such as an auditor or a register to link, whereas actually all we need is the identifying data, we don’t need the clinical data, and then we can provide a bridge file. So if we can liaise on things like methodology at the earliest point, we can address a lot of those issues.” (DP2) Abbreviations Abbreviations ADRUK : administrative data research CENTRIC : Co-Produced and stakeholder informed training for UK researchers working with routinely collected data GDPR : General Data Protection Regulation HDRUK : Health Data Research UK HRA : Health Research Authority ICO : Information Commissioner’s Office MRC : Medical Research Council NIHR : National Institute for Health Research TMRP : Trials Methodology Research Partnership (TMRP) 10 Lugg-Widger, F et al. International Journal of Population Data Science (2022) 7:1:2 Supplementary Appendix 1: Supplementary quotes from researchers and data providers Supplementary Appendix 1: Supplementary quotes from researchers and data provider Timelines “Time. Time. Time. It is almost impossible to even roughly estimate a timeframe for a project using routinely collected data. Information Governance approvals, data sharing agreements, and data transfer are all areas in which the researcher has no direct control over timeframe.” (RS58) “And that’s one of our challenges that we face, where we’d receive an application form in, and [a researcher says] ‘I need my data in two weeks’ time’. You say well sorry but it takes two weeks to create and curate the data, never mind to get it through the governance process.” (DP1) So it’s about engaging with us as a data sharing team at the earliest opportunity. You know, there’s some fantastic research ideas out there, and some fantastic people who want to do this research. But pressures of actually getting the data in a timely manner, you know, and talking to us first, and saying “I want to do this research, from your experience do you think it would be something that [data provider] could help with? How long might it take to get that data?” But also about, not assuming you can automatically have the data. It’s not a given that you can have our data (DP3) Supplementary quotes “As an example, everybody in the research environment seems to understand what it means to sponsor a study, and I’ve had numerous definitions of this and still don’t have a concrete view.” (DP2) “It’s the ability to step outside what you know, and view the same subject from the perspective of a lay person. . . it’s literally how you explain what you do to the man in the street . . . we see a lot of applications with an awful lot of Latin in, or very detailed information about the statistical analysis. . .” (DP2) Future-proofing the application “I think consent as a concept changes over time and some of it can be handled on best intent at the time of collection of consent. But there are hard and fast rules that people can’t get around now with things like DPA, GDPR things like that so it’s just having that knowledge and being aware that when you are starting to collect data or when you want to start using it for research purposes, do you do things with best intent? Do you consult with the relevant data providers or whoever they are to get it as close as possible to correct?” (DP1) “A lot of the studies are follow-up studies, so whilst they may have been having access to health records during that period, after a certain amount of years they’re then going to come into us to follow them up, to have the data. And it’s then that the consent model is a lot out of date, and it just doesn’t meet today’s standards or requirements. And that’s a big issue. . . It’s making that decision as well as whether, if there’s enough for it to stand as to meet the common law of duty, or whether you would then have to go and get section 251. And I think for some of our researchers that can be seen as. . . another obstruction, but it’s not, it has to meet, you know, it’s a legal requirement that you do have to have a legal basis to be able to have access to that”. (DP2) Training for researchers Training for researchers “The aim with the [guidance documents] is to firstly improve transparency with our customer base, so that they can understand this is what this part of the application form has to adhere to in order to meet the approval standard.” (DP2) “we offer a service where if people want a telephone call to discuss their application, or discuss the requirements of their data share, we’re quite happy to speak to people. Or if people want a hand filling in the application form, we’ll also talk through application processes” (DP3) “what we’ve done is we’ve written a guidance document, made that available within our environment for people to refer to. But you can lead a horse to water, but you can’t always get it to drink the water.” (DP4) “the amount of organisations that don’t read the guidance that we put out.” (DP3) (DP3) 11
https://openalex.org/W4308527069	https://www.nature.com/articles/s41598-022-23634-7.pdf	English	null	Investigation of the compaction process of electrical machines magnetic circuits and its detrimental effect on magnetic performances	Scientific reports	2,022	cc-by	12,430	performances Hugo Helbling1, Adrien Van Gorp2, Abdelkader Benabou1, Thierry Coorevits2, Abdelmounaïm Tounzi1, Walid Boughanmi3 & Daniel Laloy3 Hugo Helbling1, Adrien Van Gorp2, Abdelkader Benabou1, Thierry Coorevits2 Abdelmounaïm Tounzi1, Walid Boughanmi3 & Daniel Laloy3 The manufacturing processes of electrical machines may lead to significant degradation of the magnetic properties of their magnetic core (stator, rotor) performances and, as a consequence, to a decrease of their energy efficiency. While the effects of some processes (cutting, welding …) are widely discussed in the literature, this is not the case with the compaction process although it is systematically used to maintain the assembly of electrical steel sheets that compose the magnetic circuits. In addition to the conventional one, a specific compaction process exists for high-power electrical machines. After an introduction, the paper firstly deals with the two studied processes (conventional, specific). Then, an experimental mock-up to study the impact of the two configurations on the magnetic properties (iron losses, normal magnetization curve) is presented. This mock-up is the first, in the literature, that allows to study the effect of a controlled compaction mechanical stress on magnetic properties. Obtained results in both configurations highlight a magneto-mechanical effect that is not reported in the literature where these effects are commonly considered following in-plane mechanical stresses. This paper presents a magneto-mechanical model, taking into account the compaction stress effect, as well as a modelling protocol to model the effect of 3D mechanical stress on magnetic properties, which has never been done in the literature. In the context of energy transition, increasing the energy efficiency of electrical machines is a key point. This involves reducing the losses and in particular the iron loss contribution associated to the magnetic circuits. The latter are difficult to quantify accurately and there still exist variable and significant differences between the iron losses estimated by the manufacturer during the design stage and those measured on the machine once manufactured. These differences are noticeably due to the electrical steel magnetic properties degradation fol- lowing the manufacturing process. The significance and type of this effect are strongly related to the complexity of the microstructure of the magnetic materials as well as to their strong mechanical and thermal couplings1–3. In particular, it is shown in the literature that the manufacturing processes of the magnetic circuits of electrical machines can lead to significant degradation on their magnetic performances (iron losses, normal magnetization curve)4,5. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ (compressive stress or tensile stress), the level of stress and its direction with regard to the magnetic flux direction. However, for mechanical stress applied in the thickness direction of the lamination, few papers are found in the literature and the presented results vary significantly from one study to another9–11. Obviously, in this direction, one has to only consider compressive stress because tensile stress has no physical application and is against the objective of the compaction process. It is shown in10 that the application of an axial compressive stress of up to 24MPa induces an improvement (up to 8MPa) and then a degradation (beyond 8MPa) of the magnetic properties of a toroidal core composed of a single sheet. In11, the authors study the effects of localized stress, up to 10MPa, in the thickness direction of a rectangular magnetic sheet: the result is a degradation of the magnetic properties. Moreover, the observed degradations on the magnetic properties are similar, considering a magnetic flux along the rolling direction, for a stress of 0.5MPa and for a stress of 10MPa. In9, a dedicated ring core tester is developed to study the effect of axial stress on a single ring core sample. Contrary to what is observed in10, a degradation of the magnetic properties is first observed (up to 10MPa) and then a gradual improvement appears up to 30MPa.hi g p pi p g p pp p These significant variations in the results can be explained by several factors. Firstly, the possible presence of mechanical stresses in the plane of the samples, due to potential imperfection of the applied normal stress, is not verified. However, we have seen, according to the literature, that in-plane stresses can have a significant effect on magnetic properties. Noticeably in9, mechanical Finite Element (FE) simulations show an important difference between the distribution, within the studied electrical steel sheet, of the stress applied in the axial direction and the distribution of the Von Mises stress (a scalar equivalent stress considering the 3D mechanical stress distribution), thus suggesting the presence of non-negligible stresses in the plane of the sample. Then, since the studies were conducted on magnetic circuits composed of very few laminations (usually only one), these are in proportion more sensitive to the friction at the interface with device used for the compaction force application, which is more likely to induce stress in the plane of the laminations. www.nature.com/scientificreports/ Finally, the studied geometry undoubtedly has an influence on the distribution of mechanical stresses as it is shown in12. In general, the fact that existing works do not make it possible to control and/or know the 3D mechanical stress distribution makes it impossible to conclude on the effect, on magnetic properties, of mechanical stresses applied in the thickness direction or the development of generalizable and predictable magneto-mechanical models modelling the effect of compaction process on magnetic properties.ffi In that context, this paper proposes to study the effect of two different configurations of compaction pro- cess on the magnetic properties of an electrical steel stack: a conventional configuration with a homogeneous compaction and a specific one, which is mostly employed to manufacture high-power electrical machines, exhibiting heterogeneous compaction pressure. These two configurations will be firstly presented as well as the experimental mock-up developed to study their effect on magnetic properties of electrical steel laminations. Then, experimental results will be presented and discussed to move on to the modelling phase. For this part, mechanical FE simulations were performed. Moreover, magneto-mechanical models and a protocol for modeling the effect of 3D mechanical stresses on magnetic properties are described. The experimental results and those given by the modelling approach are then compared and discussed. Finally, conclusions and perspectives of this work will be given. performances While the effects of some manufacturing processes (such as cutting6, welding7, shrink-fitting8, …) are widely studied in the literature, the compaction process remains rarely studied.h y p p y The magnetic cores of electrical machines are mainly composed of stacked electrical steel sheets. In order to maintain the lamination assembly, a compaction mechanical effort is applied along the laminations thickness direction. Usually maintained by a dedicated system (clamping screws, bars welded to yokes …), this compac- tion effort implies the presence of mechanical stresses within the laminations. Depending on the configuration of the application of the compaction force, the mechanical stresses can be three-dimensional (in the plane or along the thickness direction of the sheets).l g According to the literature, mechanical stress applied in the plane of electrical steel sheets has a strong influ- ence on their magnetic properties1: this can improve or degrade them depending on the nature of the stress 1Univ. Lille, Arts et Metiers Institute of Technology, Centrale Lille, Junia, ULR 2697 - L2EP, F‑59000 Lille, France. 2Arts & Metiers Institute of Technology, Mechanics, Surfaces and Materials Processing (MSMP), Lille, France. 3JEUMONT Electric, 59460 Jeumont, France. email: Abdelkader.Benabou@univ-lille.fr Scientific Reports \| (2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 www.nature.com/scientificreports/ Experimental mock‑upi p p Compaction configurations. The conventional compaction process involves pressing a single stack of electrical steel sheets that makes up the stator or the rotor. In the case of large alternators, the magnetic circuit is usually divided into several stacks separated by interlayer sheets composed of airvent spacers. The objective of this configuration is to facilitate the cooling of the machine by thermal convection. The conventional configura- tion will be considered in the following as the homogeneous case (the compaction force is applied homogene- ously over the whole circuit). The second configuration, with airvent spacers, will be denominated as the inho- mogeneous case (the presence of airvent spacers generates localized mechanical stresses). Both configurations are presented in Fig. 1. In the inhomogeneous case, the industrial height of laminations stack is typically of few centimeters. As illustration, photos of two industrial stators with airvent spacers are given in Fig. 2. p p Compaction configurations. g g Generally, the global compaction stress (over the whole magnetic core) has the same value for the homogene- ous and inhomogeneous cases (about 1 MPa). In the inhomogeneous case, the value of the localized mechanical stress under the airvent spacers is between 5 MPa and 20 MPa, depending on the number of airvent spacers and their geometry. Design and fabrication. To characterize the magnetic properties (iron losses, normal magnetization curve) of a magnetic core in the two presented configurations, a dedicated magnetic mock up is developed. First, concerning the magnetic circuit, the selected electrical steel is a conventional material with a lamination thickness of 0.65 mm (grade M600-65A) commonly used in high-power electrical machines. Moreover, for the experimental investigations, a toroidal geometry is preferred because it is closer to the geometry of the electrical machine stator cores. Therefore, several rings were cut from the raw laminations by Wire Electrical Discharge Machining (WEDM) that is known as a non-degrading cutting method for the magnetic properties13. The final height of the magnetic core, made from stacked rings, is 42 mm. All the characteristics of the used magnetic circuit are presented in Table 1.h p The global mock-up is shown in Fig. 3, a zoom of the part composed by the magnetic circuit, the winding supports, the PVC plates, the screw and the force sensor is given in Fig. 4. Finally, the support with the spacers is presented in Fig. 5.h g Two winding supports, Fig. 4d, are placed on either side of the magnetic core, Fig. 4f. This makes possible to insert the primary and secondary windings while keeping the ability to apply the mechanical stress. The material used for the winding supports is Lab1000 that is amagnetic and electrically nonconductive. PVC plates, Fig. 4c, Scientific Reports \| (2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 www.nature.com/scientificreports/ Figure 1. The two studied configurations. Figure 2. View of two industrial stators (with airvent spacers). Laminations grade Insulation Cutting method M600-65A Coated (Alkophos method) Wire Electrical Discharge Machining (WEDM) Outer diameter (OD) Inner diameter (OD) Height 100 mm 86 mm 42 mm Figure 1. The two studied configurations. Figure 1. The two studied configurations. 3 Vol.:(0123 18983 \| https://doi.org/10.1038/s41598-022-23634-7 are also placed on both sides of the winding supports to promote the stress distribution homogeneity. These plates are also machined to have a good flatness. The whole is hold tight together with a force sensor, Fig. p p Compaction configurations. 4b, itself solidarized and centered with the fixed structure of the press, Fig. 3a. This latter is composed of metal ele- ments welded together and designed to support the mechanical levels of stress that will be applied by a hydraulic jack, Fig. 3d. For the inhomogeneous case, airvent spacers supports, Fig. 5, are machined with the same material Figure 2. View of two industrial stators (with airvent spacers). Table 1. Magnetic core characteristics. Laminations grade Insulation Cutting method M600-65A Coated (Alkophos method) Wire Electrical Discharge Machining (WEDM) Outer diameter (OD) Inner diameter (OD) Height 100 mm 86 mm 42 mm Figure 2. View of two industrial stators (with airvent spacers). Figure 2. View of two industrial stators (with airvent spacers). Figure 2. View of two industrial stators (with airvent spacers). are also placed on both sides of the winding supports to promote the stress distribution homogeneity. These plates are also machined to have a good flatness. The whole is hold tight together with a force sensor, Fig. 4b, itself solidarized and centered with the fixed structure of the press, Fig. 3a. This latter is composed of metal ele- ments welded together and designed to support the mechanical levels of stress that will be applied by a hydraulic jack, Fig. 3d. For the inhomogeneous case, airvent spacers supports, Fig. 5, are machined with the same material https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Global mock-up; (a) fixed structure of the press; (b) see Fig. 4; (c) movable steel plate; (d) hydraulic jack. Figure 3. Global mock-up; (a) fixed structure of the press; (b) see Fig. 4; (c) movable steel plate; (d) hydraulic jack. Figure 4. Zoom of the mock-up; (a) screw; (b) force sensor; (c) PVC plate; (d) winding frame; (e) airvent spacers support; (f) laminated magnetic circuit. Figure 4. Zoom of the mock-up; (a) screw; (b) force sensor; (c) PVC plate; (d) winding frame; (e) airvent spacers support; (f) laminated magnetic circuit. as the winding supports (amagnetic and electrically nonconductive). In this case, the airvent spacers supports are placed between the magnetic circuit and the winding supports, Fig. 4. For the homogeneous case, these are simply removed. Note that in Fig. 5, there is an outer ring around the spacers to promote the alignment between the different components of the mock-up. p p Compaction configurations. However, in order to facilitate the visualization of the airvent spacers, this outer ring has been removed from the view in Fig. 4. as the winding supports (amagnetic and electrically nonconductive). In this case, the airvent spacers supports are placed between the magnetic circuit and the winding supports, Fig. 4. For the homogeneous case, these are simply removed. Note that in Fig. 5, there is an outer ring around the spacers to promote the alignment between the different components of the mock-up. However, in order to facilitate the visualization of the airvent spacers, this outer ring has been removed from the view in Fig. 4. g g To validate the experimental mock-up, it is necessary to verify that, in the homogeneous case, the axial mechanical stress distribution is homogeneous within the magnetic core and that the mechanical stress distri- bution in the plane of the laminations is negligible in order to quantify only the effect of the normal mechanical stress on the magnetic properties.t g p p First, mechanical Finite Element (FE) simulations were performed with the Abaqus Software14. According to the involved levels of mechanical stress, the mechanical behavior of all the materials will remain in the elastic region. The list of materials chosen for the FE simulations and their associated mechanical properties are given in Table 2. https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ Figure 5. Airvent spacers support. Figure 5. Airvent spacers support. Table 2. Mechanical properties used for mechanical FE simulations. Part of the mock-up Material Young’s modulus (GPa) Poisson’s ratio (–) Force sensor Aluminium 70 0.34 PVC plate PVC 3 0.40 Winding support Lab1000 5.9 0.35 Magnetic laminations Steel 211 0.29 Table 2. Mechanical properties used for mechanical FE simulations. Table 3. Chosen friction coefficients for mechanical FE simulations. Materials interfaces Chosen friction coefficients Screw/force sensor 0.4 Force sensor/PVC plate 0.5 PVC plate/winding support 0.5 Winding support/lamination (Teflon) 0.05 Lamination/lamination 0.2 Table 3. Chosen friction coefficients for mechanical FE simulations. Table 3. Chosen friction coefficients for mechanical FE simulations. The friction coefficients between the different parts need to be determined because of a potential significant effect on the mechanical stress distribution. Indeed, the mechanical parameters being different for each material, the deformations will also be different. Without friction, each part moves freely without any induced mechanical stress. p p Compaction configurations. The measured diameters of the balls are very close (< 5 µm) to the theoretical diameter provided by the manufacturers. From the positions of the bearing balls, the position of each winding support can be determined by applying the least-squares method and, from these positions, the objective is to determine the kinematic torsor T to quantify the displacement of the supports. For this, winding supports are considered as non-deformable solids. The six components of the torsor T, defined at the center of each support, (1), noted (u, v, w) and (α, β, γ), represent respectively the transla- tion and rotation of the winding support according to the three axes x, y and z. The indices T and B correspond respectively to the top and bottom winding support.h in the stress distribution. For this, metrology tests were carried out, as illustrated in Fig. 11. The objective is to quantify the displacement of the two winding supports under compaction effort. As these are the parts that are the closest to the magnetic circuit, this will best reflect the mechanical behavior of the latter. First, eight bearing balls are glued to the outer diameter of the winding supports (four per support). Each ball is then measured on a high-precision coordinate measuring machine to determine its diameter (known) to confirm the measuring quality and its center position in the (x, y, z) coordinate system, Figs. 11 and 12. The measured diameters of the balls are very close (< 5 µm) to the theoretical diameter provided by the manufacturers. From the positions of the bearing balls, the position of each winding support can be determined by applying the least-squares method and, from these positions, the objective is to determine the kinematic torsor T to quantify the displacement of the supports. For this, winding supports are considered as non-deformable solids. The six components of the torsor T, defined at the center of each support, (1), noted (u, v, w) and (α, β, γ), represent respectively the transla- tion and rotation of the winding support according to the three axes x, y and z. The indices T and B correspond respectively to the top and bottom winding support.h p y p g pp The goal is to determine the six components of each support and for each compaction level. p p Compaction configurations. With friction, deformations will be more or less hindered and parasitic mechanical stresses may appear in the plane of the electrical steel sheets. However, these friction coefficients are difficult to estimate because they depend on many parameters, such as the surface state of each part. The chosen approach is therefore as follows: according to the data in the literature, mechanical FE simulations are carried out in extreme cases for the highest and lowest values of friction coefficients in order to assess the influence of these parameters on the mechanical stress distribution. In addition, a PTFE film is placed between the winding supports and the magnetic circuit to reduce friction. Simulations results show that the mechanical stress distribution within the magnetic circuit varies slighlty as a function of the friction coefficients. Therefore, the chosen friction coefficients are those com- monly found in the literature and are given in Table 3.h The Abaqus model and the coordinate system related to the magnetic circuit are respectively given in Figs. 6 and 7. Mechanical conditions of symmetries were taken into account whether axially or radially. In the following, the axial (in the thickness direction), orthoradial (in the magnetic flux direction, in the plane of the laminations) and radial (perpendicular to the magnetic flux direction, in the plane of the laminations) mechanical stresses will be respectively noted σz , σθ and σr. Note that for the magnetic circuit, the height of one mesh element corresponds to the thickness of one electri- cal steel sheet. Moreover, the simulated homogeneous configuration corresponds to an applied theoretical axial mechanical stress of 4 MPa over the whole magnetic core. The mechanical stress distributions along uz , ur and uθ are respectively given in Figs. 8, 9 and 10. First, results show that the axial stress σz is globally homogeneous and of the order of − 4 MPa. Secondly, the induced stresses in the plane of the laminations ( σθ and σr ) are very low compared to the axial one. If the results of mechanical FE simulations seem to validate the mock-up, we must be able to confirm that we are in the same configuration experimentally as any misalignment or machining inaccuracy can lead to significant error Scientific Reports \| (2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 www.nature.com/scientificreports/ Figure 6. Mock-up modeled and meshed with Abaqus. Figure 6. Mock-up modeled and meshed with Abaqus. Figure 6. Mock-up modeled and meshed with Abaqus. p p Compaction configurations. Figure 6. Mock-up modeled and meshed with Abaqus. Figure 7. Coordinated system used cutting view (a) and top view (b) of the laminated magnetic circuit. Figure 7. Coordinated system used cutting view (a) and top view (b) of the laminated magnetic circuit. in the stress distribution. For this, metrology tests were carried out, as illustrated in Fig. 11. The objective is to quantify the displacement of the two winding supports under compaction effort. As these are the parts that are the closest to the magnetic circuit, this will best reflect the mechanical behavior of the latter. First, eight bearing balls are glued to the outer diameter of the winding supports (four per support). Each ball is then measured on a high-precision coordinate measuring machine to determine its diameter (known) to confirm the measuring quality and its center position in the (x, y, z) coordinate system, Figs. 11 and 12. The measured diameters of the balls are very close (< 5 µm) to the theoretical diameter provided by the manufacturers. From the positions of the bearing balls, the position of each winding support can be determined by applying the least-squares method and, from these positions, the objective is to determine the kinematic torsor T to quantify the displacement of the supports. For this, winding supports are considered as non-deformable solids. The six components of the torsor T, defined at the center of each support, (1), noted (u, v, w) and (α, β, γ), represent respectively the transla- tion and rotation of the winding support according to the three axes x, y and z. The indices T and B correspond respectively to the top and bottom winding support.h in the stress distribution. For this, metrology tests were carried out, as illustrated in Fig. 11. The objective is to quantify the displacement of the two winding supports under compaction effort. As these are the parts that are the closest to the magnetic circuit, this will best reflect the mechanical behavior of the latter. First, eight bearing balls are glued to the outer diameter of the winding supports (four per support). Each ball is then measured on a high-precision coordinate measuring machine to determine its diameter (known) to confirm the measuring quality and its center position in the (x, y, z) coordinate system, Figs. 11 and 12. p p Compaction configurations. To address this issue, the hypothesis of small displacements is first made (very small deformations of the magnetic core). Then, the torsor of each support is calculated, from a matrix formulation of the problem, thanks to the least-squares method which allows to obtain the torsor Δ that represents the difference between TT and TB, (2). The quanti- ties Δα and Δβ are the ones of interest because they correspond to the difference in rotation of the two supports around x and y axes and therefore are an image of the heterogeneity of the compaction stress distribution with Scientific Reports \| (2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 www.nature.com/scientificreports/ Figure 8. Axial mechanical stress distribution - Homogeneous case. Figure 8. Axial mechanical stress distribution - Homogeneous case. Figure 9. Radial mechanical stress distribution - Homogeneous case. Figure 9. Radial mechanical stress distribution - Homogeneous case. regard to the lamination plane. If they are constant with the compression load, the distribution stays perfectly uniform. The idea is to determine the proportion of Δz that can be attributed to rotations Δα and Δβ for each magnetic core point. In practice, the measured Δz variation is 83.35 µm, Fig. 13a. By definition, a rotation of one milliradian induces a height variation of one millimeter over a length of one meter. Considering that the outer diameter of the magnetic circuit and the winding supports is 100 mm, the values of Δα and Δβ, Fig. 13b,c, lead respectively to a height variation of 1.9 µm and 9.5 µm that represents 2.3% and 11.4% of Δz variation at the diameter. In the worst case, this represents 14% of Δz variation. It can therefore be considered that the com- paction pressure is evenly distributed at ±7%. The experimental mock-up is therefore considered as valid if the https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ Figure 10. Orthoradial mechanical stress distribution - Homogeneous case. Figure 10. Orthoradial mechanical stress distribution - Homogeneous case. Figure 11. Principle of the metrology test; (a) centering ring; (b) bearing ball. Figure 11. Principle of the metrology test; (a) centering ring; (b) bearing ball. impact on the magnetic properties is higher than 10% for a 11MPa compaction pressure with a quite proportional evolution for other loads. impact on the magnetic properties is higher than 10% for a 11MPa compaction pressure with a quite proportional evolution for other loads. p p Compaction configurations. T = u v w α β γ (1) T = u v w α β γ (1) https://doi.org/10.1038/s41598-022-23634-7 https://doi.org/10.1038/s41598-022-23634-7 https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 12. Probe measuring the position of a bearing ball. Figure 13. Evolution of Δ torsor components; (a) Δz; (b) Δα; (c) Δβ. Figure 12. Probe measuring the position of a bearing ball. Figure 12. Probe measuring the position of a bearing ball. Figure 12. Probe measuring the position of a bearing ball. Figure 13. Evolution of Δ torsor components; (a) Δz; (b) Δα; (c) Δβ. Figure 13. Evolution of Δ torsor components; (a) Δz; (b) Δα; (c) Δβ. Figure 13. Evolution of Δ torsor components; (a) Δz; (b) Δα; (c) Δβ. (2) = uT −uB vT −vB wT −wB αT −αB βT −βB γT −γB = u v w α β γ (2) Scientific Reports \| (2022) 12:18983 \| Experimental resultsh p Homogeneous case. The magnetic core presented in Table 1 is studied in terms of its magnetic proper- ties evolution as a function of the compaction stress. The experimentally applied compaction stress levels vary from 0MPa to 20MPa. All the magnetic measurements presented in the rest of this paper are performed on the MPG200D equipment from Brockhaus Measurements in accordance with the standard IEC 60404-415 that relies on the flux metric method to determine the magnetic properties. Measurements are carried out over a frequency range from 5 Hz to 300 Hz and for peak magnetic flux density ranging from 0.1 to 1.6 T. Finally, the measured magnetic flux density is corrected by considering the compensation of the air flux due to the non-negligible height of the winding supports. To synthetize the measured B-H loops, in the following we will consider the normal magnetization curve that is obtained from the extrema Bmax-Hmax of the centered hysteresis loops.ll g max max y p As example, and to validate the air flux correction, the results obtained with the mock-up with air flux compensation are compared to those given by a reference circuit, strictly identical but conventionally wound, Fig. 14. The obtained results, Fig. 15, show, on the one hand, that the reference normal magnetization curve and the one with air flux compensation (without compaction stress) are very close and, on the other hand, that the differences between them are negligible with regards to the effect of a homogeneous compaction at a level of 4 MPa. Finally, several measurements were performed to estimate the repeatability on the experimental mock-up: the obtained repeatability is then less than 1%. p y To emphasize the effect of an increasing homogeneous compaction stress level, the normal magnetization curves and iron losses at 50 Hz are given, respectively, in Figs. 16 and 17. Results show a significant effect of the compaction stress on the normal magnetization curve mainly in the saturation knee. Moreover, for the iron Scientific Reports \| (2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 /scientificreports/ Figure 14. Reference magnetic circuit. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 14. Reference magnetic circuit. Figure 14. Reference magnetic circuit. Figure 15. Validation of the air flux compensation method. Figure 15. Validation of the air flux compensation method. Experimental resultsh The conclusions are similar with a significant and gradual degradation effect on the normal magnetization curve and a much more limited effect that saturates for iron losses, in the considered range of applied stress. It is not at all common, according to the literature, to observe such a significant degradation on the normal magnetization curve accompanied with a limited effect on iron losses, especially if one compares with the effects of mechanical stresses applied in the plane of the electrical steel sheets1. These observations will be discussed in the dedicated section of the paper. h p p A further analysis of these experimental results consists in decomposing the losses into their main physical contributions. In16, G. Bertotti proposed a decomposition of iron losses in three terms: hysteresis losses Physt (static), classical losses Pclass (dynamic) and excess losses Pexc (dynamic). The sum of these three terms consti- tutes the total iron losses. For an electrical steel sheet of thickness d and electrical conductivity σe subjected to sinusoidal magnetic flux density of frequency f and peak value Bmax , the classical losses are written as follows: Pclass = π2 6 · σe · d2 · (Bmax · f)2 . The hypothesis is made that these are not impacted (for the considered com- paction stress range) by compaction for two reasons. First, considering the Young’s modulus E of the lamina- tion, the applied mechanical stress σm and the Hooke’s law, the thickness variation d = E σm is less than 0.1%. Moreover, measurements of the electrical conductivity, using the four needles technique, were also performed on a rectangular single sheet which was submitted to different compaction stress levels (using the experimental mock-up presented in Fig. 3). Results show that the electrical conductivity is not affected by the level of the con- sidered compaction stress levels. Therefore, only the hysteresis and excess losses are potentially impacted. First, the classical losses are fitted (using experimental data without stress) according to their analytical expression. Then, the hysteresis losses are determined by an extrapolation of the total energy loss at 0 Hz and the excess losses by a power balance ( Pexc = Ptot −Physt −Pclass ). The evolutions of hysteresis losses and excess losses as a function of the compaction stress are given in Figs. 20 and 21, for 1 T peak magnetic flux density at 50 Hz. Experimental resultsh https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ /scientificreports/ 0 500 1000 1500 2000 2500 3000 H(A/m) 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0MPa 4MPa 8MPa 12MPa 16MPa 20MPa 0 500 1000 1500 H(A/m) 0.8 1 1.2 1.4 B(T) 0MPa 4MPa 8MPa 12MPa 16MPa 20MPa Figure 16. Normal magnetization curves 50 Hz - Homogeneous case. 0 0.5 1 1.5 B(T) 0 1 2 3 4 5 P(W/kg) 0MPa 4MPa 8MPa 12MPa 16MPa 20MPa Figure 17. Iron losses 50 Hz - Homogeneous case. 0 500 1000 1500 2000 2500 3000 H(A/m) 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0MPa 4MPa 8MPa 12MPa 16MPa 20MPa 0 500 1000 1500 H(A/m) 0.8 1 1.2 1.4 B(T) 0MPa 4MPa 8MPa 12MPa 16MPa 20MPa Figure 16. Normal magnetization curves 50 Hz - Homogeneous case. 0 Figure 16. Normal magnetization curves 50 Hz - Homogeneous case. 11 Vol.:(0123 2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 Figure 16. Normal magnetization curves 50 Hz - Homogeneous case. 0 0.5 1 1.5 B(T) 0 1 2 3 4 5 P(W/kg) 0MPa 4MPa 8MPa 12MPa 16MPa 20MPa Figure 17. Iron losses 50 Hz - Homogeneous case. 0 0.5 1 1.5 B(T) 0 1 2 3 4 5 P(W/kg) 0MPa 4MPa 8MPa 12MPa 16MPa 20MPa 1 Figure 17. Iron losses 50 Hz - Homogeneous case. Scientific Reports \| (2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 www.nature.com/scientificreports/ 0.5 1 1.5 B(T) -20 H/H0 (%) 0 20 40 60 80 100 1MPa 4MPa 8MPa 12MPa 16MPa 20MPa Figure 18. Relative difference on magnetic field due to compaction 50 Hz - Homogeneous case. B(T) Figure 18. Relative difference on magnetic field due to compaction 50 Hz - Homogeneous case. losses, a limited increase is visible with the compaction stress: a small increase is directly observed at 4 MPa and beyond this threshold the iron losses remain quite unchanged. To better quantify these effects, they are expressed in terms of relative variation compared to the case without stress denoted as P0 for the losses and H0 for the magnetic field. These relative variations of losses P/P0 (%) and magnetic field H/H0 (%) are determined for each level of applied stress depending on the magnetic flux density level. The relative variations of the magnetic field and iron losses are given, respectively, in Figs. 18 and 19. Experimental resultsh tion stress of 4 MPa corresponds to the magnetic circuit surface that represents the contact area with the winding frame. In the inhomogeneous case, a global compaction stress of 4 MPa implies significant localized compressive stress under the airvent spacers. Indeed, in this case, the contact area with the winding frames is the total airvent spacers area that represent approximatively 5% of the magnetic circuit surface. Therefore, the localized compres- sive stresses are of the order of 80 MPa. The followed experimental protocol is the same as for the homogeneous 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0 Figure 19. Relative difference on iron losses due to compaction 50 Hz - Homogeneous case. 0 5 10 15 20 Compaction Stress Physt (W/kg) (MPa) 0.8 0.81 0.82 0.83 0.84 0.85 Figure 20. Hysteresis loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneous case. B(T) Figure 19. Relative difference on iron losses due to compaction 50 Hz - Homogeneous case. f 0 5 10 15 20 Compaction Stress Physt (W/kg) (MPa) 0.8 0.81 0.82 0.83 0.84 0.85 Figure 20. Hysteresis loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneous case. 0 5 10 15 20 Physt (W/kg) 0.8 0.81 0.82 0.83 0.84 0.85 10 5 tion stress of 4 MPa corresponds to the magnetic circuit surface that represents the contact area with the winding frame. In the inhomogeneous case, a global compaction stress of 4 MPa implies significant localized compressive stress under the airvent spacers. Indeed, in this case, the contact area with the winding frames is the total airvent spacers area that represent approximatively 5% of the magnetic circuit surface. Therefore, the localized compres- sive stresses are of the order of 80 MPa. The followed experimental protocol is the same as for the homogeneous tion stress of 4 MPa corresponds to the magnetic circuit surface that represents the contact area with the winding frame. In the inhomogeneous case, a global compaction stress of 4 MPa implies significant localized compressive stress under the airvent spacers. Indeed, in this case, the contact area with the winding frames is the total airvent spacers area that represent approximatively 5% of the magnetic circuit surface. Therefore, the localized compres- sive stresses are of the order of 80 MPa. Experimental resultsh The global tendency is an increase of the static and dynamic losses with the compaction stress. However, this increase is mostly observed for low levels of compaction stress before exhibiting stabilization at higher levels of compaction. These results are consistent with the measurements performed on the total iron losses presented in Fig. 17 and 19. Before continuing to discuss these results, the next step is to experimentally evaluate the effect of inhomogeneous compaction, i.e. in the presence of airvent spacers, on the magnetic properties. Inhomogeneous case. In the inhomogeneous case, airvent spacers support are placed between the mag- netic core and the winding frames (Fig. 5). The mechanical compressive strength of the Lab1000 material (used for the airvent spacers and winding supports) is of about 110 MPa. In the homogeneous case, a global compac- https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ ientificreports/ tion stress of 4 MPa corresponds to the magnetic circuit surface that represents the contact area with the frame. In the inhomogeneous case, a global compaction stress of 4 MPa implies significant localized com stress under the airvent spacers. Indeed, in this case, the contact area with the winding frames is the tot spacers area that represent approximatively 5% of the magnetic circuit surface. Therefore, the localized 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0 2 4 P/P0 (%) 6 8 1MPa 4MPa 8MPa 12MPa 16MPa 20MPa Figure 19. Relative difference on iron losses due to compaction 50 Hz - Homogeneous case. 0 5 10 15 20 Compaction Stress Physt (W/kg) (MPa) 0.8 0.81 0.82 0.83 0.84 0.85 Figure 20. Hysteresis loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneou 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0 2 4 P/P0 (%) 6 8 1MPa 4MPa 8MPa 12MPa 16MPa 20MPa Figure 19. Relative difference on iron losses due to compaction 50 Hz - Homogeneous case. 6 tion stress of 4 MPa corresponds to the magnetic circuit surface that represents the contact area with the windin 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0 2 Figure 19. Relative difference on iron losses due to compaction 50 Hz - Homogeneous case. 0 5 10 15 20 Compaction Stress Physt (W/kg) (MPa) 0.8 0.81 0.82 0.83 0.84 0.85 Figure 20. Hysteresis loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneous case. Experimental resultsh The followed experimental protocol is the same as for the homogeneous tion stress of 4 MPa corresponds to the magnetic circuit surface that represents the contact area with the winding frame. In the inhomogeneous case, a global compaction stress of 4 MPa implies significant localized compressive stress under the airvent spacers. Indeed, in this case, the contact area with the winding frames is the total airvent spacers area that represent approximatively 5% of the magnetic circuit surface. Therefore, the localized compres- sive stresses are of the order of 80 MPa. The followed experimental protocol is the same as for the homogeneous https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| ientificreports/ case. The goal is to compare the evolution of magnetic properties due to the compaction process in the homo 0 5 10 15 20 Compaction Stress Pexc (W/kg) (MPa) 0.15 0.2 0.25 0.3 Figure 21. Excess loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneous case. Figure 22. Relative evolution of magnetic field - Homogeneous and inhomogeneous cases comparison. entificreports/ 0 5 10 15 20 Compaction Stress Pexc (W/kg) (MPa) 0.15 0.2 0.25 0.3 Figure 21. Excess loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneous case. www.nature.com/scientificreports/ scientificreports/ 0 5 10 15 20 Compaction Stress Pexc (W/kg) (MPa) 0.15 0.2 0.25 0.3 Figure 21. Excess loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneous case. 0 5 10 15 20 Pexc (W/kg) 0.15 0.2 0.25 0.3 Pe Figure 21. Excess loss evolution as a function of compaction stress for 1 T at 50 Hz - Homogeneous case Figure 22. Relative evolution of magnetic field - Homogeneous and inhomogeneous cases comparison. Figure 22. Relative evolution of magnetic field - Homogeneous and inhomogeneous cases comparison. Figure 22. Relative evolution of magnetic field - Homogeneous and inhomogeneous cases comparison. case. The goal is to compare the evolution of magnetic properties due to the compaction process in the homoge- neous and inhomogeneous cases, for global axial compaction pressure ranging from 0 MPa to 4 MPa.hi case. Experimental resultsh The goal is to compare the evolution of magnetic properties due to the compaction process in the homoge- neous and inhomogeneous cases, for global axial compaction pressure ranging from 0 MPa to 4 MPa.hi The relative evolutions of the magnetic field and iron losses, in the homogeneous and inhomogeneous case, are reported in Figs. 22 and 23. These results highlight a key point: for a given overall compaction pressure of 4 MPa, the inhomogeneous case is more degrading than the homogeneous case with a maximum increase in the magnetic field of 50% (against 32% for the homogeneous case) and a maximum increase in iron losses of 7.5% (against 5.5% for the homogeneous case). In addition, as this was already observed in Figs. 18 and 19, the magnetic field is significantly impacted unlike iron losses which exhibit a limited increase. Discussion. The objective is to understand the observed differences in terms of magnetic properties modifi- cations between the homogeneous case and the inhomogeneous case. For this, mechanical FE simulations were performed in Abaqus for the inhomogeneous case while considering the mechanical parameters presented in https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 23. Relative evolution of iron losses - Homogeneous and inhomogeneous cases comparison. Figure 23. Relative evolution of iron losses - Homogeneous and inhomogeneous cases comparison. Figure 23. Relative evolution of iron losses - Homogeneous and inhomogeneous cases comparison. Figure 24. Mechanical stress distribution for the inhomogeneous case (4 MPa). (a) Radial ( ur), (b) Orthoradial ( uθ) and (c) Axial ( uz). Figure 24. Mechanical stress distribution for the inhomogeneous case (4 MPa). (a) Radial ( ur), (b) Orthora ( uθ) and (c) Axial ( uz). Tables 2 and 3. Using the same coordinate system presented in Fig. 7, the mechanical stress distributions al the main directions ur , uθ and uz are respectively given in Fig. 24a–c.hi The first main result, Fig. 24a,b, is that the presence of airvent spacers implies, when applying a compaction effort, the appearance of noticeable mechanical stresses in the plane of the electrical steel sheets. According to the literature1,17, mechanical stresses in the plan have a significant effect on magnetic properties. Experimental resultsh Single Sheet Tester under tensile or compression stress (left) and its schematic drawing (right). point is the distribution of axial stress ( uz) within the magnetic circuit. This stress is localized under the airvent spacers and is significant (about − 80 MPa) compared to the overall applied compaction stress (− 4 MPa). Based on the experimental measurements presented for the homogeneous case, Figs. 18 and 19, this stress level has a highly degrading effect on the global normal magnetization curve of the magnetic core. point is the distribution of axial stress ( uz) within the magnetic circuit. This stress is localized under the airvent spacers and is significant (about − 80 MPa) compared to the overall applied compaction stress (− 4 MPa). Based on the experimental measurements presented for the homogeneous case, Figs. 18 and 19, this stress level has a highly degrading effect on the global normal magnetization curve of the magnetic core. Effect of stress in the plane of the electrical steel sheets. The next step consists in modeling the effect of the compaction process on the magnetic properties, with and without airvent spacers. For this, it is necessary to use magneto-mechanical models that take into account mechanical stresses in the three defined directions ( ur , uθ , uz ). Having already experimentally characterized the evolution of magnetic properties as a function of axial stress ( uz ), Figs. 18 and 19, it is therefore necessary to characterize the effect of mechanical stress applied in the plane of the laminations. The aim is to identify the effect of stress applied in the lamination plane (in the same direction as the magnetic field) and, by relying on an equivalent stress model18, to be able to predict the evolution of the magnetic properties under bi-axial stress in the lamination plane. p g p p p Considering the relatively limited levels of mechanical stress (tensile and compressive) existing in the lami- nation plane, one can consider an approach where the bi-axial stress (along ur and uθ ) can be replaced by an equivalent mechanical stress that would have been applied along the magnetic flux direction. In practice, such model will require only unidirectional magneto-mechanical characterizations, i.e. the stress and the magnetic flux are in the same direction, which is realizable by conventional magneto-mechanical experimental devices. Experimental resultsh These effects are described in Table 4, for the stress ranges given by the Abaqus simulations, in terms of qualitative impact on the magnetic properties.l In our case, the direction parallel to the magnetic flux is uθ and the perpendicular direction (in the plane of laminations) is ur . First, Fig. 24b shows that the mechanical stress along uθ is mainly localized under the airvent spacers along the height of the magnetic core with a compression level of about − 9 MPa, which will significantly degrade the magnetic properties (increase in iron losses, decrease in magnetic permeability). Second, in Fig. 24a, the mechanical stress along ur is divided into tensile stress area (green) and compressive stress area (blue). The average stress levels in these two areas are respectively + 4 MPa and − 2 MPa that could potentially, according to Table 4, degrade the magnetic properties. These induced mechanical stresses in the plane of the laminations allow a first analysis of the observed high level of degradation observed in the inhomogeneous case. A second https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ Table 4. Effect of mechanical stress applied in the plane of electrical steel sheet on its magnetic properties1,17. Up to a few tens of MPa, then a degradation is observed with higher stress levels. For an identical level of stress, stresses along the magnetic flux direction are quantitavely more impactful than those perpendicular1. Parallel to flux direction Perpendicular to flux direction** Tensile stress Improvement* Degradation Compressive stress Degradation Improvement* Parallel to flux direction Perpendicular to flux direction Tensile stress Improvement* Degradation Compressive stress Degradation Improvement* Table 4. Effect of mechanical stress applied in the plane of electrical steel sheet on its magnetic properties1 Up to a few tens of MPa, then a degradation is observed with higher stress levels. For an identical level o stress, stresses along the magnetic flux direction are quantitavely more impactful than those perpendicular1 Table 4. Effect of mechanical stress applied in the plane of electrical steel sheet on its magnetic properties1,17. Up to a few tens of MPa, then a degradation is observed with higher stress levels. **For an identical level of stress, stresses along the magnetic flux direction are quantitavely more impactful than those perpendicular1. Figure 25. Single Sheet Tester under tensile or compression stress (left) and its schematic drawing (right). Figure 25. Experimental resultsh In18 an equivalent scalar stress model was developed and experimentally validated for the case of two-dimensional mechanical stresses applied in the plane of the laminations. This model will be further introduced in “Equivalent scalar mechanical stress and co-simulation” section. In the present work, the experimental device used to char- acterize the unidirectional magneto-mechanical behavior has been developed by Brockhaus Measurements and is illustrated in Fig. 25. It consists in a Single Sheet Tester where the lamination strip under test is maintained at one extremity by a fixed mechanical grip and at the other extremity by a grip actuated by an hydraulic jack. This latter allows to apply tensile or compressive stresses along the magnetic flux direction while performing the magnetic characterizations.h g The magnetic measurements were performed from 5 to 300 Hz excitation frequency and from 0.1 to 1.6 T peak magnetic flux density, over the stress range [− 40 MPa; + 40 MPa]. This latter is chosen because the mechani- cal stresses in the lamination plane do not exceed 20 MPa for the considered compaction process, see Fig. 24a, b. As illustration of the measured magneto-mechanical behavior, the magnetic field and iron losses at 1T are given respectively in Figs. 26 and 27, for 50 Hz excitation frequency. These results are in accordance with the l T bl h d d f h d d l h g The magnetic measurements were performed from 5 to 300 Hz excitation frequency and from 0.1 to 1.6 T peak magnetic flux density, over the stress range [− 40 MPa; + 40 MPa]. This latter is chosen because the mechani- cal stresses in the lamination plane do not exceed 20 MPa for the considered compaction process, see Fig. 24a, b.i The magnetic measurements were performed from 5 to 300 Hz excitation frequency and from 0.1 to 1.6 T peak magnetic flux density, over the stress range [− 40 MPa; + 40 MPa]. This latter is chosen because the mechani- cal stresses in the lamination plane do not exceed 20 MPa for the considered compaction process, see Fig. 24a, b. As illustration of the measured magneto-mechanical behavior, the magnetic field and iron losses at 1T are given respectively in Figs. 26 and 27, for 50 Hz excitation frequency. Experimental resultsh These results are in accordance with the literature, Table 4, with a strong degradation of the magnetic properties under compressive stress and a slight improvement of these properties under tensile stress before observing degradation from 30 MPa.i As illustration of the measured magneto-mechanical behavior, the magnetic field and iron losses at 1T are given respectively in Figs. 26 and 27, for 50 Hz excitation frequency. These results are in accordance with the literature, Table 4, with a strong degradation of the magnetic properties under compressive stress and a slight improvement of these properties under tensile stress before observing degradation from 30 MPa.i From the results obtained for both considered configurations (axial stress only and in-plane stress only), the next step is to propose a modelling method, in the case of inhomogeneous compaction, to account for the effect of the associated three-dimensional stresses on the magnetic properties. Modelling E i l Equivalent scalar mechanical stress and co‑simulation. The equivalent scalar mechanical stress model proposed in18 has been experimentally validated for two-dimensional stresses in the lamination plane. However, it is theoretically independent from the geometric aspects of the considered material as it is based on an energetic approach deduced form the intrinsic magneto-mechanical properties of the material. In this model, a volume of magnetic and mechanical isotropic material is considered to calculate an equivalent magneto-elastic energy. Considering the geometry presented in the Fig. 7, the mechanical tensor σm , the unit vector h, which is https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ entificreports/ -40 -30 -20 -10 0 10 20 30 40 Applied stress (MPa) 50 100 150 200 250 300 350 400 450 500 Magnetic field to reach 1T (A/m) Figure 26. Evolution of the magnetic field necessary to reach 1 T as a function of applied mechanical stress. -40 -30 -20 -10 0 10 20 30 40 Applied stress (MPa) 50 100 150 200 250 300 350 400 450 500 Magnetic field to reach 1T (A/m) Figure 26. Evolution of the magnetic field necessary to reach 1 T as a function of applied mechanical stress. -40 -30 -20 -10 0 10 20 30 40 Applied stress (MPa) 50 100 150 200 250 300 350 400 450 500 Magnetic field to reach 1T (A/m) Figure 26. Evolution of the magnetic field necessary to reach 1 T as a function of applied mechanical stress. -40 -30 -20 -10 0 10 20 30 40 Applied stress (MPa) 1.2 1.4 1.6 1.8 2 2.2 2.4 Iron losses at 50Hz & 1T (W/kg) Figure 27. Evolution of the iron losses at 1 T as a function of applied mechanical stress. 10 0 10 Applied stress (MPa) Figure 26. Evolution of the magnetic field necessary to reach 1 T as a function of applied mechanical stress. re 26. Evolution of the magnetic field necessary to reach 1 T as a function of applied mechanical stress. Modelling E i l along the applied magnetic flux direction, and the analytical expression of the equivalent stress σeq are respec- tively given in (Eq. 3), (Eq. 4) and (Eq. 5). According to the literature, and to our experimental results, for mechanical stresses less than 30 MPa, if σeq increases the magnetic properties are improved, if σeq decreases, the magnetic properties will degrade. The term « −1 2 · tr(σm) » of the (Eq. 5) implies that a compressive stress applied in an orthogonal direction (in our case ur and uz ) to the magnetic flux direction (in our case uθ) will tend to improve the magnetic properties. Along ur , experimental results in the literature are in accordance with this latter consideration1. However, our experimental results for an applied stress along uz , Figs. 18 and 19, show that the applied compressive stress tends to degrade the magnetic properties and especially the normal magnetization curve. This means that this equivalent scalar stress model is not suitable for our case of laminar geometry. To continue the investigation and according to the complexity of implementation of an anisotropic three-dimensional magneto-mechanical model, an approach consisting in the decomposition of the effects (on the one hand, in the plane and, in the other hand, in the thickness direction) is proposed to correctly model the global effect of compaction, in the presence of airvent spacers, on the magnetic properties. (3) σm = σrr σrθ σrz σθr σθθ σθz σzr σzθ σzz (4) h = 0 1 0 (5) σeq = 3 2 · th · σm · h −1 2 · tr(σm) (3) σm = σrr σrθ σrz σθr σθθ σθz σzr σzθ σzz (3) (4) h = 0 1 0 (4) (5) σeq = 3 2 · th · σm · h −1 2 · tr(σm) (5) Proposed decomposition of the effects and co‑simulation protocol. Since the stresss along uz mainly impacts the normal magnetization curve, and according to the triaxial stress distribution, the choice is made to separate the effects of the stresses in the plane from those along the normal direction to the lamina- tion. It is considered that the latter stress component impacts only the normal magnetization curve and that the stresses in the plane impact only the iron losses. For the normal magnetization curve, the Langevin model is used (Eq. Modelling E i l -40 -30 -20 -10 0 10 20 30 40 Applied stress (MPa) 1.2 1.4 1.6 1.8 2 2.2 2.4 Iron losses at 50Hz & 1T (W/kg) Figure 27 Evolution of the iron losses at 1 T as a function of applied mechanical stress -40 -30 -20 -10 0 10 20 30 40 Applied stress (MPa) 1.2 1.4 1.6 1.8 2 2.2 2.4 Iron losses at 50Hz & 1T (W/kg) Figure 27. Evolution of the iron losses at 1 T as a function of applied mechanical stress. Figure 27. Evolution of the iron losses at 1 T as a function of applied mechanical stress. Figure 27. Evolution of the iron losses at 1 T as a function of applied mechanical stress. https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 28. Code_Carmel simulation - Homogeneous case 0 MPa (B = 1.1 T). Figure 28. Code_Carmel simulation - Homogeneous case 0 MPa (B = 1.1 T). along the applied magnetic flux direction, and the analytical expression of the equivalent stress σeq are respec- tively given in (Eq. 3), (Eq. 4) and (Eq. 5). According to the literature, and to our experimental results, for mechanical stresses less than 30 MPa, if σeq increases the magnetic properties are improved, if σeq decreases, the magnetic properties will degrade. The term « −1 2 · tr(σm) » of the (Eq. 5) implies that a compressive stress applied in an orthogonal direction (in our case ur and uz ) to the magnetic flux direction (in our case uθ) will tend to improve the magnetic properties. Along ur , experimental results in the literature are in accordance with this latter consideration1. However, our experimental results for an applied stress along uz , Figs. 18 and 19, show that the applied compressive stress tends to degrade the magnetic properties and especially the normal magnetization curve. This means that this equivalent scalar stress model is not suitable for our case of laminar geometry. To continue the investigation and according to the complexity of implementation of an anisotropic three-dimensional magneto-mechanical model, an approach consisting in the decomposition of the effects (on the one hand, in the plane and, in the other hand, in the thickness direction) is proposed to correctly model the global effect of compaction, in the presence of airvent spacers, on the magnetic properties. Modelling E i l This leads to an increased value of the magnetic flux density on both sides of the degraded area (near the inner and outer radii).l l y g From the magnetic flux density distribution, the iron losses are calculated in post-processing with the mag- neto-mechanical loss model taking into account the equivalent stress in the plane of the laminations. Results in terms of magnetic field and iron losses, considering a magnetic flux density of 1.1 T, are given in Table 5. They show that the proposed approach provides a good approximation of the compaction process effect on the magnetic properties. In particular, this emphasizes that the axial stress ( uz ) is mainly detrimental to the nor- mal magnetization curve and that the stresses in the plane ( ur, uz ) are impactful for the iron losses. The results are extended to three levels of magnetic flux density in Figs. 31 and 32 (white circles) demonstrating the same conclusions. Discussion. One can consider that the proposed approach is a good qualitative approximation of the impact on magnetic properties of the compaction process with very satisfactory results despite the assumptions. These latter are especially based on the decoupling of in-plane stresses from those in the thickness direction of which effects are strongly non-linear. Second, characterizations for the axial stress applied in the homogeneous case were performed up to 20 MPa, so the normal magnetization curve model was identified over this same range of stress values. In the inhomogeneous case, for a global stress of 1 MPa, the maximum σzz stresses that appear locally are of the order of 20 MPa. However, for a global applied stress of 4 MPa, this maximum stress value reaches 80 MPa. For a stress level beyond 20 MPa, the B-H model was therefore applied in a stress region where the magnetization behavior is extrapolated, which represents a strong hypothesis. It should also be noted that if no extrapolation has been made for the iron losses (because the in-plane stress levels were within the identification range of the model), their calculation is obviously influenced by the accuracy of the normal mag- netization curve modeling, which means that an overestimation of the effect on the latter can induce an overes- timation of the effect on iron losses. Modelling E i l Magnetic flux density (a), magnetic field (b) Code_Carmel - Inhomogeneous 4 MPa ( Baverage = 1.1 T). Table 5. Effect of the heterogeneous compaction on the magnetic field and iron losses at 1.1 T. Comparison between the experimental and simulation results—the relative difference (in %) is given compared to the case without compaction. Global compaction stress Magnetic field at 1.1 T Iron losses at 1.1 T Experimental Model ( σzz) Experimental Model ( σrr,σθθ) 1 MPa + 11% + 13% + 1.9% + 2.3% 4 MPa + 50% + 55% + 6.5% + 7.2% Table 5. Effect of the heterogeneous compaction on the magnetic field and iron losses at 1.1 T. Comparison between the experimental and simulation results—the relative difference (in %) is given compared to the case without compaction. Table 5. Effect of the heterogeneous compaction on the magnetic field and iron losses at 1.1 T. Comparison between the experimental and simulation results—the relative difference (in %) is given compared to the case without compaction. leading to a local variation of the magnetic field according to the local magnetic properties that are impacted by the mechanical stress under the airvent spacers, Figs. 29 and 30. Concerning the magnetic flux density distri- bution, and considering that there is no leakage flux, the global magnetic flux is conserved whatever the radial cross section of the ring core. However, as the mechanical stress is mainly located under the airvent spacers, the magnetic flux will flow around this magnetically degraded area. This leads to an increased value of the magnetic flux density on both sides of the degraded area (near the inner and outer radii).l leading to a local variation of the magnetic field according to the local magnetic properties that are impacted by the mechanical stress under the airvent spacers, Figs. 29 and 30. Concerning the magnetic flux density distri- bution, and considering that there is no leakage flux, the global magnetic flux is conserved whatever the radial cross section of the ring core. However, as the mechanical stress is mainly located under the airvent spacers, the magnetic flux will flow around this magnetically degraded area. Modelling E i l 6) and the parameters Ms , a and α are approximated by a polynomial function dependent on the stress σz along uz. (6) B = µ0 · (H + Man) Man = Ms(σz) coth He a(σz) −a(σz) He He = H + α(σz) · Man (6) For the iron losses, the coefficients kh , α , kclass and kexc of the loss model are also approximated by an analytical function dependent on the equivalent stress σeq deduced from the plane stresses σrr and σθθ. Co‑simulation protocol and FE results. Subsequently, the distribution of the three-dimensional (3D) mechanical stresses presented in “Discussion” section and the associated mesh are exported from the Abaqus simulations. This mesh is then imported in Code_Carmel, a 3D Finite Element (FE) electromagnetic calculation software. To perform the simulations, the axial stress σz is associated to each mesh element and the associated B-H curve is accounted for by the magneto-mechanical Langevin model also implemented in Code_Carmel. The homogeneous case is considered to validate the co-simulation protocol, then the configuration with air- vent spacers presented in the paper is considered. All simulations were performed with imposed magnetic flux density in the magnetic circuit. The considered global applied stress levels are 1 MPa and 4 MPa. As illustration, the simulation results obtained for an average magnetic flux density of 1.1 T are given for the homogeneous case without applied stress in Fig. 28 and for the inhomogeneous case with applied stress in Fig. 29 (1 MPa) and Fig. 30 (4 MPa). In these FE simulations, the global magnetic flux is imposed in the cross section of the ring core https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ Figure 29. Magnetic flux density (a), magnetic field (b) Code_Carmel - Inhomogeneous 1 MPa ( Baverage = 1.1 T). Figure 29. Magnetic flux density (a), magnetic field (b) Code_Carmel - Inhomogeneous 1 MPa ( Baverage = 1.1 T). Figure 29. Magnetic flux density (a), magnetic field (b) Code_Carmel - Inhomogeneous 1 MPa ( Baverage = 1.1 T). igure 29. Magnetic flux density (a), magnetic field (b) Code_Carmel - Inhomogeneous 1 MPa ( Baverage = 1 T) Figure 30. Magnetic flux density (a), magnetic field (b) Code_Carmel - Inhomogeneous 4 MPa ( Baverage = 1.1 T). Figure 30. Magnetic flux density (a), magnetic field (b) Code_Carmel - Inhomogeneous 4 MPa ( Baverage = 1.1 T). Figure 30. Modelling E i l Configuration with swapped input stresses in the material models. were dependent only on the axial stress. For this, the models have been identified according to the same proto- col previously presented. The normal magnetization curve model considering σrr and σθθ was implemented in Code_Carmel and the iron losses calculated in post-processing from the magnetic flux density distribution and the associated stress map σzz . Results are given in Table 6, showing that neglected stresses in the first simulation configuration (Table 5) do indeed have a negligible impact on the magnetic properties.f i g g g g Finally, it must be highlighted that since the effect of stress in the plane of the laminations and in the axial direction were characterized separately, it was implicitly assumed that their effects were superposable, which is not necessarily the case due to the complex coupled phenomena involved in the magneto-mechanical behavior of electrical steels. However, the proposed approach remains quite useful for assessing the global impact of the heterogeneous compaction of electrical steel laminations as commonly performed for high power electrical machines. Conclusions and perspectivesh p p The presented work deals with the experimental study of the effect of an industrial compaction process on mag- netic cores performances. This study was associated to the numerical modeling of the impact of this process. In particular, an experimental device with a capacity of 5 tons has been developed in order to apply a compaction effort with and without airvent spacers while being able to both control the mechanical stress distribution but also characterize the magnetic properties. The significant degradation effect induced by the presence of airvent spacers has been demonstrated as well as, for the first time, the particular effect of homogeneous compaction on magnetic properties. Indeed, concerning the latter, a significant deterioration of the normal magnetization curve, mainly in the saturation knee, is observed as well as a very limited effect on the iron losses, which is quite different from what is commonly observed when applying mechanical stresses in the plane of laminations. The physical origins of the behavior observed during compaction along uz are currently difficult to define. The fact of having a significant degradation on the normal magnetization curve and not on the iron losses can be explained by the appearance of a potential energy that hinders the domain walls motion in the saturation-knee region. Moreover, this result opens up new perspectives for the understanding of the magneto-mechanical behavior of electrical steel laminations for axial mechanical stresses.f Nevertheless, an approach to model the effect of compaction was proposed and validated taking into account the three-dimensional mechanical stresses present in the magnetic circuit by separating the axial stresses from the ones in the plane of the laminations. If this approach is valid in the case of compaction, it should be possible to work on a three-dimensional magneto-mechanical model. Finally, the effect of compaction and the developed modeling approach can be studied on more complex geometries such as the stator of electrical machines. Data availabilityh The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Received: 25 July 2022; Accepted: 2 November 2022 Received: 25 July 2022; Accepted: 2 November 2022 Received: 25 July 2022; Accepted: 2 November 2022 Modelling E i l For all these reasons and in order to confirm our results, a simple approach consists in performing the calculation by swapping the input data in the materials models (behavior law and iron losses) between the axial and in-plane stresses. In practice, the normal magnetization curve was dependent only on the equivalent stress determined from the stress distribution in the lamination plane whereas the iron losses https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ 0 500 1000 1500 2000 2500 3000 H(A/m) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Magnetization model ( B(T) zz) 0MPa - Experimental 1MPa - Experimental 4MPa - Experimental B-H model - 1 MPa B-H model - 4 MPa Figure 31. Comparison between experimental and numerical results on normal magnetization curve. 0 500 1000 1500 2000 2500 3000 H(A/m) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Magnetization model ( B(T) zz) 0MPa - Experimental 1MPa - Experimental 4MPa - Experimental B-H model - 1 MPa B-H model - 4 MPa Figure 31. Comparison between experimental and numerical results on normal magnetization curve. Magnetization model ( zz) Magnetization model ( zz) Figure 31. Comparison between experimental and numerical results on normal magnetization curve. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0 1 2 3 4 5 Iron loss model ( P(W/kg) rr, ) 0MPa - Experimental 1MPa - Experimental 4MPa - Experimental Losses model - 1 MPa Losses model - 4 MPa Figure 32. Comparison between experimental and numerical results on iron losses. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 B(T) 0 1 2 3 4 5 Iron loss model ( P(W/kg) rr, ) 0MPa - Experimental 1MPa - Experimental 4MPa - Experimental Losses model - 1 MPa Losses model - 4 MPa 5 Figure 32. Comparison between experimental and numerical results on iron losses. Scientific Reports \| (2022) 12:18983 \| https://doi.org/10.1038/s41598-022-23634-7 www.nature.com/scientificreports/ Table 6. Effect of the heterogeneous compaction on the magnetic field and iron losses at 1.1 T. Configuration with swapped input stresses in the material models. Global compaction stress Magnetic field at 1.1 T Iron losses at 1.1 T Experimental Model ( σrr,σθθ) Experimental Model ( σzz) 1 MPa + 11% + 0.7% + 1.9% + 0% 4 MPa + 50% + 5% + 6.5% + 0.5% Table 6. Effect of the heterogeneous compaction on the magnetic field and iron losses at 1.1 T. References f 2. de Oliveira Júnior, J., Monteiro, R., Paolinelli, S. & Cota, A. Kinetics of magnetic ageing of 2%Si non-oriented grain electrical stee Mater. Res. https://doi.org/10.1590/1980-5373-mr-2017-0575 (2017).f p g 3. Landgraf, F. J. G., Emura, M., Ito, K. & Carvalho, P. S. G. Effect of plastic deformation on the magnetic properties of non-oriented electrical steels. J. Magn. Magn. Mater. 215–216, 94–96 (2000). g 3. Landgraf, F. J. G., Emura, M., Ito, K. & Carvalho, P. S. G. Effect of plastic deformation on the magnetic properties of non-oriented electrical steels. J. Magn. Magn. Mater. 215–216, 94–96 (2000). g g ( ) 4. Nakazaki, O., Kai, Y., Todaka, T. & Enokizono, M. Iron loss properties of a practical rotating machine stator core at each manu- facturing stage. Int. J. Appl. Electromagn. Mech. https://doi.org/10.3233/JAE-2010-1099 (2010).f 5. Al-Timimy, A. et al. Considerations on the effects that core material machining has on an electrical machine’s performance. IEEE Trans. Energy Convers. 33(3), 1154–1163 (2018).f 5. Al-Timimy, A. et al. Considerations on the effects that core material machining has on an electrical machine’s performance. IEEE Trans. Energy Convers. 33(3), 1154–1163 (2018).f gy 6. Seo, U.-J., Kim, D.-J., Chun, Y.-D. & Han, P.-W. Mechanical cutting effect of electrical steel on the performance of induction motors. Energies 13(23), 6314 (2020).f 6. Seo, U.-J., Kim, D.-J., Chun, Y.-D. & Han, P.-W. Mechanical cutting effect of electrical steel on the performance of induction motors. Energies 13(23), 6314 (2020).f g ( ) ( ) 7. Sundaria, R. et al. Effects of stator core welding on an induction machine—Measurements and modeling. J. Magn. Magn. Mater 499, 166280 (2020).f 8. Bernard, L. & Daniel, L. Effect of stress on magnetic hysteresis losses in a switched reluctance motor: Application to stator and rotor shrink fitting. IEEE Trans. Magn. 51(9), 1–13 (2015). 8. Bernard, L. & Daniel, L. Effect of stress on magnetic hysteresis losses in a switched reluctance motor: Application to stator and rotor shrink fitting. IEEE Trans. Magn. 51(9), 1–13 (2015). i g g 9. Alatawneh, N., Rahman, T., Lowther, D. A. & Chromik, R. Design and analysis of a toroidal tester for the measurement of core losses under axial compressive stress. J. Magn. Magn. Mater. 432, 519–526 (2017). i g g 9. Alatawneh, N., Rahman, T., Lowther, D. A. & Chromik, R. Design and analysis of a toroidal tester for the measurement of core losses under axial compressive stress. J. Magn. Magn. Mater. Acknowledgementsh g This work was supported by the regional authority Région Hauts-de-France. Competing interests h g The authors declare no competing interests. References 432, 519–526 (2017). p J g g , ( ) 0. Yamamoto, K. & Yanase, S. Magnetic properties of non-oriented electrical steels under compressive stress normal to their surface Przeglad Elektrotechniczny (Electrical Review) (2011). https://doi.org/10.1038/s41598-022-23634-7 Scientific Reports \| (2022) 12:18983 \| www.nature.com/scientificreports/ 11. Miyagi, D. et al. Effect of compressive stress in thickness direction on iron losses of nonoriented electrical steel sheet. IEEE Trans. Magn. 46, 2040–2043 (2010). g 2. Kawabe, M. et al. Magnetic properties of particular shape specimen of nonoriented electrical steel sheet under compressive stress in thickness direction. IEEE Trans. Magn. 48(11), 3462–3465 (2012).lf g ( ) ( ) 13. Naumoski, H., Riedmüller, B., Minkow, A. & Herr, U. Investigation of the influence of different cutting procedures on the global and local magnetic properties of non-oriented electrical steel. J. Magn. Magn. Mater. 392, 126–133 (2015). 13. Naumoski, H., Riedmüller, B., Minkow, A. & Herr, U. Investigation of the influence of different cutting procedures on and local magnetic properties of non-oriented electrical steel. J. Magn. Magn. Mater. 392, 126–133 (2015). g . ABAQUS, ABAQUS Documentation, Dassault Systemes, Provid y 15. CEC IEC 60404-4. «Part 4: Methods of Measurements of d.c. Magnetic Properties of Iron Steel» (International Electrotechnical Commission, 2008). 16. G. Bertotti, Hysteresis in Magnetism: For Physicists, Material lh d l d k l 17. Mailhé, B. J., Bernard, L., Daniel, L., Sadowski, N. & Batistela, N. Modified-SST for uniaxial characterization of electrical steel sheets under controlled induced voltage and constant stress. Trans. Instrum. Meas. https://doi.org/10.1109/TIM.2020.3006682 (2020).i ( ) 18. Hubert, O. & Daniel, L. Energetical and multiscale approaches for the definition of an equivalent stress for magneto-elastic cou- plings. J. Magn. Magn. Mater. 323(13), 1766–1781 (2011). Author contributions Conceptualization: all authors contributed, Methodology: H.H., A.V.G., A.B. and A.T., Validation: H.H., Inves- tigation: H.H., A.V.G., A.B. and T.C., Writing (Original Draft): H.H., Writing (Review and Editing): A.V.G, A.B., T.C., A.T., W.B., and D.L., Supervision: A.V.G., A.B. and A.T., Project administration: A.B. Conceptualization: all authors contributed, Methodology: H.H., A.V.G., A.B. and A.T., Validation: H.H., Inves- tigation: H.H., A.V.G., A.B. and T.C., Writing (Original Draft): H.H., Writing (Review and Editing): A.V.G, A.B., T.C., A.T., W.B., and D.L., Supervision: A.V.G., A.B. and A.T., Project administration: A.B. Additional information Correspondence and requests for materials should be addressed to A.B. Correspondence and requests for materials should be addressed to A.B. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W2461183888	https://www.cifor.org/publications/pdf_files/Books/BMwangi1601.pdf	English	null	Community harvesting of trees in Peru under payment for ecosystem service schemes: A handbook illustrating results of economic games with participants in selected communities	null	2,016	cc-by	10,215	Community harvesting of trees in Peru under payment for ecosystem service schemes A handbook illustrating results of economic games with participants in selected communities Maria Claudia Lopez Sara Torres Zoila Cruz-Burga Gisella Saldarriaga María de Lourdes Espinoza Frangi Valencia Esther Mwangi Krister Andersson Community harvesting of trees in Peru under payment for ecosystem service schemes A handbook illustrating results of economic games with participants in selected communities Maria Claudia Lopez Sara Torres Zoila Cruz-Burga Gisella Saldarriaga María de Lourdes Espinoza Frangi Valencia Esther Mwangi Krister Andersson Community harvesting of trees in Peru under payment for ecosystem service schemes Maria Claudia Lopez Sara Torres Zoila Cruz-Burga Gisella Saldarriaga María de Lourdes Espinoza Frangi Valencia Esther Mwangi Krister Andersson Work Package Seven of CIFOR’s REDD+ Benefit-Sharing Project © 2016 Center for International Forestry Research © 2016 Center for International Forestry Research Content in this publication is licensed under a Creative Commons Attribution- NonCommercial-NoDerivs 3.0 Unported License http://creativecommons.org/licenses/ by-nc-nd/3.0/ López MC, Torres S, Cruz-Burga Z, Saldarriaga G, Espinoza M-L, Valencia F, Mwangi E, and Andersson K. 2016. Community harvesting of trees in Peru under payment for ecosystem service schemes: A handbook illustrating results of economic games with participants in selected communities. Bogor, Indonesia: CIFOR. ISBN 978-602-387-030-1 DOI: 10.17528/cifor/006075 Photos by Zoila Cruz-Burga. CIFOR Jl. CIFOR, Situ Gede Bogor Barat 16115 Indonesia T +62 (251) 8622-622 F +62 (251) 8622-100 E cifor@cgiar.org cifor.org We would like to thank all funding partners who supported this research through their contributions to the CGIAR Fund. For a full list of the ‘CGIAR Fund’ funding partners please see: http://www.cgiar.org/who-we-are/cgiar-fund/fund-donors-2/ Any views expressed in this book are those of the authors. They do not necessarily represent the views of CIFOR, the editors, the authors’ institutions, the financial sponsors or the reviewers. Content in this publication is licen NonCommercial-NoDerivs 3.0 Unp by-nc-nd/3.0/ López MC, Torres S, Cruz-Burga Z, Saldarriaga G K. 2016. Community harvesting of trees in Peru un illustrating results of economic games with partici ISBN 978-602-387-030-1 DOI: 10.17528/cifor/006075 Photos by Zoila Cruz-Burga. CIFOR Jl. CIFOR, Situ Gede Bogor Barat 16115 Indonesia T +62 (251) 8622-622 F +62 (251) 8622-100 E cifor@cgiar.org cifor.org We would like to thank all funding partners who the CGIAR Fund. For a full list of the ‘CGIAR Fund http://www.cgiar.org/who-we-are/cgiar-fund/fu Any views expressed in this book are those of th of CIFOR, the editors, the authors’ institutions, t Content in this publication is licensed under a Creative Commons Attribution- NonCommercial-NoDerivs 3.0 Unported License http://creativecommons.org/licenses/ by-nc-nd/3.0/ López MC, Torres S, Cruz-Burga Z, Saldarriaga G, Espinoza M-L, Valencia F, Mwangi E, and Andersson K. 2016. Community harvesting of trees in Peru under payment for ecosystem service schemes: A handbook illustrating results of economic games with participants in selected communities. Bogor, Indonesia: CIFOR. CIFOR Jl. CIFOR, Situ Gede Bogor Barat 16115 Indonesia T +62 (251) 8622-622 F +62 (251) 8622-100 E cifor@cgiar.org We would like to thank all funding partners who supported this research through their contributions to the CGIAR Fund. Community harvesting of trees in Peru under payment for ecosystem service schemes For a full list of the ‘CGIAR Fund’ funding partners please see: h // i / h / i f d/f d d / We would like to thank all funding partners who supported this research through their contributions to the CGIAR Fund. For a full list of the ‘CGIAR Fund’ funding partners please see: http://www.cgiar.org/who-we-are/cgiar-fund/fund-donors-2/ Any views expressed in this book are those of the authors. They do not necessarily represent the views of CIFOR, the editors, the authors’ institutions, the financial sponsors or the reviewers. Contents Acknowledgments v 1. Introduction 1 2. The participants 4 3. The activity representing the use of a forest 7 4. Results 14 a) Results Rounds 1 to 8 14 b) Results Rounds 9 to 16 15 c) Results Rounds 17 to 24 19 5. Experience 23 6. Conclusion 24 Acknowledgments 1. Introduction 2. The participants 3. The activity representing the use of a forest 4. Results a) Results Rounds 1 to 8 b) Results Rounds 9 to 16 c) Results Rounds 17 to 24 5. Experience 6. Conclusion Acknowledgments 1. Introduction 2. The participants 3. The activity representing the use of a forest 4. Results a) Results Rounds 1 to 8 b) Results Rounds 9 to 16 c) Results Rounds 17 to 24 5. Experience 6. Conclusion 5. Experience 6. Conclusion Figures and tables Figures and tables Figures 1. Map of Peru. 2 2. Participants responses to the question “why do you go to the forest?” The percentages for this graph were rounded. 5 3. Participants responses to “I trust most people in this community.” 6 4. Forest in the activity, the forest was represented by blocks of wood. Each block is equivalent to one tree. 7 5. Individual and group earnings in tokens per tree. 8 6. Decision card. 8 7. Average individual extraction in Peru in rounds 1 to 8. 15 8. Average Individual Extraction Rounds 9-16 in Peru. 17 9. Comparison of trees cut in rounds 1-8 versus rounds 17-24 after variations. 20 10. Average Individual Extraction in Peru from rounds 1 to 24. 21 11. Comparison of average extraction after treatments in the three countries. 22 Tables 1. Participant Characteristics by community. 4 2. Calculation Sheet. 9 3. The social dilemma introduced into the activity. 10 4. Description of variations of the activities from rounds 9-16, showing in parenthesis the label given to each variation. 11 5. Summary of the 24 rounds of the activity. 12 6. Summary of participant distribution per village and per variation. 12 7. Trees cut in the first 8 rounds of the activity. 14 8. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Peru. 17 9. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Tanzania and Indonesia. 18 10. Average number of trees cut during rounds 17 to 24 of the activity in Peru. 19 11. Average number of trees cut during rounds 17 to 24 of the activity in Tanzania and Indonesia. 21 Introduction Communities all over the world use forests as an important part of their livelihood and many have organized ways of using their forests and regulating what is harvested when, from where, and by whom. They often also have ways of distributing benefits among their members and with non-members. In many cases, these communities are also linked with external actors such as forestry agencies and conservation NGOs who also affect how resources are used and managed through the policies/laws they implement and the incentives they offer. This study seeks to understand how people make decisions regarding the use of the forest and how different policies may affect that use. In particular, we look at the effects of providing a payment to forest users to conserve the forest, often referred to as a “payment for environmental service.” A payment for environmental service as defined by Wunder (2005) is a transaction where an ecosystem service is being ‘bought’ by a buyer (or multiple buyers) from a service provider or a group of service providers, if the service provider can secure the ecosystem service. In general, the buyers of the ecosystem service are organizations external to the communities of forest users. These organizations find different ways to monitor the conservation of the forest, and make payments to the communities, these payments can be in cash or in kind, at the individual level or at the group level. In this handbook, we investigate the effects of payment for forest conservation on the forest and on users’ by implementing a set of activities simulating the use of a forest by a forest community. We look at the effectiveness of four different ways of providing the payment in four different variations of the activity, with an aditional variation in which no payment is administered. Acknowledgments This handbook has been produced with financial assistance from the European Commission and the United Kingdom Department for International Development. We are grateful to el Centro de Conservacion Investigacion y Manejo de Areas Naturales - CIMA Cordillera Azul, Conservación Internacional - CI, Asociación para la Investigación y Desarrollo Integral - AIDER, Bosques Amazónicos - BAM, Federación de Productores de Castaña de Madre de Dios - FEPROCAMD. We would also like to thank Lucero, Sol de Oro, Oriente Nuevo, Miraflores, Alto Ponaza, Planchón, Alegría, Monterrey, Sabaluyoc, Callería and Patria Nueva for their participation in this project. Additionally, Alisson Soche Forero and Paula Andrea Zuluaga provided valuable expertise for the data management process. Again, to everyone, thank you. . Any views expressed in this publication are those of the authors and do not represent the views of Center for International Forestry Research - CIFOR, the editors, the authors’ institutions, the financial sponsors. Introduction 1 Introduction Community harvesting of trees in Peru under payment for ecosystem service schemes 3 Community harvesting of trees in Peru under payment for ecosystem service schemes This project was carried out in rural communities in Peru that live near forests and use them in different ways. The communities were Alegría, Alto Ponaza, Callería, Lucero, Miraflores, Monterrey, Oriente Nuevo, Patria Nueva, Planchón, Sabaluyoc and Sol de Oro. Some, but not all of these communities participated in REDD+ projects implemented by NGOs from 2009-2014. See Figure 1 for the location of these villages. For the study we selected communities that were part of a payment for environmental services (PES) scheme (Alegría, Alto Ponaza, Callería, Oriente Nuevo, Planchón, Sol de Oro), and others that were not part of a PES program (Miraflores, Monterrey, Patria Nueva, Sabaluyoc and Lucero). The communities were also participating in the Center for International Forestry Research (CIFOR) broader research program on the efficiency, effectiveness and sustainability of REDD+. REDD+ is a program aimed at providing communities with incentives to use forests in a sustainable way in order to reduce the effects of climate change. When forests are degraded or destroyed carbon dioxide is emitted and this is thought to lead to increasing temperatures, which may eventually have negative effects on human welfare. In certain communities (Alto Ponaza, Callería, Lucero, Miraflores) we held five working sessions (one for each one of the variations mentioned earlier, and explained below). In other communities (Alegría, Monterrey, Oriente Nuevo, Patria Nueva, Planchón, Sabaluyoc, Sol de Oro) we held fewer sessions due to the size of the community (see Table 1). For all sessions we invited eight different community members. During each session, we first conducted a brief survey of each participant, followed by an activity representing the use of a forest, and after the activity we administered another survey. The purpose of the surveys1 was to gather some socio-economic information about the participants in the activity, as well as some information about the ways they interact with the forest and their perceptions after the activity. This report contains results from both the surveys and the forest activity in the 11 communities in Peru. This study was conducted not only in Peru but also in Tanzania and Indonesia. In this report, we also include some data from Tanzania and Indonesia to give some perspective to these results. 1 We applied the initial survey before the activity to exclude any influence it may have had on the opinions expressed. The post activity survey included some questions about the activity itself and others that could have influenced had they been asked before the activity. Introduction The five variations of the activity we created were: (1) allowing the community members to communicate among themselves without any payment or external organization involved; (2) having the external organization pay an equal amount directly to each member of the group, where community members were not allowed to communicate among themselves; (3) having the external organization pay an equal amount directly to each member of the group, where community members were allowed to communicate among themselves; and (4) having the organization make the payment to one person selected by the community (leader) after a group discussion, who then decides how to allocate the payment among community members; (5) having the same sort of payment described in (4), but with women making up a majority of the group. Maria Claudia Lopez, et. al. 2 Figure 1. Map of Peru. Figure 1. Map of Peru. Community harvesting of trees in Peru under payment for ecosystem service schemes Due to the fact that we had a maximum of five working sessions in each village (but in some communities only two, testing only one type of variation described in each of them) in this brief handbook we do not present results with differences between communities in Peru. This will allow us to keep the results anonymous by not mentioning what happened in each group, focusing instead on the results aggregated at the country level. The participants 2 320 people participated in the Peru study, 148 women and 172 men. The average age of the participants was 38 years with an average of 7 years of education. When asked about their finantial situation 54.38% considered themselves as average, while 40.94% considered themselves to be poor compared to other people in their community. Table 1, below presents some general information on the participants in each community where we conducted the study. Table 1. Participant Characteristics by community. Community Number of participants Average Age Number of women Number of men Average of years of education Alegría 16 48.50 9 7 7.57 Alto Ponaza 40 34.80 24 16 4.56 Callería 40 37.98 14 26 9.29 Lucero 40 39.31 21 19 6.31 Miraflores 40 33.70 15 25 5.38 Monterrey 24 43.83 9 15 8.50 Oriente Nuevo 16 33.69 3 13 7.53 Patria Nueva 40 31.83 17 23 8.90 Planchón 24 50.42 11 13 7.00 Sabaluyoc 16 41.19 13 3 6.94 Sol de Oro 24 35.25 12 12 6.25 Total 320 38.08 148 172 7.10 Table 1. Participant Characteristics by community. As mentioned earlier, participants in this study were forest users or people living close to a forest. These participants claimed to visit the forest on average 7 days a month. When they do visit it 30.94% claim to spend half a day or less, 20.31% claim to spend an hour or less, 20.63% reported going for more than a day and 16.88% go for between a half day and a whole day. 10.63% said they never visit the forest. When we asked participants about the different reasons to visit the forest, the majority mentioned recreation, followed by monitoring and hunting and/or fishing as presented in Figure 2. Survey responses do not specify what type of monitoring they do, or how that monitoring works. In addition, we do not have information about the type of recreation activities done in the forest. More fieldwork would be needed to address such questions. Community harvesting of trees in Peru under payment for ecosystem service schemes 5 Recreation 28% Why do you go to the forest? Monitoring 20% Hunting & Fishing 14% Farming 12% Timber harvesting 11% Working 9% Non forest use 5% Forest Management 1% Figure 2. Participants responses to the question “why do you go to the forest?” The percentages for this graph were rounded. Recreation 28% Why do you go to the forest? The participants 2 Monitoring 20% Hunting & Fishing 14% Farming 12% Timber harvesting 11% Working 9% Non forest use 5% Forest Management 1% Forest Management 1% Non forest use 5% Recreation 28% Figure 2. Participants responses to the question “why do you go to the forest?” The percentages for this graph were rounded. We asked participants about the importance of protecting their natural resources and nature. A total of 97.19% of them strongly agreed or agreed (60.63% and, 36.56%, respectively) with the statement: “It is important for me to care for nature and protect the natural resources in my community.” Among the participants, 57.5% (47.5% agree, 10.5% strongly agree) think that most people in the community care about the health and conditions of the ecosystem. Aditionally 59.1% (50% agree, 9.1% strongly agree) of the participants believe that their community has done a good job protecting its natural resources. The other answers to this particular question were: 20.3% somewhat agree with the statement and 9.1% somewhat disagree with it, while 11.6% disagree or strongly disagree. It is clear from this results that there is no consensus among the participants on the importance of caring for the natural resources in their communities. Unfortunately, the survey questions do not shed further light on these, although 67.2% (48.75% agree, 18.44% strongly agree) of the participants believe that the condition of the forest in their community is better than in other surronding communities. During this study we also asked about trust and cooperation within the community and found that 93.44 % (41.56% agree, 51.88% strongly agree) of the participants believe that cooperation and working together is extremely important. In this study, 50.63% of the participants trust most people in their community (42.5% agree, 8.13% strongly agree), and 32.81% (28.44% agree, 4.38% strongly agree) believe that most people in the community trust one another, (see Figure 3). Maria Claudia Lopez, et. al. 6 Figure 3. Participants responses to “I trust most people in this community.” I trust most people in this village Agree 43% Somewhat agree 19% Somewhat disagree 15% Disagree 14% Strongly agree 8% Strongly disagree 1% Disagree 14% Agree 43% Figure 3. Participants responses to “I trust most people in this community.” As part of our survey we asked some questions about payment for ecosystem services (PES). Six of the 11 communities in our study have a PES scheme implemented. The participants 2 Among these communities 1.88% of the participants knew that people in their community had received payments for conservation activities. Among the participants in the activity 0.63% had received at least one of these payments. In the communities where PES has not been implemented, 6 people in total had some awarness of the existence of payment for environmental services. We did not collect information about the type of payment or the frequency of these payments. We did not ask either about the type of ecosystem service the organization is paying for, or how the payments are being shared among community members. To answer all these questions will require further fieldwork. We also did not collect information about payments in kind, including technical assistance or other types of non-monetary payment that may explain why such a small proportion of the participants had received payments, despite living in communities where a PES is implemented. This initial result is interesting because it may indicate that most of the participants had their first experience with the motion of the monetary payment during the forest activity described below. i including technical assistance or other types of non-monetary payment that may explain why such a small proportion of the participants had received payments, despite living in communities where a PES is implemented. This initial result is interesting because it may indicate that most of the participants had their first experience with the motion of the monetary payment during the forest activity described below. Community harvesting of trees in Peru under payment for ecosystem service schemes Community harvesting of trees in Peru under payment for ecosystem service schemes After each round of the activity, the monitor collected the decision cards from the 8 participants, and announced in public how many trees were cut from the forest by the group, and how many trees were left standing in the group forest. Additionally, the monitor announced the earnings all participants received from the trees that were not harvested but left standing in the group forest. Then each participant individually and privately, calculated his or her earnings in tokens for that round based on the tokens earned from cutting trees, plus the tokens earned for the trees left standing in the group forest. All this information was recorded on a calculation sheet (Table 2) that participants had with them at all times. This process was repeated for 24 rounds. Table 2. Calculation Sheet. Round number Trees you cut from the forest Earnings in tokens for the trees you cut (for each tree you will receive 5 tokens) (A5) Number of trees cut by the entire group (announced by the monitor) Earnings in tokens for the trees left in the forest (announced by the monitor) (80-C) Tokens earned in this round (=B+D) 1 2 Table 2. Calculation Sheet. The activity representing the use of a forest 3 The team carried out this decision-making activity (commonly called economic games) in Peru, Indonesia and Tanzania. This activity portrays a scenario where a group of forest users must decide how to use a common forest. The group consists of eight people sharing a forest with 80 trees (see Figure 4). Each participant was invited to participate in the activity only once, and each activity consisted of 24 rounds. Each round represents a day spent harvesting wood. During each round, each participant had to choose how many trees from 0 to 10 he/she wanted to cut from a shared forest containing 80 trees. This decision was made in private and without communicating with other participants in the group. Figure 4. Forest in the activity, the forest was represented by blocks of wood. Each block is equivalent to one tree. Figure 4. Forest in the activity, the forest was represented by blocks of wood. Each block is equivalent to one tree. The monitor explained that a participant will get five tokens for each tree he/she cuts from the forest, whereas for each tree left standing in the forest each one of the participants in the group would get one token (see Figure 5). At the end of the 24 rounds, the monitor calculated the total number of tokens collected by each participant, paying each participant 0.027 cents of Nuevos Soles for each token earned during the activity. Maria Claudia Lopez, et. al. 8 Individual earnings per tree cut Group earnings per tree left standing Figure 5. Individual and group earnings in tokens per tree. 1 1 1 1 1 1 1 1 1 1 1 1 1 gs per tree cut Group earnings per tree left standing Individual earnings per tree cut Figure 5. Individual and group earnings in tokens per tree. Participants made their decisions in private, and reported their decisions to the monitor by filling out a decision card (Figure 6). Participants made their decisions in private, and reported their decisions to the monitor by filling out a decision card (Figure 6). Figure 6. Decision card. Decision card Participant number: Round number: Please mark with an X the trees you want to cut from 0 to 10. 0 - 10 1 2 3 4 5 6 7 8 9 10 Figure 6. Decision card. 9 9 Explanation of the activity This basic activity simulates a cooperation dilemma where at the individual level it is often viewed by participants to be in their best interest to cut as many trees as possible, but at the group level it is better to leave the trees standing in the common forest. In other words, there is a tension between what an individual sees as best for him or herself and what is best for the group overall. However, if everybody in the group follows the individual strategy, then in the end the group would not earn as many tokens as they could if they were not cutting trees and they will end up destroying the group forest. As shown in Table 3, if nobody cuts trees from the forest, then the earnings for each individual from the trees left in the forest are 80 tokens, and the total earnings for the group are 808=640. This is the way to earn more tokens as a group and we call this a social optimum. If every participant cuts one tree, then the earnings for each individual are 5 tokens from the tree cut + 72 tokens from the trees left in forest, thus 77 tokens per participant. The total earnings for the group in this case are 616 (778), which is substantially less than what the group could have earned if everyone had abstained from harvesting trees. Thus, if one individual cuts 10 trees and the rest of the participants do not cut any trees, that individual’s earnings are 50 from the trees cut + 70 from the trees Maria Claudia Lopez, et. al. 10 10 left standing in the forest= 120 tokens. In this particular case the total earnings for the group are 610, (120+707). This example shows how for the individual it may be better to cut all trees, but by doing so the individual is affecting the total earnings of the group. In this activity, all participants face the temptation to harvest some trees while other players refrain from harvesting all together. The example illustrated yields the highest possible individual earnings (for the individual cutting all 10 trees), but that comes with a cost in earnings to the rest of the group (because the other players in the group are not cutting any trees). Explanation of the activity In this particular case, we say that this individual is free-riding on the effort of others in the group to conserve the forest. If every participant decides to do the same, and to cut all trees they are allowed to cut, then each individual’s earnings are 50 tokens, and the total earnings made by the group are 400 tokens (508). In this case none of the participants cooperate, and this will be damaging for the forest and also for the individual and group earnings. This situation is known as a Nash equilibrium, a situation in which no participant can benefit from doing something different while the other participants keep doing what they are doing. In this case in which the whole group is cutting 80 trees, the only way a participant can benefit, in terms of earnings, from doing something different (cutting fewer trees) is if others cut fewer tress as well. If everyone follows this individualistic strategy the forest will disappear rapidly and participants will earn very little income from all that harvesting. The cooperation dilemma manifests itself in this activity then by representing the advantages for the group and for the forest of not cutting trees (the social optimum), but showing how difficult it is to get there if there is a lack of commitment to the group and trust among its members. If participants start free- riding, then it will be more likely to end up in a situation like the one described in the Nash equilibrium. Table 3. The social dilemma introduced into the activity. Trees cut by individual Trees cut by 7 other players Earnings for individual by the trees cut Earnings for the individual from trees left standing in the forest Total Earnings for individual from trees cut+ trees left standing in the forest Total earnings for the group 0 0 0 80 80 808=640 1 7 5 72 77 778=616 10 0 50 70 120 120+707=610 10 70 50 0 50 50*8=400 Table 3. The social dilemma introduced into the activity. For the first eight rounds of the activity participants make their decisions as described above. Each participant decided how many trees to cut from the shared Community harvesting of trees in Peru under payment for ecosystem service schemes 11 11 forest, without any way to communicate with other group members or any type of external organization looking or controlling the group decision-making. Explanation of the activity From rounds 9 to 16, we included five different possible variations to the basic activity. Each group participated in only one of these five possible variations (variations are explained in Table 4). As mentioned earlier, in four communities in Peru we conducted the five variations of the activity once, and in one Table 4. Description of variations of the activities from rounds 9-16, showing in parenthesis the label given to each variation. Variation for rounds 9 to 16 Description Communication (COMM) Participants were allowed to talk among themselves before making decisions each round. The decisions remained private. Bonus (BONUS) In this variation an organization offers a bonus to the group to abstain from cutting trees from the forest. This bonus is offered every round. But if the organization finds out that the group is cutting trees, it will not pay the bonus. The organization cannot perfectly monitor whether or not the group is cutting trees, but each tree cut increases the probability that the organization will find out that trees are being cut. If the group cuts more than 40 trees the organization will always know they cut trees. The bonus is 160 tokens, and it is distributed equally among all participants. No communication was allowed. Bonus and communication (BONUS+COMM) In this variation, participants participated in the “Bonus” variation described above, but additionally they had the opportunity to communicate for 5 minutes with each other before making any decision. Bonus and communication with leader (BONUS+LEADER) In this variation, an organization offers a bonus to the group not to cut trees from the forest. This bonus is offered every round. If the organization finds out that the group is cutting trees, it will not pay the bonus. The organization cannot perfectly monitor whether or not the group is cutting trees, but each tree cut increases the probability the organization will find out that trees are being cut. If the group cuts more than 40 trees the organization will always know they cut trees. Participants could communicate and had to elect a participant that would be in charge of distributing the bonus of 160 tokens, if a bonus is given. The leader was free to distribute the bonus in any possible way. Group members are not informed of how the bonus is distributed. Bonus and communication with leader and a majority of women in the group. Explanation of the activity (BONUS+LEADER+MAJORITY) This variation is the same as “Bonus and communication with leader,” but the majority of the participants were women. ble 4. Description of variations of the activities from rounds 9-16, showing in arenthesis the label given to each variation. Bonus (BONUS) Bonus and communication (BONUS+COMM) Bonus and communication with leader (BONUS+LEADER) Bonus and communication with leader (BONUS+LEADER) Bonus and communication with leader and a majority of women in the group. (BONUS+LEADER+MAJORITY) Bonus and communication with leader and a majority of women in the group. (BONUS+LEADER+MAJORITY) Bonus and communication with leader and a majority of women in the group. (BONUS+LEADER+MAJORITY) This variation is the same as “Bonus and communication with leader,” but the majority of the participants were women. Maria Claudia Lopez, et. al. 12 12 community we did only four activities. In three communities we did only three activities and in three communities we did only two activities. community we did only four activities. In three communities we did only three activities and in three communities we did only two activities. In rounds 17- 24, regardless of the variation of the activity they were participating in, all groups went back to participating in the activity under the same conditions as in rounds 1 to 8; thus there was no communication or any type of bonus or organization monitoring their actions. However at this point they have experienced the effects of making decisions under the variations to the game in rounds 9-16. In rounds 17- 24, regardless of the variation of the activity they were participating in, all groups went back to participating in the activity under the same conditions as in rounds 1 to 8; thus there was no communication or any type of bonus or organization monitoring their actions. However at this point they have experienced the effects of making decisions under the variations to the game in rounds 9-16. Table 5 summarizes the different components of the activity across the 24 rounds that were played in each community. In rounds 1-8 we did not have any type of variation in the activity (pre-variations rounds); rounds 9-16 introduced the 5 variations explained above (one per group); finally in rounds 17-24 the groups were not subject to any variations, but they experienced the withdrawal of the variation they previously experienced in rounds 9-16, (post-variations rounds). Table 5. Summary of the 24 rounds of the activity. Results 4 Results 4 Explanation of the activity Rounds 1-8 Pre-variations Rounds 9-16 Variations Rounds 17-24 Post-variations No communication. No Bonus COMM No communication. No Bonus BONUS COMM+BONUS BONUS+LEADER BONUS+LEADER+MAJORITY Table 5. Summary of the 24 rounds of the activity. Table 6 lists the villages where each variation was conducted, and the breakdown of the number of participants per variation, including the number of women and men in each group. Table 6. Summary of participant distribution per village and per variation. Community Variation Number of participants Number of women Number of men Alegría BONUS 8 4 4 COMM+BONUS 8 4 4 Alto Ponaza BONUS 8 6 2 COMM+BONUS 8 2 6 COMM 8 6 2 BONUS+LEADER 8 4 4 BONUS+LEADER+MAJORITY 8 4 4 Table 6. Summary of participant distribution per village and per variation. continued on next page Community harvesting of trees in Peru under payment for ecosystem service schemes Community harvesting of trees in Peru under payment for ecosystem service schemes 13 13 Community Variation Number of participants Number of women Number of men Callería BONUS 8 0 8 COMM+BONUS 8 7 1 COMM 8 8 0 BONUS+LEADER 8 1 7 BONUS+LEADER+MAJORITY 8 6 2 Lucero BONUS 8 4 4 COMM+BONUS 8 4 4 COMMUNICATION 8 5 3 BONUS+LEADER 8 3 5 BONUS+LEADER+MAJORITY 8 5 3 Miraflores BONUS 8 1 7 COMM+BONUS 8 2 6 COMM 8 2 6 BONO+LEADER 8 2 6 BONUS+LEADER+MAJORITY 8 8 0 Monterrey BONUS 8 2 6 COMM+BONUS 8 0 8 BONUS+LEADER+MAJORITY 8 6 2 Oriente Nuevo COMM+BONUS 8 3 5 BONUS+LEADER 8 0 8 Patria Nueva BONUS 8 4 4 COMM+BONUS 16 4 12 COMM 8 2 6 BONUS+LEADER+MAJORITY 8 7 1 Planchón COMM 8 1 7 BONUS+LEADER 8 4 4 BONUS+LEADER+MAJORITY 8 6 2 Sabaluyoc COMM 8 5 3 BONUS+LEADER 8 8 0 Sol de Oro BONUS 8 3 5 COMM 8 4 4 BONUS+LEADER+MAJORITY 8 5 3 Total 320 152 168 Table 6. Continued a) Results Rounds 1 to 8 Table 7. Trees cut in the first 8 rounds of the activity. Round Peru Indonesia Tanzania 1 3.58 3.31 2.09 2 3.66 3.52 2.27 3 3.78 3.92 2.14 4 3.99 4.03 2.33 5 4.12 4.02 2.08 6 4.15 3.70 2.32 7 4.19 3.87 2.26 8 4.16 4.06 2.29 Table 7. Trees cut in the first 8 rounds of the activity. Table 7 and Figure 7 (below) present the average extraction decisions during the first eight rounds, in Peru and compare them to the extraction decisions of Indonesia and Tanzania. We used the data of Indonesia and Tanzania only as a reference for decisions made by the participants in Peru. We see that the average extraction during these rounds in Peru was 3.95, showing that participants were not playing so as to optimize the group earnings, which results from cutting 0 trees; but neither where they playing as in the Nash Equilibrium individualistic scenario, which would have been to cut 10 trees each. It is important to remember that participants during these 8 rounds did not have any way to coordinate their decisions because they were not able to communicate. The average extraction levels in Tanzania were 2.22 and in Indonesia were 3.80. So the extraction levels in Peru were higher than in the other two countries, but similar to the ones from Indonesia. Figure 7 offers a graphical representation showing that in Peru the extraction levels increased gradually over time. Community harvesting of trees in Peru under payment for ecosystem service schemes 15 15 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 1 2 3 4 5 6 7 8 Tanzania Peru Indonesia Average Individual Extraction Figure 7. Average individual extraction in Peru in rounds 1 to 8. Average Individual Extraction Figure 7. Average individual extraction in Peru in rounds 1 to 8. Three variations provided participants the possibility to communicate and receive the bonus: COMM+BONUS, BONUS+LEADER, BONUS+LEADER+MAJORITY. COMM+BONUS is a variation that combines the characteristics of COMM and BONUS, so one might expect this variation to be more efficient than these other two variations alone. However this was not the case in Peru. The average extraction level during this variation was 2.83, so the extraction levels were slightly higher than for the BONUS alone, and slightly smaller than for the COMM alone. This result is somewhat surprising because it appears to suggest that the fact participants were able to discuss and to get the bonus was not enough to lead them to coordinate their actions to extract fewer trees from the forest in order to receive the bonus, at least not when you compared to the results when they could only communicate, or those when they could only get the bonus. Additionally, with COMM+BONUS if participants got the bonus the organization was distributing it in equal shares among all participants, meaning that participants did not have to worry about how to share the bonus. Apparently the fact that the organization was offering the bonus and was distributing it to all community members was not as effective in increasing cooperation compared to just COMM or BONUS. The variation BONUS+LEADER was the variation leading to the least extraction (among all variations used in rounds 9 to 16), thus increasing the cooperation among participants. In this case the average extraction of trees was 1.81. This variation resulted in a reduction of almost 2 trees with respect to rounds 1-8, and of 1 tree with respect to COMM+BONUS. Cooperation increased in Peru when participants were able to select a leader to distribute the bonus. This result is very interesting if we compare it with COMM+BONUS since in this variation the leaders controlled the bonus, and its distribution was kept private (only the leader knew how she/he was allocating the bonus), therefore the leaders could have distributed it in ways that might not have seemed fair to all the participants. This result is even more interesting when we overlay the levels of trust in others inside the community reported in the survey. The result in this variation suggest that participants trusted the leaders they were choosing and, even more importantly, they trusted them more than the external organization to allocate the bonus. b) Results Rounds 9 to 16 Table 8 and Figure 8 (below) present an overview of the average extraction levels in rounds 9 to 16 with all the variations tested. The variation COMM resulted in an average extraction level of 2.89 during these 8 rounds, reducing the extraction level with respect to the first eight rounds by 1.06 trees. It is important to note that during the COMM variation participants were able to communicate but they did not receive any bonus. During the communication process participants were free to discuss any topic, but decisions had to be made in private. So even if they made agreements during the communication process, those agreements were not monitored or enforced. They could only “see” the results of the communication when the monitor announced the total number of trees extracted by the group. In the BONUS variation, the average extraction level in these 8 rounds was 2.72, which is lower than what was extracted in the first 8 rounds by 1.23 trees. The extraction levels with this variation are not very different from the ones with COMM. Thus, the efficiency of COMM and BONUS in promoting cooperation seems to be very similar in terms of extraction levels. However the BONUS provided providing this extra payment to maintain the forest, which in this particular case produced the same results as allowing participants to communicate while making decisions in private. An interesting difference in these two variations is found in round 16, where in COMM the results were very similar to the rest of the rounds (average of 2.86), while in the BONUS variation, the extraction levels increased in the last rounds. In fact in the last round the average extraction was 3.34. Are these results showing that a bonus may not be required to get people to conserve their resources? How often can communities in Peru discuss and agree on ways to manage their resources? More fieldwork is needed to answer these questions. Maria Claudia Lopez, et. al. 16 Three variations provided participants the possibility to communicate and receive the bonus: COMM+BONUS, BONUS+LEADER, BONUS+LEADER+MAJORITY. Three variations provided participants the possibility to communicate and receive the bonus: COMM+BONUS, BONUS+LEADER, BONUS+LEADER+MAJORITY. 0.00 0.50 1.50 1.00 2.50 2.00 3.00 3.50 4.00 9 10 11 12 13 14 15 16 Average individual extraction peru (rounds 9-16) BONUS COMM + BONUS COMM BONUS + LEADER BONUS + LEADER + MAJORITY When we look more closely at the selection of the leader in variations BONUS+LEADER and BONUS+LEADER+MAJORITY in one group the same person was elected for six rounds (in variation BONUS+LEADER+MAJORITY), Table 8. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Peru. Round BONUS COMM+ BONUS COMM BONUS+ LEADER BONUS+ LEADER+ MAJORITY 9 2.30 2.90 2.63 1.54 2.28 10 2.67 2.67 2.64 1.70 2.14 11 2.48 2.92 3.09 1.75 3.22 12 2.66 2.93 2.70 1.93 2.30 13 2.45 2.76 2.84 1.98 3.17 14 3.17 2.86 2.94 1.89 1.69 15 2.72 3.14 3.42 1.61 2.39 16 3.34 2.43 2.86 2.11 2.23 Figure 8. Average Individual Extraction Rounds 9-16 in Peru. 0.00 0.50 1.50 1.00 2.50 2.00 3.00 3.50 4.00 9 10 11 12 13 14 15 16 Average individual extraction peru (rounds 9-16) BONUS COMM + BONUS COMM BONUS + LEADER BONUS + LEADER + MAJORITY When we look more closely at the selection of the leader in variations BONUS+LEADER and BONUS+LEADER+MAJORITY in one group the same person was elected for six rounds (in variation BONUS+LEADER+MAJORITY), Table 8. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Peru. Round BONUS COMM+ BONUS COMM BONUS+ LEADER BONUS+ LEADER+ MAJORITY 9 2.30 2.90 2.63 1.54 2.28 10 2.67 2.67 2.64 1.70 2.14 11 2.48 2.92 3.09 1.75 3.22 12 2.66 2.93 2.70 1.93 2.30 13 2.45 2.76 2.84 1.98 3.17 14 3.17 2.86 2.94 1.89 1.69 15 2.72 3.14 3.42 1.61 2.39 16 3.34 2.43 2.86 2.11 2.23 Table 8. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Peru. 0.00 0.50 1.50 1.00 2.50 2.00 3.00 3.50 4.00 9 10 11 12 13 14 15 16 Average individual extraction peru (rounds 9-16) BONUS COMM + BONUS COMM BONUS + LEADER BONUS + LEADER + MAJORITY Average individual extraction peru (rounds 9-16) Average individual extraction peru (rounds 9-16) Figure 8. Average Individual Extraction Rounds 9-16 in Peru. Three variations provided participants the possibility to communicate and receive the bonus: COMM+BONUS, BONUS+LEADER, BONUS+LEADER+MAJORITY. It may be the case that an equal distribution of the bonus was not more appealing for them than a distribution made based on other criteria that they might have discussed in the activity. The variation BONUS+LEADER+MAJORITY is identical to BONUS+ LEADER but in the former the majority of the group members are women. The average extraction level in this variation is 2.43. Although BONUS+LEADER+ MAJORITY led to lower extraction levels than COMM+BONUS, it was not as effective as BONUS+LEADER, and the only difference compared to that variation was the number of women in the group. This result corroborates some of the results found for BONUS+LEADER, discussed earlier. The fact that a leader was elected Community harvesting of trees in Peru under payment for ecosystem service schemes 17 to distribute the bonus had a positive impact on the decisions of the participants. Additionally, Figure 8 shows that BONUS+LEADER+MAJORITY produced fluctuations in average extraction levels, which ranged from 1.69 to 3.22 compared to fluctuations in BONUS+LEADER, which ranged from 1.54 to 2.11. Does this mean that it was more difficult to reach an agreement when more women were in the group? Or was it because the leaders in these groups were not distributing the bonus in a fair way? Is it that women in these communities are not as accustomed to making this type of decisions? Further research is needed to explore these questions. that it was more difficult to reach an agreement when more women were in the gro Or was it because the leaders in these groups were not distributing the bonus in a fa way? Is it that women in these communities are not as accustomed to making this t of decisions? Further research is needed to explore these questions. Table 8. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Peru. Round BONUS COMM+ BONUS COMM BONUS+ LEADER BONUS+ LEADER+ MAJORITY 9 2.30 2.90 2.63 1.54 2.28 10 2.67 2.67 2.64 1.70 2.14 11 2.48 2.92 3.09 1.75 3.22 12 2.66 2.93 2.70 1.93 2.30 13 2.45 2.76 2.84 1.98 3.17 14 3.17 2.86 2.94 1.89 1.69 15 2.72 3.14 3.42 1.61 2.39 16 3.34 2.43 2.86 2.11 2.23 Figure 8. Average Individual Extraction Rounds 9-16 in Peru. Three variations provided participants the possibility to communicate and receive the bonus: COMM+BONUS, BONUS+LEADER, BONUS+LEADER+MAJORITY. When we look more closely at the selection of the leader in variations When we look more closely at the selection of the leader in variations BONUS+LEADER and BONUS+LEADER+MAJORITY in one group the same person was elected for six rounds (in variation BONUS+LEADER+MAJORITY), Maria Claudia Lopez, et. al. 18 while another group in the same variation had only two leaders throughout the eight rounds (each for four rounds). One group in the BONUS+LEADER variation had the same leader elected for five rounds. Clearly, different groups developed different strategies to choose their leader. A significant fact is that a woman leader was choosen in only 45 (37.5%) of the rounds, and only five of those female leaders were chosen as leaders twice. No woman were elected leader more than two times in a group. Even in the BONUS+LEADER+MAJORITY variation, which required having a majority of women, in the group, a woman was elected to lead in only 34 (53.1%) rounds. This is much higher than the LEADER+COMM variation, which only had woman leaders in 11 (19.6%) rounds. The fact that women were not elected to be leaders as often may be a reflection of what happens to them in real life: women may not have the chance to participate in many decision making processes related to forest or resource management in their communities. The bonus was paid in 75 (62.5%) out of 120 rounds by the organization. When the bonus was distributed by the leader in one (1.3%) round the leader distributed the bonus unequally keeping nothing for him/herself. During 5 (6.7%) rounds the leader kept more for him/herself than was distributed to others. In nine (12%) rounds the leaders distributed the bonus not in an equal way, but they did not keep more for themselves. Finally in 60 (80%) rounds the leaders distributed the bonus equally providing 20 tokens to each player. Table 9. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Tanzania and Indonesia. Community harvesting of trees in Peru under payment for ecosystem service schemes 19 Community harvesting of trees in Peru under payment for ecosystem service schemes When we compare the results of Peru to those of Tanzania and Indonesia we see that the average extraction levels in Tanzania were much lower than in Peru and Indonesia for all variations. Comparing Peru and Indonesia, we see that some variations were more effective in Peru (BONUS, COMM, BONUS+LEADER) and some more effective in Indonesia (BONUS+COMM, BONUS+LEADER+MAJORITY), and we do not yet have a very strong explanation for that result. The variation that was most effective in Peru, BONUS+LEADER had an average of 1.81 in Peru compared to 2.34 in Indonesia and 1.63 in Tanzania. At the same time, the least effective variation in Peru, COMM led to an average of 2.89 in Peru and 3.29 and 1.60, respectively in Indonesia and Tanzania (see Table 8 and Table 9). Not only do the results vary by country, but the variations that were more effective in Indonesia and Tanzania were not the more effective in Peru. This result shows that variations that may be very efficient in one location may not be as efficient in another one. This may be related to the way in which resources are managed in the different locations, and also perhaps significantly, to cultural and gender differences associated with the forest management. Three variations provided participants the possibility to communicate and receive the bonus: COMM+BONUS, BONUS+LEADER, BONUS+LEADER+MAJORITY. Round BONUS COMM+ BONUS COMM BONUS+ LEADER BONUS+ LEADER+ MAJORITY Indonesia 9 2.61 2.19 4.47 3.17 1.92 10 2.58 2.42 3.64 2.09 1.41 11 3.05 2.66 2.78 2.63 1.63 12 3.53 2.92 2.92 2.36 2.30 13 3.72 2.17 3.38 2.14 1.59 14 2.91 2.28 3.30 1.81 1.53 15 3.11 2.34 3.11 3.05 1.67 16 3.14 2.36 2.70 1.50 1.41 Tanzania 9 0.59 0.73 1.48 1.11 1.92 10 0.89 0.77 1.55 1.51 1.41 11 1.20 0.85 1.90 1.49 1.63 12 1.14 1.02 1.76 1.44 2.30 13 1.33 0.81 1.67 1.64 1.59 14 1.08 0.94 1.54 2.05 1.53 15 1.47 0.94 1.40 1.93 1.67 16 1.56 0.93 1.49 1.90 1.41 Table 9. Average of trees cut during rounds 9-16 of the activity for each one of the variations in Tanzania and Indonesia. 19 Table 10 and Figure 9 show the results of the forest activity once the variations were removed from the activity. In almost all variations the average extraction levels were lower than in the first 8 rounds (see Figure 9); however the variation after BONUS+LEADER+MAJORITY led to some extraction levels very similar to (and even greater than) the ones found at the very beginning of the forest activity (see for example round 6 compared to round 22). On average the extraction level in rounds 17-24 was 4.02 whereas the average extraction level in the first 8 rounds was 3.95. c) Results Rounds 17 to 24 Table 10. Average number of trees cut during rounds 17 to 24 of the activity in Peru. Round After BONUS After COMM+ BONUS After COMMM After BONUS+ LEADER After BONUS+ LEADER+ MAJORITY 17 2.55 2.71 2.48 2.05 3.58 18 3.42 3.21 2.75 2.29 3.45 19 3.66 3.13 2.97 2.34 4.11 20 3.78 3.65 2.88 2.43 4.11 21 3.20 3.21 2.84 2.11 4.25 22 3.42 3.49 2.92 2.34 3.89 23 3.27 3.25 2.92 2.20 4.31 24 3.83 3.63 3.03 2.70 4.44 Table 10. Average number of trees cut during rounds 17 to 24 of the activity in Peru. ble 10. Average number of trees cut during rounds 17 to 24 of the activity P Table 10 and Figure 9 show the results of the forest activity once the variations were removed from the activity. In almost all variations the average extraction levels were lower than in the first 8 rounds (see Figure 9); however the variation after BONUS+LEADER+MAJORITY led to some extraction levels very similar to (and even greater than) the ones found at the very beginning of the forest activity (see for example round 6 compared to round 22). On average the extraction level in rounds 17-24 was 4.02 whereas the average extraction level in the first 8 rounds was 3.95. Table 10 and Figure 9 show the results of the forest activity once the variations were removed from the activity. In almost all variations the average extraction levels were lower than in the first 8 rounds (see Figure 9); however the variation after BONUS+LEADER+MAJORITY led to some extraction levels very similar to (and even greater than) the ones found at the very beginning of the forest activity (see for example round 6 compared to round 22). On average the extraction level in rounds 17-24 was 4.02 whereas the average extraction level in the first 8 rounds was 3.95. Maria Claudia Lopez, et. al. 20 Figure 9. Comparison of trees cut in rounds 1-8 versus rounds 17-24 after variations. 0 1 2 3 4 5 6 7 8 17 18 19 20 21 22 23 24 1 2 3 4 5 Before Variations vs. After Variations After BONUS After COMM + BONUS After COMM After BONUS + LEADER After BONUS + LEADER + MAJORITY Average Individual Extraction Before Variations Before Variations vs. After Variations Figure 9. Comparison of trees cut in rounds 1-8 versus rounds 17-24 after variations. c) Results Rounds 17 to 24 Almost all average extraction levels were higher in rounds 17 to 24 than in rounds 9 to 16. In the case of BONUS+LEADER+MAJORITY the levels increase on average of more than one tree above the rounds 9-16 results. However, after COMM is an important exception, that had showing a slight reduction in the extraction level from 2.89 to 2.85 in rounds 17 to 24. This is an important result since it shows that the benefits of communication in Peru lasted even after the variation ended, somehow the fact that participants were able to communicate without any monetary incentive was sufficient for them to change their behavior even after they could no longer communicate. These results may reflect the fact that participants are not used to getting money to conserve their forest, but they do engage in some activities to conserve the forest. Figure 10 shows a graphical representation of the average individual extraction throughout the 24 rounds of the forest activity in Peru. As described before, all variations lead to less extraction in rounds 9 to 16 compared to rounds 1-8. Thus all variations were effective at reducing the number of trees cut. At the same time, some variations clearly worked better than others at achieving reductions in cutting. In rounds 17 to 24 the extraction levels did not return to the levels observed in rounds 1 to 8 when no variations were available, but they did increase from levels when the variations were in place. There are two notable exceptions to this: the BONUS+LEADER+MAJORITY began an upward trend after round 18 that ended with more extraction than even before the variation. The other exception was the variation COMM which maintained the lower levels achieved during the variation. Community harvesting of trees in Peru under payment for ecosystem service schemes 21 21 Figure 10. Average Individual Extraction in Peru from rounds 1 to 24. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Pre-variation Variation Post-variation Average individual extraction troughout the game BONUS COMM+ BONUS COMM BONUS+ LEADER BONUS+ LEADER+ MAJORITY Average individual extraction troughout the game Figure 10. Average Individual Extraction in Peru from rounds 1 to 24. Table 11. c) Results Rounds 17 to 24 Average number of trees cut during rounds 17 to 24 of the activity in Tanzania and Indonesia. Round After BONUS After COMM+ BONUS After COMM After BONUS+ LEADER After BONUS+ LEADER+ MAJORITY Indonesia 17 2.23 2.73 3.03 2.81 2.47 18 2.84 2.98 3.30 3.03 2.50 19 3.08 3.02 3.63 3.09 2.03 20 3.06 2.84 3.31 3.41 2.23 21 2.78 3.30 3.22 3.25 2.22 22 2.94 3.38 3.33 3.84 1.81 23 2.77 3.00 3.31 3.34 1.77 24 2.92 3.59 3.52 3.52 1.91 Tanzania 17 1.15 0.96 1.70 1.90 1.57 18 1.69 1.14 1.57 2.15 1.93 19 1.59 1.08 1.68 1.99 2.01 20 1.77 0.96 1.55 1.81 1.70 21 1.97 1.21 1.39 1.96 1.94 22 1.61 1.02 1.48 1.83 2.05 23 1.53 1.21 1.73 2.00 1.98 24 1.71 1.36 1.55 2.35 1.46 Table 11. Average number of trees cut during rounds 17 to 24 of the activity in Tanzania and Indonesia. Maria Claudia Lopez, et. al. 22 22 When we compare the results of Peru vs. those of Tanzania and Indonesia we find that Tanzania consistently cut less trees than the other two countries (see Figure 11). The difference is at greater than one. Among all the after variations, Peru showed the highest levels of extraction in after BONUS, after COMM+BONUS, and after BONUS + LEADER+ MAJORITY. 0.0 1.0 2.0 3.0 4.0 5.0 BONUS COMM+ BONUS COMM BONUS+ LEADER BONUS+ LEADER+ MAJORITY After variation comparison Peru Indonesia Tanzania Figure 11. Comparison of average extraction after treatments in the three countries. Figure 11. Comparison of average extraction after treatments in the three countries. Experience 5 After participating in the forest activity 98.13% of the participants mentioned they had learned something new, and 99.38% stayed that day enjoyed participating in this study. Aditionally participants who played with one of the BONUS variations were asked whether or not they thought the distribution of the bonus was fair. Of these, 44.9% thought it was good and 24.6% thought it was equally distributed, 7.8% felt the distribution of the bonus resembled reality, and a 5.1% felt it was unfair. When we disaggregated the answers between participants whose bonus was distributed by the organization (BONUS+COMM) compared to those who had a chosen leader (BONUS+LEADER+MAJORITY and COMM+ LEADER) we found that 0% of the individuals in groups whose bonus was distributed by the organization thought the bonus was unfair, while 10.8% of those who chose a leader found it to be unfair. So even though trust on the leader seemed to be an important factor when making the decisions, and does increasing cooperation, in the end of the activity some people thought the distribution was unfair. The result for the BONUS+COMM is interesting because even though participants noticed that the distribution was fair, that did not lead to high more extraction levels in the game compared to BONUS+LEADER+MAJORITY and COMM+ LEADER. Conclusion 6 A noteworthy result for Peru was the effect of communication. Participants in that variation decreased their extraction levels in rounds 9- 16 with respect to rounds 1-8, and it was the variation that led to lower cooperation in rounds 9-16, but among all variations those groups maintained (and even slightly reduced) their extraction levels in rounds 17 to 24. The effects of communication were not as significant in reducing cutting as other variations when it was in place, but they lasted for more than 8 rounds, persisting even after the communication ceased. It is even more relevant, that this persistence was only observed in the variation where communication took place alone and not in combination with one of the bonus. This suggest that the bonus was not a necessary mechanism to get participants to agree on the importance of conservation. It is possible that the results from the forest activity also reflect a reality that came to light in the survey, namely that even in places where PES is implemented participants are not receiving monetary payments. Our survey did not explore other types of payments such as in kind payment or technical assistance thus we do not have enough information about the type of PES these communities are receiving. This may be reflected in the extraction levels observed in some of the variations involving a bonus. It is also possible that this played a role in the results described earlier for the COMM variation and its persistent effects even after the communication was removed. In general extraction levels in Peru are similar to those in Indonesia, but very different from Tanzania. The variation leading to lowest extraction levels was COMM+ LEADER, but the levels of extraction were not as low in comparisson to Tanzania. Some of the results may be associated with the fact that women in these rural areas of Peru are not used to participating in decision making scenarios such as the ones portrayed by the forest activity and aditional fieldwork will be needed to better understand this findings. This research was carried out by CIFOR as part of the CGIAR Research Program on Forests, Trees and Agroforestry (CRP-FTA). This collaborative program aims to enhance the management and use of forests, agroforestry and tree genetic resources across the landscape from forests to farms. Conclusion 6 CIFOR leads CRP-FTA in partnership with Bioversity International, CATIE, CIRAD, the International Center for Tropical Agriculture and the World Agroforestry Centre. blog.cifor.org Fund Center for International Forestry Research (CIFOR) Center for International Forestry Research (CIFOR) CIFOR advances human well-being, environmental conservation and equity by conducting research to help shape policies and practices that affect forests in developing countries. CIFOR is a member of the CGIAR Consortium. Our headquarters are in Bogor, Indonesia, with offices in Asia, Africa and Latin America. Center for International Forestry Research (CIFOR) CIFOR advances human well-being, environmental conservation and equity by conducting research to help shape policies and practices that affect forests in developing countries. CIFOR is a member of the CGIAR Consortium. Our headquarters are in Bogor, Indonesia, with offices in Asia, Africa and Latin America. Center for International Forestry Research (CIFOR) CIFOR advances human well-being, environmental conservation and equity by conducting research to help shape policies and practices that affect forests in developing countries. CIFOR is a member of the CGIAR Consortium. Our headquarters are in Bogor, Indonesia, with offices in Asia, Africa and Latin America.
https://openalex.org/W4253283155	https://bmccardiovascdisord.biomedcentral.com/track/pdf/10.1186/s12872-020-01434-z	English	null	The COngenital HeARt Disease in adult and Pulmonary Hypertension (COHARD-PH) registry: a descriptive study from single-center hospital registry of adult congenital heart disease and pulmonary hypertension in Indonesia	Research Square (Research Square)	2,019	cc-by	8,956	(2020) 20:163 (2020) 20:163 Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 https://doi.org/10.1186/s12872-020-01434-z Open Access The COngenital HeARt Disease in adult and Pulmonary Hypertension (COHARD-PH) registry: a descriptive study from single- center hospital registry of adult congenital heart disease and pulmonary hypertension in Indonesia The COngenital HeARt Disease in adult and Pulmonary Hypertension (COHARD-PH) registry: a descriptive study from single- center hospital registry of adult congenital heart disease and pulmonary hypertension in Indonesia Lucia Kris Dinarti, Anggoro Budi Hartopo, Arditya Damar Kusuma, Muhammad Gahan Satwiko, Muhammad Reyhan Hadwiono, Aditya Doni Pradana and Dyah Wulan Anggrahini* * Correspondence: kris_dinarti@ugm.ac.id; a_bhartopo@ugm.ac.id; wulan.anggrahini@ugm.ac.id gg @ g Department of Cardiology and Vascular Medicine, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada – Dr. Sardjito Hospital, Yogyakarta, Indonesia Abstract Backgrounds: The COngenital HeARt Disease in adult and Pulmonary Hypertension (COHARD-PH) registry is the first registry for congenital heart disease (CHD) and CHD-related pulmonary hypertension (PH) in adults in Indonesia. The study aims to describe the demographics, clinical presentation, and hemodynamics data of adult CHD and CHD-related PH in Indonesia. Methods: The COHARD-PH registry is a hospital-based, single-center, and prospective registry which includes adult patients with CHD and CHD-related PH. The patients were enrolled consecutively. For this study, we evaluated the registry patients from July 2012 until July 2019. The enrolled patients underwent clinical examination, electrocardiography, chest x-ray, 6-min walking test, laboratory measurement, and transthoracic and transesophageal echocardiography. Right heart catheterization was performed to measure hemodynamics and confirm the diagnosis of pulmonary artery hypertension (PAH). Results: We registered 1012 patients during the study. The majority were young, adult females. The majority of CHD was secundum ASD (73.4%). The main symptom was dyspnea on effort. The majority of patients (77.1%) had already developed signs of PH assessed by echocardiography. The Eisenmenger syndrome was encountered in 18.7% of the patients. Based on the right heart catheterization, 66.9% of patients had developed PAH. Patients with PAH were significantly older, had lower peripheral oxygen saturation, had lower 6-min walking distance, and higher NTproBNP. The NTproBNP level independently predicted the development of PAH among CHD. (Continued on next page) Background countries, the situation regarding the adult CHD- related PH in developing countries are very different [11–13]. The COngenital HeARt Disease in adult and Pul- monary Hypertension (COHARD-PH) registry was initiated in 2012 to be the first registry done in Indonesia to describe adult CHD and CHD-related PH populations. This hospital-based registry is performed in Dr. Sardjito Hos- pital, Jogjakarta, Indonesia, which is a national referral hos- pital for cardiovascular disease in the region. The current study aims to describe the prevalence, demographics, clin- ical presentation, and hemodynamics characteristics of adult patients with CHD and CHD-related PH registered in the COHARD-PH registry. The prevalence of adult congenital heart disease (CHD) in developed countries continues to rise due to improved survival attributed to successful surgical and medical man- agement in childhood [1]. As a result, an increasing popu- lation of children with CHD is surviving into adulthood [2]. In less developed countries, a significant number of adults with CHD seeking medical help because of the emerging symptoms and signs of complications. One of the devastating complications of CHD is pul- monary hypertension (PH) which occurs in about 10% of the CHD populations [3]. PH is defined as an increase in mean pulmonary artery pressures (mPAP) ≥25 mmHg at rest [4]. Based on the current clinical classification of PH, CHD may cause pulmonary artery hypertension (PAH) which is defined as a group 1 in this classification [4]. The PAH or PH group 1 is a clinical group which is characterised by hemodynamic parameters as pre-capillary PH (mPAP ≥25 mmHg with pulmonary artery wedge pres- sure (mPAWP) ≤15 mmHg) and pulmonary vascular resist- ance (PVR) > 3 Wood units (WU) [4]. The hemodynamic measurement by right heart catheterization (RHC) is mandatory to diagnose PAH and to assess the recommen- dation fordefect closure. The implications of CHD-related PH are limited functional capacity, increased risk of ar- rhythmias, right heart failure, and increased mortality [5]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 11 Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 (Continued from previous page) Conclusions: The COHARD-PH registry is the first Indonesian adult-CHD and CHD-related PH registry. The demographics, clinical presentation, and hemodynamics dataof this registry reflect the situation in developing countries which needs to be compared with similar registries from developed countries. Keywords: Registry, Adult congenital heart disease, Atrial septal defects, Pulmonary hypertension Methods Subjects The COngenital HeARt Disease in adult and Pulmonary Hypertension (COHARD-PH) registry, is a single-center, observational, and prospective registry which enrolls adult patients with CHD and CHD-related PH. The adult patients presented in Dr. Sardjito Hospital, Jogja- karta, Indonesia with suspected CHD and CHD-related PH undergo a series of examinations to confirm the CHD and CHD-related PH diagnosis. The subjects are enrolled consecutively from outpatient clinics and in- patient wards. The enrollment and follow-up have been performed from July 2012 until the present. This study evaluated the patients of COHARD-PH registry enrolled from July 2012 until July 2019. This registry enrolled adult patients with age ≥18 years old. The populational based registries in developed countries indicate that the prevalence of CHD-related PH is approxi- mately 5–10% [6–8]. The CHD-related PH is a result of the systemic-to-pulmonary shunt at both the pre-tricuspid (atrial septal defect (ASD)) and the post-tricuspid levels (such as ventricle septal defect (VSD), patent ductus arter- iosus (PDA), and aortopulmonary (AP) window) which cause the chronic increased flow to the pulmonary vessels. Its consequences are endothelial dysfunction, pulmonary vascular remodeling, increased pulmonary artery pressure and increased pulmonary vascular resistance [9]. Procedures Patients were interviewed, underwent physical examin- ation, electrocardiography (ECG) examination, and chest x-ray examination. The suspected CHD patients contin- ued for transthoracic echocardiography (TTE) as the initial examination to confirm the diagnosis of CHD. By TTE, the probability of PH was assessed based on current guidelines [4]. The bubble test was performed in selected cases if the TTE examination was dubious regarding septal defects/shunts. Transoesofageal echocardiography (TOE) was performed in patients with confirmed ASD and VSD by TTE examination. The TTE and TOE examination were done with G.E Vivid 7 (G.E Healthcare, U.S.A), G.E Vivid S6 (G.E Healthcare, U.S.A) or Phillips HD 15 Indonesia, a developing country and one of the most populous countries in the world, until recently did not have a national registry in regards to the CHD-related PH in adults. The prevalence and inci- dence of CHD-related PH are still unknown; never- theless in clinical practice, adult patients with undetected and delayed diagnosis of CHD are fre- quent [10]. Compared with registries from developed Page 3 of 11 Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 Page 3 of 11 using standard procedures in non-sedated patients. The purpose of RHC was to measure hemodynamics, diagnose pulmonary artery hypertension (PAH) and decide the closure procedure for septal defects/ shunts. The cardiac output was determined by indirect Fick method, as per hospital protocol. The flow ratio was calculated with the formula: pulmonary blood flow (Qp)/sys- temic blood flow (Qs) = (aorta saturation - mixed vein (MV) saturation)/(pulmonary vein (PV) saturation-pulmonary ar- tery (PA) saturation). An MV saturation was calculated from: ((3 x superior vena cava saturation) + inferior vena cava saturation)/4. The pulmonary vascular resistance index (PVRi) was derived from the formula: (mPAP – mean left atrial pressure (mLAP) (or mPAWP)/Qp. A Qp was calcu- lated from the formula: O2 consumption (ml/min)/ (1.36x10xhemoglobin level x ((PV saturation-PA satur- ation)/100). The PVR was calculated from PVRi/body sur- face area. The PAH diagnosis was established when mPAP ≥25 mmHg, PVR > 3 WU and PAWP or mLAP ≤15 mmHg [4]. The diagnosis of Eisenmenger syndrome is established hemodynamically when Qp/Qs < 1 and PVRi > 8 WU.m2 [4]. The vasoreactivity test was performed in selected pa- tients (discretion by cardiologist consultants). The vasoreac- tivity result was assessed based on current guideline (reduced in PVR > 20% and final PVRi < 6 WU.m2). Procedures The correctability of shunt was defined as patients with suitable anatomy of defects (surgery and/or device), Qp:Qs > 2 and PVRi < 6 WU.m2. Figure 1 shows the flowchart of (Philips N.V, The Netherland). The image acquisitions were made by three experience sonographers. The valid- ation and confirmation of TTE and TOE examinations were performed by cardiologist consultants in our center dedicated to the registry. The cardiologist consultants had been tested for interobserver variability coefficients with results > 80% in agreement [14]. The image ac- quisition, validation and confirmation were in accord- ance with European Association of Echocardiography and American Society of Echocardiography guidelines prevailed in our hospital practice. The 6-min walking test to measure the distant of walking was performed for baseline of the registry. Patients with simple defects such as ASD, VSD, PDA, AVSD, patent foramen ovale (PFO), and AP window were included in this study. The combined defects and other defect types were categorized as multiple defects. Patients with high probability of PH by TTE without confirmed CHD were excluded from the COHARD-PH registry (they were included in another PH registry). Complex CHD patients were excluded from the registry. The signs of Eisenmenger syndrome (desaturasion and bidirectional shunt from TTE) were noted and later con- firmed by RHC. Right heart catheterisation (RHC) was subsequently performed in all patients after being confirmed as CHD by TTE and TOE and enrolled for the registry. The RHC was performed by cardiologist consultants Fig. 1 The flowchart of patients enrollment of COHARD-PH registry Fig. 1 The flowchart of patients enrollment of COHARD-PH registry Fig. 1 The flowchart of patients enrollment of COHARD-PH registry Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 Dinarti et al. Data collection The research assistants dedicated to the registry col- lected and compiled the data and subsequently input the data to the electronic case report form of the COHARD- PH registry database. The baseline characteristics of patients were collected, comprising demographic data and clinical data. The ECG and chest X-ray results were documented. The TTE and TOE data were collected, comprising the type of CHD, dimension of right atrium (RA) and right ventricle (RV), left ventricle ejection frac- tion, tricuspid valvular regurgitation gradient (TVRG), tri- cuspid annular plane systolic excursion (TAPSE) and estimated mPAP. The 6-min walking distance was col- lected. The laboratory data were also compiled. The RHC data were collected, comprising mPAP, PAWP, mRAP, PVRi, mLAP, flow ratio, and oxygen saturations. a data of 736 patients; b data of 616 patients; c data of 965 patients; d data of 574 patients; e data of 586; f data of 405 patients Thesignedinformedconsentswereacquiredforeachpa- tienttobeincludedintheregistry.TheMedicalandHealth Research Ethics Committee of the Faculty of Medicine, Public Health and Nursing Universitas Gadjah Mada and Dr.SardjitoHospitalhadapprovedtheregistryprotocol. The majority of patients were females, which accounted for 78.5% of all patients (as shown in Fig. 2). Normal and underweight body mass categories were predomin- ant. Mean peripheral oxygen saturation was 95.5%. The WHO functional class was predominantly class II (43.0% of patients), only the minority of patients had worse WHO functional class (10.0% class III and 1.1% class IV). The mean 6-min walking distance was 356.5 m. The increased probability of PH by TTE examination was pre- dominant (77.1%).The signs of Eisenmenger syndrome were encountered in 18.7% of patients. The laboratory re- sults showed mean hemoglobin level was 13.8 g/dL, hematocrit 41.9% and median NTproBNP level 370.9 pg/ mL. The main symptoms were dyspnea on effort (35.9%), easily fatigued (16.3%), chest pain/discomfort (10.8%) and palpitations (9.3%). As many as 9.4% of patients did not report any symptoms during first enrollment. The main symptoms of patients are depicted in Fig. 3. Procedures BMC Cardiovascular Disorders (2020) 20:163 Page 4 of 11 Table 1 Demography, Clinical and Laboratory Characteristics of COHARD-PH Registry Patients Characteristics Total (n = 1012) Age at First Enrollment (years) [mean ± SD] 34.7 ± 13.2 Body Mass Index [mean ± SD] 19.9 ± 7.7 Underweight [n(%)] 417 (41.2) Blood Pressure (mmHg) [mean ± SD] Systolic 111.9 ± 16.4 Diastolic 72.9 ± 11.4 Oxygen Saturation (%)[mean ± SD]a 95.5 ± 5.3 6 Minute Walk Distance (meter)[mean ± SD]b 356.5 ± 99.9 WHO Functional Class [n(%)]c I 418 (41.9) II 435 (43.0) III 101 (10.0) IV 11 (1.1) Hemoglobin (g/dL) [mean ± SD]d 13.8 ± 2.2 Hematocrit (%) [mean ± SD]e 42.1 ± 16.2 NTproBNP (pg/mL) [median (IQR)]f 370.9 (132.3–1625.0) Pulmonary hypertension (by TTE) [n(%)] 780 (77.1) Eisenmenger Syndrome [n (%)] 189 (18.7) Post Defect Closure [n (%)] 24 (2.9) a data of 736 patients; b data of 616 patients; c data of 965 patients; d data of 574 patients; e data of 586; f data of 405 patients COHARD-PH registry enrollment from July 2012 until July 2019. COHARD-PH registry enrollment from July 2012 until July 2019. The blood sample was collected from each patient by venipuncture in peripheral veins and during RHC. The blood sample was centrifuged and stored in −80° for fur- ther analysis. The hemoglobin and hematocrit levels were measured with routine hemocytometer. The NTproBNP measurement was performed using a electrochemilumi- nescence immunoassay (ElecsysProBNP II) and a Cobas e immunoassay analyzer (Roche Diagnostics, Germany). Statistics analysis We performed the descriptive analysis of the data. The continuous data were presented in mean and standard deviation (SD) or median and interquartile range (IQR) depending on normality data distribution after tested with the Shapiro Wilk or Kolmogorov Smirnov test. The categorical data were presented in percentage. The com- parison between two groups was conducted with Stu- dent T test and Chi-square test according to the type of data. The comparison among groups was conducted with one-way ANOVA test. A multivariable analysis was performed with logistic regression test to assess the independent predictor(s) among covariables. A value of p < 0.05 was set as statistically significant. The majority of CHD type was secundum ASD (73.4%). Other CHD types were perimembranous VSD (9.0%), PDA (5.8%), doubly-committed subarterial (DCSA) VSD (3.6%), sinus venosus ASD (2.0%), primum ASD (1.3%), PFO (0.8%), AVSD (0.3%) and AP window (0.1%). The pa- tients with multiple defects accounted for 0.9% of all pa- tients. The majority of patients had undergone RA and Results From July 2012 until July 2019, we have registered data from 1012 patients who have the confirmed diagnosis as septal defects/shunts CHD. The clinical characteristics of the patients are shown in Table 1. The mean age of the patients at first diagnosis/enrollment was 34.7 years. Page 5 of 11 Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 (2020) 20:163 RV dilatation, with mean RA diameter of 45.6 mm and RV diameter of 42.1 mm. The mean mPAP based on TTE examination was 36.1 mmHg. The mean TVRG was 61.6 mmHg. The mean TAPSE was 24.3 mm. The mean left ventricle ejection fraction was 68.1%. Table 2 shows the results of TTE and TOE procedures. The RHC had been performed in 614 subjects (60.7%). Among 1012 patients, 103 patients did not undergo RHC examination and 295 patients were on a waiting list to get RHC performed. The RHC was not performed in 103 patients due to: (1) patients had already had clos- ure of defects (24 patients), (2) patients died before scheduled for RHC (39 patients), (3) patients refused the Fig. 2 The proportion of sex in COHARD-PH registry. The majority were female patients (78.5%) Fig. 2 The proportion of sex in COHARD-PH registry. The majority were female patients (78.5%) Fig. 2 The proportion of sex in COHARD-PH registry. The majority were female patients (78.5%) RV dilatation, with mean RA diameter of 45.6 mm and RV diameter of 42.1 mm. The mean mPAP based on TTE examination was 36.1 mmHg. The mean TVRG was 61.6 mmHg. The mean TAPSE was 24.3 mm. The mean left ventricle ejection fraction was 68.1%. Table 2 shows the results of TTE and TOE procedures. RV dilatation, with mean RA diameter of 45.6 mm and RV diameter of 42.1 mm. The mean mPAP based on TTE examination was 36.1 mmHg. The mean TVRG was 61.6 mmHg. The mean TAPSE was 24.3 mm. The mean left ventricle ejection fraction was 68.1%. Table 2 shows the results of TTE and TOE procedures. The RHC had been performed in 614 subjects (60.7%). Among 1012 patients, 103 patients did not undergo RHC examination and 295 patients were on a waiting list to get RHC performed. Results The RHC was not performed in 103 patients due to: (1) patients had already had clos- ure of defects (24 patients), (2) patients died before scheduled for RHC (39 patients), (3) patients refused the The RHC had been performed in 614 subjects (60.7%). Among 1012 patients, 103 patients did not undergo RHC examination and 295 patients were on a waiting list to get RHC performed. The RHC was not performed in 103 patients due to: (1) patients had already had clos- ure of defects (24 patients), (2) patients died before scheduled for RHC (39 patients), (3) patients refused the Fig. 3 The percentage of symptoms of patients. Dyspnea on effort in 363 patients (35.9%), easily fatigued in 165 patients (16.3%), chest pain/ discomfort in 109 patients (10.8%), palpitation in 94 patients (9.3%), cough in 63 patients (6.2%), headache in 15 patients (1.5%), leg swelling in 10 patients (1.0%), dizziness/syncope in 7 patients (0.7%), and other symptoms in 12 patients (1.2). Asymptomatic patients were 95 (9.4%) and indeterminate were 79 patients (7.8%) Fig. 3 The percentage of symptoms of patients. Dyspnea on effort in 363 patients (35.9%), easily fatigued in 165 patients (16.3%), chest pain/ discomfort in 109 patients (10.8%), palpitation in 94 patients (9.3%), cough in 63 patients (6.2%), headache in 15 patients (1.5%), leg swelling in 10 patients (1.0%), dizziness/syncope in 7 patients (0.7%), and other symptoms in 12 patients (1.2). Asymptomatic patients were 95 (9.4%) and indeterminate were 79 patients (7.8%) Fig. 3 The percentage of symptoms of patients. Dyspnea on effort in 363 patients (35.9%), easily fatigued in 165 patients (16.3%), chest pain/ discomfort in 109 patients (10.8%), palpitation in 94 patients (9.3%), cough in 63 patients (6.2%), headache in 15 patients (1.5%), leg swelling in 10 patients (1.0%), dizziness/syncope in 7 patients (0.7%), and other symptoms in 12 patients (1.2). Asymptomatic patients were 95 (9.4%) and indeterminate were 79 patients (7.8%) Dinarti et al. Results BMC Cardiovascular Disorders (2020) 20:163 Page 6 of 11 Table 2 Echocardiography Characteristics of COHARD-PH Registry Patients Echocardiographic Findings Total (n = 1012) Congenital anomaly [n (%)] Primum ASD 13 (1.3) Secundum ASD 743 (73.4) Sinus venosus ASD 20 (2.0) Multiple ASD 5 (0.5) Patent foramen ovale 8 (0.8) Perimembranous VSD 91 (9.0) Doubly-committed subarterial VSD 36 (3.6) Atrioventricular septal defect 3 (0.3) Patent ductus arteriosus 59 (5.8) Aortopulmonary window 1 (0.1) Multiple defects 9 (0.9) Postclosure ASD 18 (1.8) Postclosure VSD 5 (0.5) Postclosure PDA 1 (0.1) mPAP (mmHg) [mean ± SD]a 35.3 ± 15.7 TVRG (mmHg) [mean ± SD]b 61.6 ± 34.3 RA diameter (mm) [mean ± SD]c 45.6 ± 8.8 RV diameter (mm) [mean ± SD]d 42.1 ± 9.0 TAPSE (mm) [mean ± SD] 24.3 ± 5.8 Left ventricle EF (%)[mean ± SD] 68.1 ± 8.8 adata from 828 subjects; b data from 810 subjects; c data from 843 subjects; d data 902 subjects ASD atrial septal defect, VSD ventricular septal defect, mPAP mean pulmonary artery pressure, TVRG tricuspid valve regurgitation gradient, RA right atrium, RV right ventricle, TAPSE tricuspid annular plane systolic excursion, EF ejection fraction Table 2 Echocardiography Characteristics of COHARD-PH Registry Patients Echocardiographic Findings Total (n = 1012) Congenital anomaly [n (%)] Primum ASD 13 (1.3) Secundum ASD 743 (73.4) Sinus venosus ASD 20 (2.0) Multiple ASD 5 (0.5) Patent foramen ovale 8 (0.8) Perimembranous VSD 91 (9.0) Doubly-committed subarterial VSD 36 (3.6) Atrioventricular septal defect 3 (0.3) Patent ductus arteriosus 59 (5.8) Aortopulmonary window 1 (0.1) Multiple defects 9 (0.9) Postclosure ASD 18 (1.8) Postclosure VSD 5 (0.5) Postclosure PDA 1 (0.1) mPAP (mmHg) [mean ± SD]a 35.3 ± 15.7 TVRG (mmHg) [mean ± SD]b 61.6 ± 34.3 RA diameter (mm) [mean ± SD]c 45.6 ± 8.8 RV diameter (mm) [mean ± SD]d 42.1 ± 9.0 TAPSE (mm) [mean ± SD] 24.3 ± 5.8 Left ventricle EF (%)[mean ± SD] 68.1 ± 8.8 adata from 828 subjects; b data from 810 subjects; c data from 843 subjects; d data 902 subjects ASD atrial septal defect, VSD ventricular septal defect, mPAP mean pulmonary artery pressure, TVRG tricuspid valve regurgitation gradient, RA right atrium, RV right ventricle TAPSE tricuspid annular plane systolic excursion EF Table 3 Hemodynamic Data from Right Heart Catheterizationof COHARD-PH Registry Patients Right Heart Catheterization Result Total (n = 614) mPAP (mmHg) [median (IQR)] 34.0 (23.0–56.0) PVRi (Wood Unit.m2 )[median (IQR)] 3.3 (1.6–11.6) mRAP (mmHg) [median (IQR)]a 9.0 (6.0–13.0) PAWP (mmHg) [median (IQR)]b 10.0 (7.0–13.0) mLAP (mmHg) [median (IQR)]c 10.0 (7.0–14.0) Flow Ratio [median (IQR)] 2.3 (1.4–3.4) Aorta saturation (%)[mean ± SD]d 91.9 ± 8.2 SVC saturation (%)[mean ± SD]e 62.9 ± 9.6 IVC saturation (%)[mean ± SD]f 72.8 ± 9.9 Left atrial saturation (%)[mean ± SD]g 91.7 ± 5.7 Right atrial saturation (%)[mean ± SD]h 77.6 ± 9.9 Right ventricle saturation (%)[mean ± SD]h 80.2 ± 11.6 Pulmonary artery saturation (%)[mean ± SD]i 81.4 ± 10.0 Pulmonary vein saturation (%)[mean ± SD]j 94.1 ± 5.9 PAH [n (%)] 411 (66.9) Vasoreactivity test [n (%)]k -Vasoreactive 43 (23.1) -Non vasoreactive 143 (76.9) Correctable [n (%)] 363 (59.1) a data of 563 patients; b data of 219 patients; c data of 517 patients; d data of 467 patients; e data of 507 patients; f data of 457 patients; g data of 418 patients; h data of 456 patients; i data of 499 patients; j data of 410 patients; k performed in 186 patients mPAP mean pulmonary artery pressure, PVRi pulmonary vascular resistance index, mRAP mean right atrial pressure, PAWP pulmonary artery wedge pressure, mLAP mean left atrial pressure, SVC superior vena cava, IVC inferior vena cava, PAH pulmonary arteryhypertension Table 3 Hemodynamic Data from Right Heart Catheterizationof COHARD-PH Registry Patients Right Heart Catheterization Result Total (n = 614) mPAP (mmHg) [median (IQR)] 34.0 (23.0–56.0) PVRi (Wood Unit.m2 )[median (IQR)] 3.3 (1.6–11.6) mRAP (mmHg) [median (IQR)]a 9.0 (6.0–13.0) PAWP (mmHg) [median (IQR)]b 10.0 (7.0–13.0) mLAP (mmHg) [median (IQR)]c 10.0 (7.0–14.0) Flow Ratio [median (IQR)] 2.3 (1.4–3.4) Aorta saturation (%)[mean ± SD]d 91.9 ± 8.2 SVC saturation (%)[mean ± SD]e 62.9 ± 9.6 IVC saturation (%)[mean ± SD]f 72.8 ± 9.9 Left atrial saturation (%)[mean ± SD]g 91.7 ± 5.7 Right atrial saturation (%)[mean ± SD]h 77.6 ± 9.9 Right ventricle saturation (%)[mean ± SD]h 80.2 ± 11.6 Pulmonary artery saturation (%)[mean ± SD]i 81.4 ± 10.0 Pulmonary vein saturation (%)[mean ± SD]j 94.1 ± 5.9 PAH [n (%)] 411 (66.9) Vasoreactivity test [n (%)]k -Vasoreactive 43 (23.1) -Non vasoreactive 143 (76.9) Correctable [n (%)] 363 (59.1) a data of 563 patients; b data of 219 patients; c data of 517 patients; d data of 467 patients; e data of 507 patients; f data of 457 patients; g data of 418 patients; h data of 456 patients; i data of 499 patients; j data of 410 patients; k performed in 186 patients mPAP mean pulmonary artery pressure, PVRi pulmonary vascular resistance index, mRAP mean right atrial pressure, PAWP pulmonary artery wedge pressure, mLAP mean left atrial pressure, SVC superior vena cava, IVC inferior vena cava, PAH pulmonary arteryhypertension a data of 563 patients; b data of 219 patients; c data of 517 patients; d data of 467 patients; e data of 507 patients; f data of 457 patients; g data of 418 patients; h data of 456 patients; i data of 499 patients; j data of 410 patients; k performed in 186 patients 5.5 vs. Results 97.4 ± 3.2%, p < 0.001), lower 6-min walking distance (336.3 ± 99.7 vs. 393.9 ± 82.1 m, p < 0.001), worse WHO functional class (WHO III-IV: 14.2% vs. 5.0%, p < 0.001), higher hemoglobin level (14.1 ± 2.2 vs. 13.5 ± 1.9 g/dL, p = 0.006), higher hematocrit level (42.2 ± 6.5 vs. 40.2 ± 4.9%, p < 0.001) and higher NTproBNP level (median: 774.0 vs. 121.5 pg/mL, p < 0.001). The proportion of ASD was pre- dominant in patients with PAH (89.3%), followed by PDA (5.1%) and VSD (4.1%). Among the patients with multiple defects, the majority had developed PAH (4 of 5 patients) and all subjects with AP window and AVSD had PAH. Multivariable analysis showed that only NTproBNP level independently predicts the PAH in patients with CHD (OR 1.003, 95% CI: 1.001–1.004, p = 0.001), as shown in Table 5. Table 6 shows the difference of characteristics among patients based on WHO functional class (total amount 602 patients). Worse WHO functional class (class III-IV) was marked by the least peripheral oxygen saturation, the least 6-min walk distance and the highest NTproBNP level. Based on echocardiography examination, worse WHO functional class was associated with increased RHC examination (n = 10) and (4) patients did not re- spond to RHC schedule (30 patients). Patients who did not undergo RHC examination were mostly lost to follow-up from COHARD-PH registry and did not con- tinue regular visits to our hospital. Patients who were on a waiting list were managed based on clinical symptoms and probability of PH based on echocardiography signs. The RHC results confirmed that 411 patients (66.9%) had developed PAH. The hemodynamics data from RHC showed median mPAP was 34.0 mmHg, PVRi 3.3 Wood Unit.m2, PAWP 10.0 mmHg, and flow ratio 2.3. The vasoreactivity test was performed in 186 patients and indicated that 43 patients (23.1%) had vasoreactive re- sponse. As many as 363 patients (59.1%) had correctable criteria for defect closure. Table 3 shows the result of RHC procedure. 5.5 vs. 97.4 ± 3.2%, p < 0.001), lower 6-min walking distance (336.3 ± 99.7 vs. 393.9 ± 82.1 m, p < 0.001), worse WHO functional class (WHO III-IV: 14.2% vs. 5.0%, p < 0.001), higher hemoglobin level (14.1 ± 2.2 vs. 13.5 ± 1.9 g/dL, p = 0.006), higher hematocrit level (42.2 ± 6.5 vs. Results 40.2 ± 4.9%, p < 0.001) and higher NTproBNP level (median: 774.0 vs. 121.5 pg/mL, p < 0.001). The proportion of ASD was pre- dominant in patients with PAH (89.3%), followed by PDA (5.1%) and VSD (4.1%). Among the patients with multiple defects, the majority had developed PAH (4 of 5 patients) and all subjects with AP window and AVSD had PAH. Multivariable analysis showed that only NTproBNP level independently predicts the PAH in patients with CHD (OR 1.003, 95% CI: 1.001–1.004, p = 0.001), as shown in Table 5. RHC examination (n = 10) and (4) patients did not re- spond to RHC schedule (30 patients). Patients who did not undergo RHC examination were mostly lost to follow-up from COHARD-PH registry and did not con- tinue regular visits to our hospital. Patients who were on a waiting list were managed based on clinical symptoms and probability of PH based on echocardiography signs. The RHC results confirmed that 411 patients (66.9%) had developed PAH. The hemodynamics data from RHC showed median mPAP was 34.0 mmHg, PVRi 3.3 Wood Unit.m2, PAWP 10.0 mmHg, and flow ratio 2.3. The vasoreactivity test was performed in 186 patients and indicated that 43 patients (23.1%) had vasoreactive re- sponse. As many as 363 patients (59.1%) had correctable criteria for defect closure. Table 3 shows the result of RHC procedure. Table 6 shows the difference of characteristics among patients based on WHO functional class (total amount 602 patients). Worse WHO functional class (class III-IV) was marked by the least peripheral oxygen saturation, the least 6-min walk distance and the highest NTproBNP level. Based on echocardiography examination, worse WHO functional class was associated with increased Table 4 shows the comparison of clinical and laboratory parameters between patients with CHD-related PAH and those without PAH. Patients with PAH had significantly older age at first diagnosis (36.4 ± 12.9 vs. 32.2 ± 12.0 years old, p < 0.001), lower peripheral oxygen saturation (94.8 ± Dinarti et al. Results BMC Cardiovascular Disorders (2020) 20:163 Page 7 of 11 Table 4 Characteristics of Patients based on the PAH Diagnosis by RHC (n = 614) Characteristics No PAH (n = 203) PAH (n = 411) P value Age at Enrollment (years) [mean ± SD] 32.2 ± 12.0 36.4 ± 12.9 < 0.001 Gender [n (%)] Males 42 (20.7) 75 (18.2) 0.469 Females 161 (79.3) 336 (81.8) Body Mass Index [mean ± SD] 20.2 ± 3.4 19.5 ± 6.9 0.136 Blood Pressure (mmHg) [mean ± SD] Systolic 111.4 ± 15.1 110.7 ± 18.8 0.678 Diastolic 73.4 ± 10.9 72.9 ± 11.9 0.698 Oxygen Saturation (%)[mean ± SD]a 97.4 ± 3.2 94.8 ± 5.5 < 0.001 6 Minute Walk Distance (meter)[mean ± SD]b 393.9 ± 82.1 336.3 ± 99.7 < 0.001 WHO Functional Class [n(%)]c < 0.001 I 122 (60.4) 136 (34.0) II 70 (34.7) 207 (51.8) III-IV 10 (5.0) 57 (14.2) Hemoglobin (g/dL) [mean ± SD]d 13.5 ± 1.9 14.1 ± 2.2 0.006 Hematocrit (%) [mean ± SD]e 40.2 ± 4.9 42.2 ± 6.5 < 0.001 NTproBNP (pg/mL) [median (IQR)]f 121.5 (57.1–218.1) 774.0 (242.8–2022.3) < 0.001 Congenital Anomalies [n (%)] 0.005 ASD 166 (81.8) 367 (89.3) VSD 26 (12.8) 17 (4.1) PDA 10 (4.9) 21 (5.1) Multiple defect 1 (0.5) 4 (1.0) AP window 0 (0) 1 (0.2) AVSD 0 (0) 1 (0.2) a data of 488 patients, b data of 442 patients, c data of 602 patients, d data of 439 patients, e data of 444 patients, f data of 294 patients, PAH pulmonary artery hypertension, ASD: atrial septal defect, VSD ventricular septal defect, PDA patent ductus arteriosus, AP aortopulmonal, AVSD atrioventricular septal defects, WHO world health organisation a data of 488 patients, b data of 442 patients, c data of 602 patients, d data of 439 patients, e data of 444 patients, f data of 294 patients, PAH pulmonary artery hypertension, ASD: atrial septal defect, VSD ventricular septal defect, PDA patent ductus arteriosus, AP aortopulmonal, AVSD atrioventricular septal defects, WHO world health organisation a data of 488 patients, b data of 442 patients, c data of 602 patients, d data of 439 patients, e data of 444 patients, f data of 294 patients, PAH pulmonary artery hypertension, ASD: atrial septal defect, VSD ventricular septal defect, PDA patent ductus arteriosus, AP aortopulmonal, AVSD atrioventricular septal defects, WHO world health organisation mPAP, higher TVRG, larger RA and RV diameters, lower TAPSE and lower LVEF. Results Based on RHC results, worse WHO functional class was related with higher mPAP and increased PVRi. The ASD patients were the majority among those with worse WHO functional class (95.5%). development of PAH in ASD patients may be associated with defect size and shunt flow. We analysed the difference of minimal and maximal diameter of ASD defect based on echocardiography examination between ASD patients with PAH and those with no PAH. The ASD patients with PAH had larger minimal defect diameter as compared to those without PAH (2.3 ± 0.8 vs. 1.9 ± 1.5 cm, p < 0.001) and lar- ger maximal defect diameter (2.6 ± 0.9 vs. 2.2 ± 1.8 cm, p = 0.001) (as shown in Fig. 4). There was no significant differ- ence in the Qp/Qs ratio based on TTE and flow ratio based on RHC results between ASD patients with PAH and those without PAH (as shown in Fig. 5). g The predominance of ASD patients in the COHARD-PH registry was in accord with previous reports. The Table 5 Multivariable Analysis to Predict the PAH Covariables* OR 95%CI P value Age at Enrollment 1.020 0.986–1.055 0.258 Oxygen Saturation 0.829 0.686–1.001 0.052 6 Minute Walk Distance 0.997 0.993–1.001 0.206 WHO Functional Class (III-IV) 1.874 0.322–10.886 0.484 Hemoglobin (g/dL) 1.019 0.581–1.789 0.947 Hematocrit (%) 0.952 0.781–1.160 0.627 NTproBNP (pg/mL) 1.003 1.001–1.004 0.001 Congenital Anomalies (ASD) 0.778 0.219–2.767 0.698 Covariables included were those with p < 0.05 from Table 4 Table 5 Multivariable Analysis to Predict the PAH C bl OR CI P There was an incremental increase of the proportion of PAH according to age range, with the highest propor- tion of PAH in the age group between 51 and 60 years old (Table 7). Covariables included were those with p < 0.05 from Table 4 Covariables included were those with p < 0.05 from Table 4 Discussion We report the first hospital-based registry of adults with CHD and CHD-related PH in Indonesia which is Dinarti et al. Discussion BMC Cardiovascular Disorders (2020) 20:163 Page 8 of 11 Table 6 Characteristics of Patients based on the WHO Functional Class (n = 602) Characteristics WHO class I (n = 258) WHO class II (n = 277) WHO class III-IV (n = 67) P value Age at Enrollment (years) [mean ± SD] 34.5 ± 12.9 34.9 ± 12.8 36.6 ± 12.2 0.498 Gender [n (%)] Males 55 (21.3) 53 (19.1) 7 (10.4) 0.131 Females 203 (78.7) 224 (80.9) 60 (89.6) Body Mass Index [mean ± SD] 19.9 ± 3.4 19.2 ± 3.9 20.9 ± 14.7 0.064 Oxygen Saturation (%)[mean ± SD]a 96.9 ± 3.2 95.4 ± 5.2 92.6 ± 8.1 < 0.001 6 Minute Walk Distance (meter)[mean ± SD]b 403.9 ± 80.2 333.1 ± 88.4 276.6 ± 104.6 < 0.001 Hemoglobin (g/dL) [mean ± SD]c 13.8 ± 2.0 13.9 ± 2.1 14.0 ± 2.5 0.569 Hematocrit (%) [mean ± SD]d 41.4 ± 5.3 41.6 ± 6.4 41.9 ± 7.6 0.783 NTproBNP (pg/mL) [median (IQR)]e 178.0 (91.9–718.6) 556.9 (159.6–1687.0) 929.1 (383.0–2882.0) 0.022 Congenital Anomaly [n (%)] 0.001 ASD (n = 523) 207 (80.2) 252 (91.0) 64 (95.5) VSD (n = 41) 29 (11.2) 9 (3.2) 3 (4.5) PDA (n = 31) 21 (8.1) 10 (3.6) 0 (0) Others (n = 7) 1 (0.5) 6 (2.2) 0 (0) Echocardiography parameters mPAP (mmHg) [mean ± SD]f 32.6 ± 14.7 37.3 ± 15.2 43.5 ± 15.0 < 0.001 TVRG (mmHg) [mean ± SD]g 50.7 ± 32.7 66.5 ± 34.1 80.8 ± 35.4 < 0.001 RA diameter (mm) [mean ± SD]h 44.8 ± 7.5 46.6 ± 8.1 47.4 ± 8.6 0.017 RV diameter (mm) [mean ± SD]i 39.8 ± 7.7 44.4 ± 8.4 45.4 ± 7.5 < 0.001 TAPSE (mm) [mean ± SD]j 25.4 ± 5.3 25.2 ± 5.8 22.7 ± 5.4 0.002 Left ventricle EF (%)[mean ± SD]k 68.9 ± 7.4 69.7 ± 9.1 66.1 ± 11.6 0.011 RHC parameters mPAP (mmHg) [median (IQR)]l 28.0 (20.3–46.0) 40.0 (26.0–58.0) 52.5 (32.8–68.5) < 0.001 PVRi (Wood Unit.m2) [median (IQR)]m 2.2 (1.4–5.3) 3.8 (1.5–13.5) 10.2 (2.1–21.6) < 0.001 Flow Ratio [median (IQR)]n 2.5 (1.7–3.6) 2.4 (1.5–3.4) 1.8 (1.0–3.1) 0.115 a data of 488 patients;b data of 442 patients; c data of 439 patients;d data of 444 patients;e data of 294 patients;f data of 504 patients;g data of 519 patients; h data of 542 patients; i data of 566 patients; j data of 572 patients; k data of 581 patients; l data of 582 patients; m data of 515 patients;n data of 530 patients; WHO world health organisation, PAH pulmonary artery hypertension, ASD atrial septal defect, VSD ventricular septal defect, PDA patent ductus arteriosus, AP aortopulmonal, AVSD atrioventricular septal defects, mPAP mean pulmonary artery pressure, TVRG tricuspid valve regurgitation gradient, RA right atrium, RV right ventricle, TAPSE tricuspid annular plane systolic excursion, EF ejection fraction, mPAP mean pulmonary artery pressure, PVRi pulmonary vascular resistance index a data of 488 patients;b data of 442 patients; c data of 439 patients;d data of 444 patients;e data of 294 patients;f data of 504 patients;g data of 519 patients; h data of 542 patients; i data of 566 patients; j data of 572 patients; k data of 581 patients; l data of 582 patients; m data of 515 patients;n data of 530 patients; WHO world health organisation, PAH pulmonary artery hypertension, ASD atrial septal defect, VSD ventricular septal defect, PDA patent ductus arteriosus, AP aortopulmonal, AVSD atrioventricular septal defects, mPAP mean pulmonary artery pressure, TVRG tricuspid valve regurgitation gradient, RA right atrium, RV right ventricle, TAPSE tricuspid annular plane systolic excursion, EF ejection fraction, mPAP mean pulmonary artery pressure, PVRi pulmonary vascular resistance index In our registry, the majority of CHD is ASD. Discussion This is similar with another study in an adult registry, that showed ASD is the most common [1]. Most patients come to our medical facility when they already had a complaint, with shortness of breath and easily fatigued among the most common complaints. These symptoms are similar to previous studies where the patients are already in an advanced condition and had limited activ- ity [13, 15]. Since the most prevalent CHD in our regis- try is ASD, the patients remained asymptomatic for decades. Therefore, the symptoms that appear later will urge the patients to consult doctor and visit hospital. The main symptoms of patients were associated with the development of PAH, which was later confirmed by RHC procedure. We excluded patients with complex adult CHDs from the analysis because most of them were already diagnosed in childhood and the proportion comprised of the complete diagnostic work-up. The COHARD-PH registry is a single-center registry in Dr. Sardjito Hospital, a national referral center for cardiovascular disease in Indonesia. The COHARD- PH registry was started in July 2012 and continues until the present day. Within the duration of 7 years, July 2012 – July 2019, the number of patients en- rolled were 1012 adults with CHD. The majority of CHD in this registry are ASD, followed by VSD and PDA. Young adult females (ages between 21 and 40 years old) are predominant in our registry. Most pa- tients are symptomatic, with the majority in WHO functional class II. By echocardiography, the preva- lence of increased probability of PH is 77.1%.With the further confirmation by RHC measurement, the COHARD-PH registry shows that 66.9% subjects had developed PAH. Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 Page 9 of 11 Fig. 4 The PAH development associated with defect diameters in ASD. Minimum defect (2.3 ± 0.8 vs. 1.9 ± 1.5 cm, p < 0.001) and maximum defect (2.6 ± 0.9 vs. 2.2 ± 1.8 cm, p = 0.001) between ASD patients with PAH (n = 341) and those without PAH (n = 157) Fig. 4 The PAH development associated with defect diameters in ASD. Minimum defect (2.3 ± 0.8 vs. 1.9 ± 1.5 cm, p < 0.001) and maximum defect (2.6 ± 0.9 vs. 2.2 ± 1.8 cm, p = 0.001) between ASD patients with PAH (n = 341) and those without PAH (n = 157) ig. Discussion 4 The PAH development associated with defect diameters in ASD. Minimum defect (2.3 ± 0.8 vs. 1.9 ± 1.5 cm, p < 0.00 efect (2.6 ± 0.9 vs. 2.2 ± 1.8 cm, p = 0.001) between ASD patients with PAH (n = 341) and those without PAH (n = 157) of PH and PAH cannot accurately be determined. Fur- thermore, complex adult CHDs have distinct character- istics in association with development of PH and PAH. of our patients were untreated cases, and probably un- detected cases in their childhood period. They presented to our hospital late in their delayed and progressed diseases. The early finding of CHD-related PAH is often not easy to recognize due to the unknown precise period of PAH [11]. The chronic systemic-to-pulmonary shunt is a congenital malformation causing blood overflow in the pulmonary vasculature from infancy, and if left un- treated may give rise to PAH in adult life. The majority Our registry shows that patients are predominantly young adult females. This observation is consistent with other registries [3, 6, 7, 13, 16–18]. The mean age of pa- tients in our registry during first enrollment is 34.7 years old. The fact is that most cases are ASDs, in which at Fig. 5 The PAH development did not associate with shunt flow in ASD. The Qp:Qs ratio by TTE (3.6 ± 2.2 vs. 3.3 ± 2.2, p = 0.118) between ASD patients with PAH (n = 306) and those without PAH (n = 138) and flow ratio by RHC (2.6 ± 1. 3vs. 2.8 ± 1.4, p = 0.080), between ASD patients with PAH (n = 326) and those without PAH (n = 137) Fig. 5 The PAH development did not associate with shunt flow in ASD. The Qp:Qs ratio by TTE (3.6 ± 2.2 vs. 3.3 ± 2.2, p = 0.118) between ASD patients with PAH (n = 306) and those without PAH (n = 138) and flow ratio by RHC (2.6 ± 1. 3vs. 2.8 ± 1.4, p = 0.080), between ASD patients with PAH (n = 326) and those without PAH (n = 137) Page 10 of 11 Dinarti et al. Discussion BMC Cardiovascular Disorders (2020) 20:163 Table 7 The Age Distribution Based of CHD-associated PAH Age (years) Congenital heart diseases (n (%)) PAH (n (%)) 17–20 72 (11.7) 41 (56.9) 21–30 194 (31.6) 121 (62.4) 31–40 156 (25.4) 101 (64.7) 41–50 108 (17.6) 80 (74.1) 51–60 61 (9.9) 51 (83.6) > 61 23 (3.7) 17 (73.9) Total 614 411 CHD congenital heart disease, PAH pulmonary artery hypertension patients in which PAH develops or persists after closure of the defect [17, 18]. The large defects with prevalent systemic-to-pulmonary shunts have better survival as compared with other clinical types [12, 17]. Limitation The COHARD-PH registry is a hospital-based registry therefore patients who enrolled were those who devel- oped symptoms. Although they cannot represent the en- tire population in the community, the registry reflects real world conditions of earlier undetected congenital heart disease in Indonesia. The procedure for hemodynamic evaluation, i.e. RHC, was limited by hos- pital standard procedures and timeframe, therefore not all patients in the registry underwent complete RHC. However, the fact that the majority of patients had undergone RHC is an accomplishment of this registry. younger age there had been no complaints. In ASD clin- ical presentation, the pulmonary hypercirculation and right heart volume overload induce PAH after a longer period of time, which is different from VSD or PDA [6]. The patients with VSD and PDA are symptomatic in earlier years of age and have more evident signs, prob- ably before reaching adulthood, therefore they are mostly detected in childhood and adolescent period [12]. Our hospital registry in pediatric patients indicated that VSD has the most prevalence in childhood [19]. More- over, 72.7% of patients are asymptomatic for a long period of time (> 2 decades). Mostly in the third decades of life, the PAH complications start to clinically manifest and urge the patients to visit the hospital. Abbreviations l AP: Aortopulmonary; ASD: Atrial septal defect; CHD: Congenital heart disease; COHARD-PH: The COngenital HeARt Disease in adult and Pulmonary Hypertension; DCSA: Doubly-committed subarterial; ECG: Electrocardiography; IQR: Interquartile range; mLAP: Mean left atrial pressure; mPAP: Mean pulmonary artery pressures; mRAP: Mean right atrial pressure; PDA: Patent ductus arteriosus; PFO: Patent foramen ovale; PH: Pulmonary hypertension; PVR: Pulmonary vascular resistance; PVRi: Pulmonary vascular resistance index; RA: Right atrial; RHC: Right heart catheterization; RV: Right ventricle; Qp: Pulmonary blood flow; Qs: Systemic blood flow; SD: Standard deviation; TOE: Transoesofageal echocardiography; TTE: Transthoracal echocardiography; TVRG: Tricuspid valvular regurgitation gradient; VSD: Ventricle septal defect; WU: Wood units Acknowledgements A h k l d d Authors acknowledged the research assistants who support and maintain the COHARD-PH registry database: Arina Prihesti MD, Theresia Dwiamelia MD, Athanasius Wrin Hudoyo MD, Aristida Cahyono MD, Reza Pandu Aji MD, Monika Setiawan MD, Zaki Horison Islami MD, Vera Dewanto MD, Dimas Setiadji MD, and Armalya Pritazahra MD. Authors thank the echo-lab sonographers and cath-lab nurses and radiographers who assist the acquisiton of COHARD-PH data. Authors express gratitude to Professor Noriaki Emoto MD, PhD from Kobe University/Kobe Pharmaceutical University, Japan for his mentorship during the Bilateral Exchange Program Japan JSPS/Indonesia DGHE Joint Research in 2014-2017 which enabled further collaboration in CHD-related PH research. Authors acknowledged the English Language Editing Center (Klinik Bahasa) in Faculty of Medicine, Public Health and Nursing Universitas Gadjah Mada) that provided assistance of English languange usage in the manuscript. The patients with PAH have worse clinical characteris- tics as compared to those without PAH. They are in young adult age and older than patients without PAH. Their functional capacities, measured by WHO class, 6- min walking distance and peripheral oxygen saturation, are worse. The NTproBNP levels, the sole biomarker for prognostication of PH, are higher in patients with PAH as compared with those without. Among CHD-associated PAH, it should be noted that four different clinical sub- groups have been proposed which reflect different patho- physiology and prognosis [4, 12, 17]. Patients with small defect-associated PAH had similar outcome with Eisen- menger syndrome which was better than the outcome of Conclusions The COHARD-PH registry is the first reported Indonesian adult CHD and CHD-related PH registry. The demograph- ics data, clinical presentation, and hemodynamics data of this hospital-based registry are indicative of the real world situation in developing countries which needs to be com- pared with other CHD-related PH and PH registries, both from developed and other developing countries. g The echocardiographic data showed that the majority of the patients are categorized in increased probability of PH and confirmed PAH by RHC, which is a gold stand- ard for diagnosis of PH and PAH. Almost 70% of our pa- tients have already developed PAH based on RHC. These data are much higher than data from other regis- tries, especially registries from developed countries. The striking difference is likely due to late presentation and selection bias, because the patients are enrolled at our hospital mostly due to signs and symptoms they suffer. Currently, in Indonesia there is no screening and early detection of asymptomatic CHD, therefore many pa- tients are undetected until they come to visit medical fa- cilities due to complications. Funding h 12. Vijarnsorn C, Durongpisitkul K, Chungsomprasong P, Bositthipichet D, Ketsara S, Titaram Y, et al. Contemporary survival of patients with pulmonary arterial hypertension and congenital systemic to pulmonary shunts. PLoS OnePLoS One. 2018;13:e0195092. The COHARD-PH registry received fundings from: (1) Dana Masyarakat Faculty of Medicine, Public Health and Nursing UGM fiscal years 2013–2018 (for Principal Investigator: Lucia Kris Dinarti), (2) Hospital Research Fund from Dr. Sardjito Hospital fiscal year 2016, 2017, 2018 (for Principal Investigator: Lucia Kris Dinarti), (3) UGM Research Grant PKLN 2018 (1666/UN1/DITLIT/DIT- LIT/LT/2018) (for Principal Investigator: Anggoro Budi Hartopo), and (4) UGM Penelitian Dasar Unggulan Perguruan Tinggi 2018 (53/UN1/DITLIT/DIT-LIT/LT/ 2018) and 2019 (2609/UN1.DITLIT/DIT-LIT/LT/2019) (Principal Investigator: Lucia Kris Dinarti). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. 13. Kaymaz C, Mutlu B, Küçükoğlu MS, Kaya B, Akdeniz B, Kılıçkıran Avcı B, et al. Preliminary results from a nationwide adult cardiology perspective for pulmonary hypertension: RegiStry on clInical outcoMe and sUrvival in pulmonaRy hypertension groups (SIMURG). Anatol J Cardiol. 2017;18:242–50. 13. Kaymaz C, Mutlu B, Küçükoğlu MS, Kaya B, Akdeniz B, Kılıçkıran Avcı B, et al. Preliminary results from a nationwide adult cardiology perspective for pulmonary hypertension: RegiStry on clInical outcoMe and sUrvival in pulmonaRy hypertension groups (SIMURG). Anatol J Cardiol. 2017;18:242–50. 14. Dinarti LK, Hartopo AB, Anggrahini DW, Sadewa AH, Setianto BY, Wahab AS. Profile of endothelin-1, nitric oxide, and prostacyclin levels in pulmonary arterial hypertension related to uncorrected atrial septal defect: result from a single center study in Indonesia. Cardiol Res Pract. 2020:1–10. https://doi. org/10.1155/2020/7526508. Availability of data and materials 15. Wasywich CA, Cicovic A, McWilliams T, Coverdale HA, Stewart C, Whyte K. Building a pulmonary vascular service: the 12-year experience and outcomes of the Auckland pulmonary arterial hypertension clinic. Intern Med JIntern Med J. 2013;43:635–42. 15. Wasywich CA, Cicovic A, McWilliams T, Coverdale HA, Stewart C, Whyte K. Building a pulmonary vascular service: the 12-year experience and outcomes of the Auckland pulmonary arterial hypertension clinic. Intern Med JIntern Med J. 2013;43:635–42. The datasets used and/or analysed during the current study are de-identified and available from the corresponding author on reasonable request. 16. Humbert M, Sitbon O, Chaouat A, Bertocchi M, Habib G, Gressin V, et al. Pulmonary arterial hypertension in France: results from a national registry. Am J Respir Crit Care MedAm J Respir Crit Care Med. 2006;173:1023–30. Consent for publication N l bl 18. Alonso-Gonzalez R, Lopez-Guarch CJ, Subirana-Domenech MT, Ruíz JM, González IO, Cubero JS. Et al; REHAP investigators. Pulmonary hypertension and congenital heart disease: an insight from the REHAP National Registry. Int J CardiolInt J Cardiol. 2015;184:717–23. Authors’ contributions LKD, ABH and DWA initiated the registry, conceptualized the study outline, drafted and revised the manuscript. ADK and MGS drafted manuscript and performed statistics analysis. MRH and ADP collected data, maintained eCRF and drafted the manuscript. All authors have read and approved the final manuscript. Page 11 of 11 Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 Dinarti et al. BMC Cardiovascular Disorders (2020) 20:163 Ethics approval and consent to participate The Medical and Health Research Ethics Committee Faculty of Medicine, Public Health and Nursing Universitas Gadjah Mada approved this study. All patients provided written informed consent prior to enrollment in accordance with the 1975 Declaration of Helsinki. 17. Manes A, Palazzini M, Leci E, Bacchi Reggiani ML, Branzi A, Galiè N. 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https://openalex.org/W2004692083	https://journals.iucr.org/e/issues/2008/03/00/rk2073/rk2073.pdf	English	null	8-Hydroxy-8-phenyl-2,3,7,8-tetrahydro-6<i>H</i>-1,4-dioxino[2,3-<i>f</i>]isoindol-6-one	Acta crystallographica. Section E	2,008	cc-by	3,627	Table 1 Hydrogen-bond geometry (A˚ , ). Table 1 Hydrogen-bond geometry (A˚ , ). Received 29 December 2007; accepted 28 January 2008 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A O2—H2 O3i 0.82 1.95 2.725 (3) 158 N7—H7 O2ii 0.86 2.09 2.922 (3) 161 C5—H5 O4iii 0.93 2.52 3.404 (3) 160 C19—H19 O2 0.93 2.40 2.734 (4) 101 Symmetry codes: (i) x; y; z þ 1; (ii) x; y þ 3 2; z 1 2; (iii) x; y þ 1; z þ 1. Key indicators: single-crystal X-ray study; T = 291 K; mean (C–C) = 0.004 A˚; R factor = 0.054; wR factor = 0.147; data-to-parameter ratio = 13.8. In the title compound, C16H13NO4, the indole system is essentially planar, whereas the dioxane ring adopts a twist conformation. The molecules are linked into chains by —O— H O C— hydrogen bonds and these chains are linked into rods by means of N—H O hydrogen bonds. Exept for weak C—H O interactions between the rods, no other inter- molecular contacts of interest are present. Data collection: CAD-4 Software (Enraf–Nonius, 1989); cell reﬁnement: CAD-4 Software; data reduction: XCAD4 (Harms & Wocadlo, 1995); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: DIAMOND (Brandenburg, 2000); software used to prepare material for publication: WinGX (Farrugia, 1999). Related literature For details of the appropriate nitrile hydrolysis, see: Moorthy & Singhal (2005). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: RK2073). Experimental Crystal data C16H13NO4 Mr = 283.27 Monoclinic, P21=c a = 8.6001 (17) A˚ b = 27.005 (5) A˚ c = 5.7221 (5) A˚ = 92.602 (10) V = 1327.6 (4) A˚ 3 Brandenburg, K. (2000). DIAMOND. Release 2.1d. Crystal Impact GbR, Bonn, Germany. Enraf–Nonius (1989). CAD-4 Software. Version 5.0. Enraf–Nonius, Delft, The Netherlands. Farrugia, L. J. (1999). J. Appl. Cryst. 32, 837–838. Harms, K. & Wocadlo, S. (1995). XCAD4. University of Marburg, Germany. Moorthy, J. N. & Singhal, N. (2005). J. Org. Chem. 70, 1926–1929. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. organic compounds Z = 4 Cu K radiation = 0.85 mm1 T = 291 (2) K 0.08 0.06 0.04 mm Data collection Enraf–Nonius CAD-4 diffractometer Absorption correction: none 2892 measured reﬂections 2653 independent reﬂections 1784 reﬂections with I > 2(I) Rint = 0.025 2 standard reﬂections frequency: 120 min intensity decay: none Reﬁnement R[F 2 > 2(F 2)] = 0.054 wR(F 2) = 0.147 S = 1.05 2653 reﬂections 192 parameters H-atom parameters constrained max = 0.23 e A˚ 3 min = 0.24 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 T = 291 (2) K 0.08 0.06 0.04 mm 8-Hydroxy-8-phenyl-2,3,7,8-tetrahydro- 6H-1,4-dioxino[2,3-f]isoindol-6-one Reﬁnement R[F 2 > 2(F 2)] = 0.054 wR(F 2) = 0.147 S = 1.05 2653 reﬂections 192 parameters H-atom parameters constrained max = 0.23 e A˚ 3 min = 0.24 e A˚ 3 Viktor A. Tafeenko, Leonid A. Aslanov,* Mahmud I. Khasanov and Sergei S. Mochalov Correspondence e-mail: aslanov@struct.chem.msu.ru S2. Experimental A mixture of (1) (1 g, 0.0038 mol), concentrated sulfuric acid (1 ml) and trifluoroacetic acid (4 ml) was boiled under reflux. with stirring, for 5 h. The solution was then poured into ice-water (75 ml). The resulting white precipitate was filtered off, washed with water and recrystallized from acetone. S1. Comment To investigate mechanisms of intra- and intermolecular reactions of ortho-substituted benzenes we intended to synthesize novel ortho-acyl-substituted benzamides by hydrolysis (Moorthy & Singhal, 2005) of appropriate nitriles. In the case of hydrolysis of 7-benzoyl-2,3-dihydro-1,4-benzodioxine-6-carbonitrile (1) the compound 7-benzoyl-2,3-dihydro-1,4- benzodioxine-6-carboxamide (2) was expected to be produced (Fig. 1). Both the elemental analysis and mass spectroscopic data (M+ 283) of the compound we obtained, were in good agreement with structure (2), but 1H NMR data were not. Although 13 protons were identified in the 1H NMR spectrum, an expected signal for the NH2 group was absent. In addition, two single signals were detected in the 1H NMR spectrum, each corresponding to one proton of large difference in chemical shift (6.70 and 9.02). To determine the structure of the compound, we carried out an X-ray crystallographic analysis, which revealed that hydrolysis of (1), under the conditions specified by Moorthy & Singhal, did not produce the expected compound (2); instead the product was an isomer of compound (2), viz. 8-hydroxy-8- phenyl-2,3,7,8-tetrahydro-6H-[1,4]dioxino [2,3-f]isoindol-6-one, (3) (Fig. 1). The dihedral angle between the planes defined by the atoms C5/C9/C10/C11/C12/C13 (plane 1) and C8/N7/C6/C10/C11 (plane 2) (Fig. 2) is 1.64 (9)°. The 6-membered dioxane ring adopts a twist conformation, with atoms C3 and C2 displaced out of plane 1 by 0.375 (4) and -0.273 (3) Å, respectively, compared with displacements of -0.012 (3) and 0.010 (3) Å for O4 and O1, respectively (Fig. 2). The torsion angle O2—C8—C14—C19 has rather a small value [16.7 (3)°]. This results from the intramolecular hydrogen bond C19—H19···O2. The packing motif, as shown in Fig.3, can be described as follows: molecules are linked by hydrogen bonds in head-to-tail fashion through oxy- and keto-groups to form infinite chains. The two adjacent chains are linked by N7—H7···O2ii hydrogen bonds, forming infinite rods running along the c axis. Neighbouring rods interact via centrosymmetric C5—H5···O4iii hydrogen bonds. Symmetry codes are listed in Table 1. References Experimental Crystal data b = 27.005 (5) A˚ c = 5.7221 (5) A˚ = 92.602 (10) V = 1327.6 (4) A˚ 3 o548 o548 Tafeenko et al. Acta Cryst. (2008). E64, o548 doi:10.1107/S1600536808003012 supporting information Acta Cryst. (2008). E64, o548 [doi:10.1107/S1600536808003012] Acta Cryst. (2008). E64, o548 [doi:10.1107/S1600536808003012] Acta Cryst. (2008). E64, o548 [doi:10.1107/S1600536808003012] Acta Cryst. (2008). E64, o548 [doi:10.1107/S1600536808003012] S3. Refinement The positions of the H atoms were determined from Fourier difference maps; they were then placed in calculated positions and allowed to ride on their parent atoms [C—H = 0.93–0.97 Å, O—H = 0.82 Å and N—H = 0.86 Å]. Uiso(H) = xUeq(parent atom), where x = 1.5 for attached O and 1.2 for C and N. Acta Cryst. (2008). E64, o548 sup-1 supporting information Figure 1 Hydrolysis of (1) did not produce the expected compound, (2) but rather an isomer of (2), viz. compound (3). Figure 1 s of (1) did not produce the expected compound, (2) but rather an isomer of (2), viz. compound (3). Hydrolysis of (1) did not produce the expected compound, (2) but rather an isomer of (2), viz. compound (3). Hydrolysis of (1) did not produce the expected compound, (2) but rather an isomer of (2), viz. compound (3). sup-2 Acta Cryst. (2008). E64, o548 supporting information Figure 2 The molecular structure of the title compound, with displacement ellipsoids drawn at the 50% probability level. The Figure 2 g The molecular structure of the title compound, with displacement ellipsoids drawn at the 50% probability level. The dashed line indicates an intramolecular hydrogen bond. g The molecular structure of the title compound, with displacement ellipsoids drawn at the 50% probability level. The dashed line indicates an intramolecular hydrogen bond. g The molecular structure of the title compound, with displacement ellipsoids drawn at the 50% probability level. The dashed line indicates an intramolecular hydrogen bond. Figure 3 The packing motif of the crystal structure. Hydrogen atoms not involved in hydrogen bonds (dashed lines) have been omitted for clarity. Symmetry codes: (i) x, y, z + 1; (ii) x, -y + 3/2, z - 1/2; (iii) -x, -y + 1, -z + 1. Figure 3 E64, o548 sup-3 supporting information 8-Hydroxy-8-phenyl-2,3,7,8-tetrahydro-6H Crystal data C16H13NO4 Mr = 283.27 Monoclinic, P21/c Hall symbol: -P 2ybc a = 8.6001 (17) Å b = 27.005 (5) Å c = 5.7221 (5) Å β = 92.602 (10)° V = 1327.6 (4) Å3 Z = 4 Data collection Enraf–Nonius CAD-4 diffractometer Radiation source: fine-focus sealed tube Graphite monochromator Non–profiled ω scans 2892 measured reflections 2653 independent reflections 1784 reflections with I > 2σ(I) Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.054 wR(F2) = 0.147 S = 1.05 2653 reflections 192 parameters 0 restraints Primary atom site location: structure-invarian direct methods Secondary atom site location: difference Four map 8-Hydroxy-8-phenyl-2,3,7,8-tetrahydro-6H-1,4-dioxino[2,3-f]isoindol-6-one Crystal data C16H13NO4 Mr = 283.27 Monoclinic, P21/c Hall symbol: -P 2ybc a = 8.6001 (17) Å b = 27.005 (5) Å c = 5.7221 (5) Å β = 92.602 (10)° V = 1327.6 (4) Å3 Z = 4 F(000) = 592 Dx = 1.417 Mg m−3 Melting point = 485–486 K Cu Kα radiation, λ = 1.54184 Å Cell parameters from 25 reflections θ = 26–42° µ = 0.85 mm−1 T = 291 K Prism, colorless 0.08 × 0.06 × 0.04 mm F(000) = 592 Dx = 1.417 Mg m−3 Melting point = 485–486 K Cu Kα radiation, λ = 1.54184 Å Cell parameters from 25 reflections θ = 26–42° µ = 0.85 mm−1 T = 291 K Prism, colorless 0.08 × 0.06 × 0.04 mm Rint = 0.025 θmax = 73.9°, θmin = 3.3° h = −10→10 k = 0→32 l = 0→7 2 standard reflections every 120 min intensity decay: none 2 standard reflections every 120 min intensity decay: none Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.061P)2 + 0.4104P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.23 e Å−3 Δρmin = −0.24 e Å−3 Extinction correction: SHELXL97 (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.0051 (6) Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.061P)2 + 0.4104P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.23 e Å−3 Δρmin = −0.24 e Å−3 Extinction correction: SHELXL97 (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.0051 (6) ρ Extinction correction: SHELXL97 (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.0051 (6) Special details Figure 3 g The packing motif of the crystal structure. Hydrogen atoms not involved in hydrogen bonds (dashed lines) have been omitted for clarity. Symmetry codes: (i) x, y, z + 1; (ii) x, -y + 3/2, z - 1/2; (iii) -x, -y + 1, -z + 1. g The packing motif of the crystal structure. Hydrogen atoms not involved in hydrogen bonds (dashed lines) have been omitted for clarity. Symmetry codes: (i) x, y, z + 1; (ii) x, -y + 3/2, z - 1/2; (iii) -x, -y + 1, -z + 1. The packing motif of the crystal structure. Hydrogen atoms not involved in hydrogen bonds (dashed lines) have been omitted for clarity. Symmetry codes: (i) x, y, z + 1; (ii) x, -y + 3/2, z - 1/2; (iii) -x, -y + 1, -z + 1. Acta Cryst. (2008). Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. sup-4 Acta Cryst. (2008). E64, o548 Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq O1 0.3338 (2) 0.50617 (7) 1.1457 (3) 0.0654 (5) O2 0.3093 (2) 0.70474 (6) 0.9980 (3) 0.0549 (5) onal atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) U /U sup-4 Acta Cryst. (2008). Special details E64, o548 supporting information H2 0.2370 0.6906 1.0585 0.082 O3 0.1250 (2) 0.65694 (7) 0.3005 (3) 0.0568 (5) O4 0.1351 (3) 0.48070 (7) 0.7532 (4) 0.0737 (6) N7 0.2877 (2) 0.68919 (8) 0.5922 (3) 0.0498 (5) H7 0.2972 0.7182 0.5321 0.060* C2 0.2445 (4) 0.46150 (12) 1.1374 (6) 0.0810 (10) H2A 0.1463 0.4671 1.2100 0.097* H2B 0.3003 0.4360 1.2259 0.097* C3 0.2138 (5) 0.44427 (12) 0.8942 (7) 0.0878 (11) H3A 0.3117 0.4361 0.8260 0.105* H3B 0.1510 0.4144 0.8956 0.105* C5 0.1581 (3) 0.56216 (9) 0.6119 (4) 0.0536 (6) H5 0.0937 0.5541 0.4826 0.064* C6 0.2011 (3) 0.65333 (9) 0.4881 (4) 0.0467 (6) C8 0.3642 (3) 0.67510 (9) 0.8154 (4) 0.0455 (6) C9 0.3521 (3) 0.58711 (9) 1.0021 (4) 0.0498 (6) H9 0.4163 0.5955 1.1313 0.060* C10 0.2183 (3) 0.60935 (9) 0.6404 (4) 0.0467 (6) C11 0.3138 (3) 0.62151 (9) 0.8321 (4) 0.0446 (5) C12 0.1965 (3) 0.52745 (10) 0.7811 (5) 0.0542 (6) C13 0.2925 (3) 0.53960 (9) 0.9759 (4) 0.0510 (6) C14 0.5399 (3) 0.68000 (9) 0.8127 (4) 0.0458 (6) C15 0.6197 (3) 0.66247 (10) 0.6231 (4) 0.0555 (6) H15 0.5636 0.6499 0.4937 0.067* C16 0.7790 (3) 0.66338 (11) 0.6228 (5) 0.0645 (7) H16 0.8301 0.6514 0.4946 0.077* C17 0.8631 (4) 0.68214 (12) 0.8136 (5) 0.0683 (8) H17 0.9712 0.6829 0.8143 0.082* C18 0.7876 (3) 0.69959 (11) 1.0011 (5) 0.0678 (8) H18 0.8449 0.7121 1.1297 0.081* C19 0.6268 (3) 0.69889 (10) 1.0022 (4) 0.0561 (7) H19 0.5767 0.7112 1.1307 0.067* O4 0.1351 (3) 0.48070 (7) 0.7532 (4) 0.0737 (6) N7 0.2877 (2) 0.68919 (8) 0.5922 (3) 0.0498 (5) H7 0.2972 0.7182 0.5321 0.060* C2 0.2445 (4) 0.46150 (12) 1.1374 (6) 0.0810 (10) H2A 0.1463 0.4671 1.2100 0.097* H2B 0.3003 0.4360 1.2259 0.097* C3 0.2138 (5) 0.44427 (12) 0.8942 (7) 0.0878 (11) H3A 0.3117 0.4361 0.8260 0.105* H3B 0.1510 0.4144 0.8956 0.105* C5 0.1581 (3) 0.56216 (9) 0.6119 (4) 0.0536 (6) H5 0.0937 0.5541 0.4826 0.064* C6 0.2011 (3) 0.65333 (9) 0.4881 (4) 0.0467 (6) C8 0.3642 (3) 0.67510 (9) 0.8154 (4) 0.0455 (6) C9 0.3521 (3) 0.58711 (9) 1.0021 (4) 0.0498 (6) H9 0.4163 0.5955 1.1313 0.060* C10 0.2183 (3) 0.60935 (9) 0.6404 (4) 0.0467 (6) C11 0.3138 (3) 0.62151 (9) 0.8321 (4) 0.0446 (5) C12 0.1965 (3) 0.52745 (10) 0.7811 (5) 0.0542 (6) C13 0.2925 (3) 0.53960 (9) 0.9759 (4) 0.0510 (6) C14 0.5399 (3) 0.68000 (9) 0.8127 (4) 0.0458 (6) C15 0.6197 (3) 0.66247 (10) 0.6231 (4) 0.0555 (6) H15 0.5636 0.6499 0.4937 0.067* C16 0.7790 (3) 0.66338 (11) 0.6228 (5) 0.0645 (7) H16 0.8301 0.6514 0.4946 0.077* C17 0.8631 (4) 0.68214 (12) 0.8136 (5) 0.0683 (8) H17 0.9712 0.6829 0.8143 0.082* C18 0.7876 (3) 0.69959 (11) 1.0011 (5) 0.0678 (8) H18 0.8449 0.7121 1.1297 0.081* C19 0.6268 (3) 0.69889 (10) 1.0022 (4) 0.0561 (7) H19 0.5767 0.7112 1.1307 0.067* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1 0.0765 (13) 0.0525 (11) 0.0666 (12) −0.0020 (9) −0.0039 (10) 0.0182 (9) O2 0.0654 (12) 0.0516 (10) 0.0488 (10) −0.0046 (8) 0.0138 (8) −0.0083 (8) O3 0.0575 (11) 0.0718 (12) 0.0406 (9) −0.0020 (9) −0.0038 (8) 0.0054 (8) O4 0.0847 (15) 0.0480 (11) 0.0870 (15) −0.0136 (10) −0.0131 (12) 0.0040 (10) N7 0.0603 (13) 0.0463 (11) 0.0421 (10) −0.0032 (9) −0.0049 (9) 0.0071 (9) C2 0.097 (3) 0.0529 (18) 0.093 (2) −0.0106 (16) 0.0003 (19) 0.0230 (17) C3 0.107 (3) 0.0513 (18) 0.104 (3) −0.0006 (18) −0.011 (2) 0.0065 (18) C5 0.0578 (15) 0.0547 (15) 0.0480 (13) −0.0073 (12) −0.0020 (11) −0.0051 (12) C6 0.0464 (13) 0.0567 (15) 0.0371 (11) 0.0029 (11) 0.0021 (10) 0.0008 (10) C8 0.0575 (14) 0.0446 (13) 0.0343 (11) −0.0017 (11) −0.0001 (10) −0.0005 (9) C9 0.0564 (14) 0.0511 (14) 0.0417 (12) −0.0030 (11) −0.0005 (10) 0.0034 (11) C10 0.0512 (13) 0.0500 (14) 0.0388 (11) −0.0035 (11) 0.0017 (10) −0.0004 (10) sup-5 Acta Cryst. Special details E64, o548 supporting information C11 0.0505 (13) 0.0459 (13) 0.0373 (12) −0.0014 (10) 0.0022 (10) 0.0010 (10) C12 0.0608 (16) 0.0454 (14) 0.0565 (15) −0.0078 (12) 0.0027 (12) −0.0041 (12) C13 0.0556 (14) 0.0469 (14) 0.0506 (13) −0.0001 (11) 0.0042 (11) 0.0074 (11) C14 0.0567 (14) 0.0436 (13) 0.0370 (11) −0.0025 (11) 0.0011 (10) 0.0021 (10) C15 0.0610 (16) 0.0649 (17) 0.0407 (12) −0.0025 (13) 0.0027 (11) −0.0039 (12) C16 0.0606 (16) 0.074 (2) 0.0594 (17) 0.0007 (14) 0.0109 (13) −0.0030 (15) C17 0.0570 (16) 0.076 (2) 0.0716 (19) −0.0002 (14) 0.0011 (14) 0.0022 (16) C18 0.0635 (18) 0.076 (2) 0.0624 (17) −0.0039 (15) −0.0152 (14) −0.0050 (15) C19 0.0672 (17) 0.0599 (16) 0.0404 (12) 0.0001 (13) −0.0040 (11) −0.0032 (11) Geometric parameters (Å, º) O1—C13 1.362 (3) C8—C11 1.515 (3) O1—C2 1.430 (4) C8—C14 1.518 (3) O2—C8 1.415 (3) C9—C11 1.374 (3) O2—H2 0.8200 C9—C13 1.387 (3) O3—C6 1.236 (3) C9—H9 0.9300 O4—C12 1.375 (3) C10—C11 1.380 (3) O4—C3 1.424 (4) C12—C13 1.396 (3) N7—C6 1.344 (3) C14—C19 1.386 (3) N7—C8 1.460 (3) C14—C15 1.393 (3) N7—H7 0.8600 C15—C16 1.370 (4) C2—C3 1.480 (5) C15—H15 0.9300 C2—H2A 0.9700 C16—C17 1.378 (4) C2—H2B 0.9700 C16—H16 0.9300 C3—H3A 0.9700 C17—C18 1.363 (4) C3—H3B 0.9700 C17—H17 0.9300 C5—C12 1.376 (4) C18—C19 1.384 (4) C5—C10 1.382 (3) C18—H18 0.9300 C5—H5 0.9300 C19—H19 0.9300 C6—C10 1.477 (3) C13—O1—C2 114.4 (2) C13—C9—H9 120.9 C8—O2—H2 109.5 C11—C10—C5 121.3 (2) C12—O4—C3 113.5 (2) C11—C10—C6 108.5 (2) C6—N7—C8 114.7 (2) C5—C10—C6 130.2 (2) C6—N7—H7 122.6 C9—C11—C10 121.1 (2) C8—N7—H7 122.6 C9—C11—C8 129.1 (2) O1—C2—C3 111.7 (3) C10—C11—C8 109.8 (2) O1—C2—H2A 109.3 O4—C12—C5 117.7 (2) C3—C2—H2A 109.3 O4—C12—C13 121.2 (2) O1—C2—H2B 109.3 C5—C12—C13 121.0 (2) C3—C2—H2B 109.3 O1—C13—C9 116.9 (2) H2A—C2—H2B 107.9 O1—C13—C12 122.7 (2) O4—C3—C2 112.1 (3) C9—C13—C12 120.4 (2) O4—C3—H3A 109.2 C19—C14—C15 117.8 (2) C2—C3—H3A 109.2 C19—C14—C8 121.7 (2) O4—C3—H3B 109.2 C15—C14—C8 120.3 (2) supporting information C11 0.0505 (13) 0.0459 (13) 0.0373 (12) −0.0014 (10) 0.0022 (10) 0.0010 (10) C12 0.0608 (16) 0.0454 (14) 0.0565 (15) −0.0078 (12) 0.0027 (12) −0.0041 (12) C13 0.0556 (14) 0.0469 (14) 0.0506 (13) −0.0001 (11) 0.0042 (11) 0.0074 (11) C14 0.0567 (14) 0.0436 (13) 0.0370 (11) −0.0025 (11) 0.0011 (10) 0.0021 (10) C15 0.0610 (16) 0.0649 (17) 0.0407 (12) −0.0025 (13) 0.0027 (11) −0.0039 (12) C16 0.0606 (16) 0.074 (2) 0.0594 (17) 0.0007 (14) 0.0109 (13) −0.0030 (15) C17 0.0570 (16) 0.076 (2) 0.0716 (19) −0.0002 (14) 0.0011 (14) 0.0022 (16) C18 0.0635 (18) 0.076 (2) 0.0624 (17) −0.0039 (15) −0.0152 (14) −0.0050 (15) C19 0.0672 (17) 0.0599 (16) 0.0404 (12) 0.0001 (13) −0.0040 (11) −0.0032 (11) sup-6 Acta Cryst. Special details (2008). Special details (2008). E64, o548 supporting information pp g C2—C3—H3B 109.2 C16—C15—C14 121.5 (3) H3A—C3—H3B 107.9 C16—C15—H15 119.2 C12—C5—C10 117.9 (2) C14—C15—H15 119.2 C12—C5—H5 121.0 C15—C16—C17 119.6 (3) C10—C5—H5 121.0 C15—C16—H16 120.2 O3—C6—N7 126.1 (2) C17—C16—H16 120.2 O3—C6—C10 127.7 (2) C18—C17—C16 119.9 (3) N7—C6—C10 106.23 (19) C18—C17—H17 120.0 O2—C8—N7 110.3 (2) C16—C17—H17 120.0 O2—C8—C11 112.82 (19) C17—C18—C19 120.7 (3) N7—C8—C11 100.69 (18) C17—C18—H18 119.7 O2—C8—C14 108.89 (19) C19—C18—H18 119.7 N7—C8—C14 112.20 (19) C18—C19—C14 120.4 (3) C11—C8—C14 111.8 (2) C18—C19—H19 119.8 C11—C9—C13 118.2 (2) C14—C19—H19 119.8 C11—C9—H9 120.9 C13—O1—C2—C3 40.4 (4) C3—O4—C12—C5 162.9 (3) C12—O4—C3—C2 45.2 (4) C3—O4—C12—C13 −18.1 (4) O1—C2—C3—O4 −57.6 (4) C10—C5—C12—O4 179.4 (2) C8—N7—C6—O3 179.2 (2) C10—C5—C12—C13 0.3 (4) C8—N7—C6—C10 −1.7 (3) C2—O1—C13—C9 167.3 (3) C6—N7—C8—O2 −117.5 (2) C2—O1—C13—C12 −13.5 (4) C6—N7—C8—C11 1.8 (3) C11—C9—C13—O1 179.3 (2) C6—N7—C8—C14 120.9 (2) C11—C9—C13—C12 0.1 (4) C12—C5—C10—C11 −0.1 (4) O4—C12—C13—O1 1.5 (4) C12—C5—C10—C6 178.4 (3) C5—C12—C13—O1 −179.5 (2) O3—C6—C10—C11 179.9 (2) O4—C12—C13—C9 −179.3 (2) N7—C6—C10—C11 0.8 (3) C5—C12—C13—C9 −0.4 (4) O3—C6—C10—C5 1.3 (4) O2—C8—C14—C19 16.7 (3) N7—C6—C10—C5 −177.8 (3) N7—C8—C14—C19 139.1 (2) C13—C9—C11—C10 0.2 (4) C11—C8—C14—C19 −108.6 (3) C13—C9—C11—C8 −178.9 (2) O2—C8—C14—C15 −167.4 (2) C5—C10—C11—C9 −0.2 (4) N7—C8—C14—C15 −45.1 (3) C6—C10—C11—C9 −178.9 (2) C11—C8—C14—C15 67.2 (3) C5—C10—C11—C8 179.0 (2) C19—C14—C15—C16 0.5 (4) C6—C10—C11—C8 0.3 (3) C8—C14—C15—C16 −175.6 (2) O2—C8—C11—C9 −64.5 (3) C14—C15—C16—C17 −0.2 (4) N7—C8—C11—C9 178.0 (2) C15—C16—C17—C18 0.1 (5) C14—C8—C11—C9 58.6 (3) C16—C17—C18—C19 −0.3 (5) O2—C8—C11—C10 116.3 (2) C17—C18—C19—C14 0.5 (5) N7—C8—C11—C10 −1.2 (3) C15—C14—C19—C18 −0.6 (4) C14—C8—C11—C10 −120.5 (2) C8—C14—C19—C18 175.3 (2) Hydrogen-bond geometry (Å, º) D H···A D H H···A D···A D H···A sup-7 Acta Cryst. (2008). Symmetry codes: (i) x, y, z+1; (ii) x, −y+3/2, z−1/2; (iii) −x, −y+1, −z+1. ry codes: (i) x, y, z+1; (ii) x, −y+3/2, z−1/2; (iii) −x, −y+1, −z+1. Special details E64, o548 N7—C8—C14 112.20 (19) C18—C19—C14 120.4 (3) C11—C8—C14 111.8 (2) C18—C19—H19 119.8 C11—C9—C13 118.2 (2) C14—C19—H19 119.8 C11—C9—H9 120.9 C13—O1—C2—C3 40.4 (4) C3—O4—C12—C5 162.9 (3) C12—O4—C3—C2 45.2 (4) C3—O4—C12—C13 −18.1 (4) O1—C2—C3—O4 −57.6 (4) C10—C5—C12—O4 179.4 (2) C8—N7—C6—O3 179.2 (2) C10—C5—C12—C13 0.3 (4) C8—N7—C6—C10 −1.7 (3) C2—O1—C13—C9 167.3 (3) C6—N7—C8—O2 −117.5 (2) C2—O1—C13—C12 −13.5 (4) C6—N7—C8—C11 1.8 (3) C11—C9—C13—O1 179.3 (2) C6—N7—C8—C14 120.9 (2) C11—C9—C13—C12 0.1 (4) C12—C5—C10—C11 −0.1 (4) O4—C12—C13—O1 1.5 (4) C12—C5—C10—C6 178.4 (3) C5—C12—C13—O1 −179.5 (2) O3—C6—C10—C11 179.9 (2) O4—C12—C13—C9 −179.3 (2) N7—C6—C10—C11 0.8 (3) C5—C12—C13—C9 −0.4 (4) O3—C6—C10—C5 1.3 (4) O2—C8—C14—C19 16.7 (3) N7—C6—C10—C5 −177.8 (3) N7—C8—C14—C19 139.1 (2) C13—C9—C11—C10 0.2 (4) C11—C8—C14—C19 −108.6 (3) C13—C9—C11—C8 −178.9 (2) O2—C8—C14—C15 −167.4 (2) C5—C10—C11—C9 −0.2 (4) N7—C8—C14—C15 −45.1 (3) C6—C10—C11—C9 −178.9 (2) C11—C8—C14—C15 67.2 (3) C5—C10—C11—C8 179.0 (2) C19—C14—C15—C16 0.5 (4) C6—C10—C11—C8 0.3 (3) C8—C14—C15—C16 −175.6 (2) O2—C8—C11—C9 −64.5 (3) C14—C15—C16—C17 −0.2 (4) N7—C8—C11—C9 178.0 (2) C15—C16—C17—C18 0.1 (5) C14—C8—C11—C9 58.6 (3) C16—C17—C18—C19 −0.3 (5) O2—C8—C11—C10 116.3 (2) C17—C18—C19—C14 0.5 (5) N7—C8—C11—C10 −1.2 (3) C15—C14—C19—C18 −0.6 (4) C14—C8—C11—C10 −120.5 (2) C8—C14—C19—C18 175.3 (2) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A O2—H2···O3i 0.82 1.95 2.725 (3) 158 sup-7 Acta Cryst. (2008). E64, o548 Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A O2—H2···O3i 0.82 1.95 2.725 (3) 158 sup-7 Acta Cryst. (2008). E64, o548 supporting information N7—H7···O2ii 0.86 2.09 2.922 (3) 161 C5—H5···O4iii 0.93 2.52 3.404 (3) 160 C19—H19···O2 0.93 2.40 2.734 (4) 101 Symmetry codes: (i) x, y, z+1; (ii) x, −y+3/2, z−1/2; (iii) −x, −y+1, −z+1. sup-8 Acta Cryst. (2008). E64, o548
https://openalex.org/W4255098484	https://europepmc.org/articles/pmc2969932?pdf=render	Latin	null	4-[(4-Fluorobenzylidene)amino]-3-[1-(4-isobutylphenyl)ethyl]-1<i>H</i>-1,2,4-triazole-5(4<i>H</i>)-thione	Acta crystallographica. Section E	2,009	cc-by	3,771	organic compounds organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Hoong-Kun Fun,a‡ Wan-Sin Loh,a A. C. Vinayakab and B. Kallurayab Cg1 is the centroid of the C11– C16 i Experimental aX-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia, and bDepartment of Studies in Chemistry, Mangalore University, Mangalagangotri, Mangalore 574 199, India Correspondence e-mail: hkfun@usm.my aX-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia, and bDepartment of Studies in Chemistry, Mangalore University, Mangalagangotri, Mangalore 574 199, India Correspondence e-mail: hkfun@usm.my Experimental Crystal data C21H23FN4S Mr = 382.49 Triclinic, P1 a = 5.7883 (1) A˚ b = 9.9001 (1) A˚ c = 18.4972 (3) A˚ = 98.132 (1) = 97.087 (1) = 105.997 (1) V = 993.90 (3) A˚ 3 Z = 2 Mo K radiation = 0.19 mm1 T = 100 K 0.46 0.20 0.07 mm Data collection Bruker SMART APEXII CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2005) Tmin = 0.919, Tmax = 0.987 31031 measured reﬂections 7460 independent reﬂections 5798 reﬂections with I > 2(I) Rint = 0.037 Reﬁnement R[F 2 > 2(F 2)] = 0.048 wR(F 2) = 0.135 S = 1.06 7460 reﬂections 336 parameters All H-atom parameters reﬁned max = 0.63 e A˚ 3 min = 0.29 e A˚ 3 Received 28 July 2009; accepted 30 July 2009 Key indicators: single-crystal X-ray study; T = 100 K; mean (C–C) = 0.002 A˚; R factor = 0.048; wR factor = 0.135; data-to-parameter ratio = 22.2. Data collection Bruker SMART APEXII CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2005) Tmin = 0.919, Tmax = 0.987 31031 measured reﬂections 7460 independent reﬂections 5798 reﬂections with I > 2(I) Rint = 0.037 Reﬁnement R[F 2 > 2(F 2)] = 0.048 wR(F 2) = 0.135 S = 1.06 7460 reﬂections 336 parameters All H-atom parameters reﬁned max = 0.63 e A˚ 3 min = 0.29 e A˚ 3 In the title compound, C21H23FN4S, the benzene rings of the isobutylphenyl and ﬂuorobenzene units form dihedral angles of 75.89 (7) and 13.26 (7), respectively, with the triazole ring. An intramolecular C—H S hydrogen-bonding contact generates an S(6) ring motif. In the crystal packing, pairs of N—H S hydrogen bonds link neighbouring molecules into inversion dimers, forming R2 2(8) ring motifs. The crystal structure is further stabilized by C—H interactions. ‡ Thomson Reuters ResearcherID: A-3561-2009. Hoong-Kun Fun,a‡ Wan-Sin Loh,a A. C. Vinayakab and B. Kallurayab Hoong-Kun Fun,a‡ Wan-Sin Loh,a A. C. Vinayakab and B. Kallurayab Experimental Crystal data C21H23FN4S Mr = 382.49 Triclinic, P1 a = 5.7883 (1) A˚ b = 9.9001 (1) A˚ c = 18.4972 (3) A˚ = 98.132 (1) = 97.087 (1) = 105.997 (1) V = 993.90 (3) A˚ 3 Z = 2 Mo K radiation = 0.19 mm1 T = 100 K 0.46 0.20 0.07 mm Data collection Bruker SMART APEXII CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 2005) Tmin = 0.919, Tmax = 0.987 31031 measured reﬂections 7460 independent reﬂections 5798 reﬂections with I > 2(I) Rint = 0.037 Reﬁnement R[F 2 > 2(F 2)] = 0.048 wR(F 2) = 0.135 S = 1.06 7460 reﬂections 336 parameters All H-atom parameters reﬁned max = 0.63 e A˚ 3 min = 0.29 e A˚ 3 Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N3—H1N3 S1i 0.85 (2) 2.43 (2) 3.2763 (12) 172.3 (18) C7—H7A S1 0.96 (2) 2.50 (2) 3.2415 (13) 133.2 (16) C4—H4A Cg1ii 1.01 (2) 2.85 (2) 3.6276 (16) 133.8 (17) Symmetry codes: (i) x 1; y þ 1; z; (ii) x; y 1; z. Fun, H.-K., Jebas, S. R., Razak, I. A., Sujith, K. V., Patil, P. S., Kalluraya, B. & Dharmaprakash, S. M. (2008). Acta Cryst. E64, o1076–o1077. Fun, H.-K., Jebas, S. R., Sujith, K. V. & Kalluraya, B. (2009a). Acta Cryst. E65, o1149–o1150. Fun, H.-K., Jebas, S. R., Sujith, K. V. & Kalluraya, B. (2009b). Acta Cryst. E65, o1242–o1243. Go¨knur, A., Birsen, T. & Mevlu¨t, E. (2005). Arch. Pharm. Res. 28, 438–442. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Related literature For pharmacological activity of triazoles, see: Go¨knur et al. (2005). For the anti-tumor activity of triazole derivatives, see: Demirbas et al. (2002, 2004). For the synthesis of related heterocyclic compounds, see: Fun et al. (2008, 2009a). For a related structure, see: Fun et al. (2009b). For hydrogen-bond motifs, see: Bernstein et al. (1995). For the stability of the temperature controller used for the data collection, see: Cosier & Glazer (1986). Table 1 Hydrogen-bond geometry (A˚ , ). Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N3—H1N3 S1i 0.85 (2) 2.43 (2) 3.2763 (12) 172.3 (18) C7—H7A S1 0.96 (2) 2.50 (2) 3.2415 (13) 133.2 (16) C4—H4A Cg1ii 1.01 (2) 2.85 (2) 3.6276 (16) 133.8 (17) Symmetry codes: (i) x 1; y þ 1; z; (ii) x; y 1; z. Cg1 is the centroid of the C11– C16 ring. Data collection: APEX2 (Bruker, 2005); cell reﬁnement: SAINT (Bruker, 2005); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL and PLATON (Spek, 2009). HKFand WSL thank Universiti Sains Malaysia (USM) for a Research University Golden Goose Grant (1001/PFIZIK/ 811012). WSL thanks USM for a student assistantship. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: TK2518). Fun et al. o2079 Fun et al. o2079 Acta Cryst. (2009). E65, o2079–o2080 doi:10.1107/S160053680903030X Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. Bruker (2005). APEX2, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Cosier, J. & Glazer, A. M. (1986). J. Appl. Cryst. 19, 105–107. Demirbas, N., Alpay-Karaog˘lu, S., Demirbas, A. & Sancak, K. (2004). Eur. J. Med. Chem. 39, 793–804. Demirbas, N., Ug˘urluog˘lu, R. & Demirbasx, A. (2002). Bioorg. Med. Chem. 10, 3717–3723. Acta Cryst. (2009). E65, o2079–o2080 Fun, H.-K., Jebas, S. R., Razak, I. A., Sujith, K. V., Patil, P. S., Kalluraya, B. & Dharmaprakash, S. M. (2008). Acta Cryst. E64, o1076–o1077. Fun, H.-K., Jebas, S. R., Sujith, K. V. & Kalluraya, B. (2009b). Acta Cryst. E65, o1242–o1243. ( ) Fun, H.-K., Jebas, S. R., Sujith, K. V. & Kalluraya, B. (2009a). Acta Cryst. E65, o1149–o1150. Demirbas, N., Ug˘urluog˘lu, R. & Demirbasx, A. (2002). Bioorg. Med. Chem. 10, 3717–3723. Go¨knur, A., Birsen, T. & Mevlu¨t, E. (2005). Arch. Pharm. Res. 28, 438–442. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Demirbas, N., Alpay-Karaog˘lu, S., Demirbas, A. & Sancak, K. (2004). Eur. J. Med. Chem. 39, 793–804. References Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. g Bruker (2005). APEX2, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. Cosier, J. & Glazer, A. M. (1986). J. Appl. Cryst. 19, 105–107. Demirbas, N., Alpay-Karaog˘lu, S., Demirbas, A. & Sancak, K. (2004). Eur. J. Med. Chem. 39, 793–804. Demirbas, N., Ug˘urluog˘lu, R. & Demirbasx, A. (2002). Bioorg. Med. Chem. 10, 3717–3723. o2080 Fun et al. C21H23FN4S Acta Cryst. (2009). E65, o2079–o2080 4-[(4-Fluorobenzylidene)amino]-3-[1-(4-isobutylphenyl)ethyl]-1H-1,2,4-triazole-5(4H)-thione H.-K. Fun, W.-S. Loh, A. C. Vinayaka and B. Kalluraya Comment 1,2,4-Triazoles and their derivatives represent a rapidly developing field in modern heterocyclic chemistry. Similarly, ibupro- fen belongs to the class of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) with diverse pharmacological activities. The analgesic, anti-asthematic, diuretic, anti-hypertensive and anti-inflammatory properties associated with these drugs have made them important chemotherapeutic agents (Göknur et al., 2005). Our earlier studies involved the synthesis of heterocyc- lic compounds incorporating ibuprofen and 1,2,4-triazole fragments in the structures (Fun et al., 2008, 2009a). Schiff base derivatives of 1,2,4-triazole are known to possess anti-tumor activity (Demirbas et al., 2004). Similarly, some Schiff base derivatives of acetic acid hydrazides containing 1,2,4-triazol-5-one ring have displayed anti-tumoral activity only against breast cancer, while 2-phenyl ethylidenamino and 2-phenyl ethylamino derivatives of 4-amino-1,2,4-triazol-5-ones have been found to be effective towards non-small cell lung cancer, cranial neural crest cancer, and breast cancer (Demirbas et al., 2002). In this connection and in continuation of our interest in the synthesis of chemically and biologically important heterocycles, we now report a substituted 1,2,4-triazole Schiff base carrying the ibuprofen moiety, (I). In (I), Fig. 1, the triazole ring (C8/C9/N2–N4) is approximately planar with a maximum deviation of 0.009 (1)° at atom N2. The dihedral angles formed by the triazole ring with C1–C6 and C11–C16 benzene rings are 13.26 (7) and 75.89 (7)°, respectively. Bond lengths and angles are comparable to a closely related structure (Fun et. al., 2009b). An intramolecular C7—H7A···S1 hydrogen bond generates an S(6) ring motif (Bernstein et al., 1995), Fig. 1. In the crystal packing (Fig. 2), pairs of N3—H1N3···S1 hydrogen bonds link molecules into dimers forming R22(8) ring motifs; these stack along the a axis. The crystal structure is further stabilized by C—H···π interactions (Table 1). Experimental Compound (I) was obtained by refluxing 4-amino-5-[1-(4-isobutylphenyl)ethyl]-4H-1,2,4-triazole-3-thiol (0.01 mol) and 4-fluorobenzaldehyde (0.01 mol) in ethanol (50 ml) with 3 drops of concentrated sulfuric acid for 6 h. The solid product obtained was collected by filtration, washed with ethanol and dried. Crystals were obtained from the slow evaporation of an ethanol solution of (I). All H atoms were located from difference Fourier maps and allowed to refine freely [N—H = 0.85 (2) Å; range of C—H = 0.91 (2) - 1.07 (2) Å]. supplementary materials supplementary materials Acta Cryst. (2009). E65, o2079-o2080 [ doi:10.1107/S160053680903030X ] Acta Cryst. (2009). E65, o2079-o2080 [ doi:10.1107/S160053680903030X ] Refinement All H atoms were located from difference Fourier maps and allowed to refine freely [N—H = 0.85 (2) Å; range of C—H = 0.91 (2) - 1.07 (2) Å]. sup-1 supplementary materials Fig. 1. The molecular structure of (I), showing 50% probability displacement ellipsoids and the atom numbering scheme. The intramolecular interaction is shown by a dashed line. Fig. 2. The crystal packing of (I), viewed down the a axis, showing the R22(8) ring motifs. In- termolecular hydrogen bonds are shown by dashed lines. 4-[(4-Fluorobenzylidene)amino]-3-[1-(4-isobutylphenyl)ethyl]-1H- 1,2,4-triazole-5(4H)-thione 4-[(4-Fluorobenzylidene)amino]-3-[1-(4-isobutylphenyl)ethyl]-1H- 1,2,4-triazole-5(4H)-thione Crystal data C21H23FN4S Z = 2 Mr = 382.49 F000 = 404 Triclinic, P1 Dx = 1.278 Mg m−3 Hall symbol: -P 1 Mo Kα radiation, λ = 0.71073 Å a = 5.7883 (1) Å Cell parameters from 8265 reflections b = 9.9001 (1) Å θ = 2.3–32.9º c = 18.4972 (3) Å µ = 0.19 mm−1 α = 98.132 (1)º T = 100 K β = 97.087 (1)º Plate, colourless γ = 105.997 (1)º 0.46 × 0.20 × 0.07 mm V = 993.90 (3) Å3 Data collection Bruker SMART APEXII CCD area-detector diffractometer 7460 independent reflections Radiation source: fine-focus sealed tube 5798 reflections with I > 2σ(I) Monochromator: graphite Rint = 0.037 T = 100 K θmax = 33.1º φ and ω scans θmin = 1.1º Absorption correction: multi-scan (SADABS; Bruker, 2005) h = −8→8 Tmin = 0.919, Tmax = 0.987 k = −13→15 31031 measured reflections l = −28→28 Data collection Bruker SMART APEXII CCD area-detector diffractometer 7460 independent reflections Radiation source: fine-focus sealed tube 5798 reflections with I > 2σ(I) Monochromator: graphite Rint = 0.037 T = 100 K θmax = 33.1º φ and ω scans θmin = 1.1º Absorption correction: multi-scan (SADABS; Bruker, 2005) h = −8→8 Tmin = 0.919, Tmax = 0.987 k = −13→15 31031 measured reflections l = −28→28 sup-2 supplementary materials Refinement Refinement on F2 Secondary atom site location: difference Fourier map Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites R[F2 > 2σ(F2)] = 0.048 All H-atom parameters refined wR(F2) = 0.135 w = 1/[σ2(Fo 2) + (0.0657P)2 + 0.2752P] where P = (Fo 2 + 2Fc 2)/3 S = 1.06 (Δ/σ)max = 0.001 7460 reflections Δρmax = 0.63 e Å−3 336 parameters Δρmin = −0.29 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites All H-atom parameters refined w = 1/[σ2(Fo 2) + (0.0657P)2 + 0.2752P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.63 e Å−3 Δρmin = −0.29 e Å−3 Special details Experimental. The crystal was placed in the cold stream of an Oxford Cyrosystems Cobra open-flow nitrogen cryostat (Cosier & Glazer, 1986) operating at 100.0 (1) K. Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq S1 −0.23157 (6) 0.40057 (3) 0.062645 (17) 0.02273 (9) F1 1.07698 (17) 0.46076 (11) 0.34727 (5) 0.0384 (2) N2 0.09265 (19) 0.67553 (11) 0.11123 (6) 0.0200 (2) N3 −0.2432 (2) 0.66569 (12) 0.04603 (6) 0.0240 (2) N4 −0.1117 (2) 0.80841 (12) 0.06196 (6) 0.0262 (2) C1 0.7243 (2) 0.63478 (14) 0.23907 (7) 0.0230 (2) C2 0.9179 (2) 0.61374 (15) 0.28295 (8) 0.0265 (3) C3 0.8872 (3) 0.48191 (16) 0.30420 (8) 0.0275 (3) C4 0.6725 (3) 0.37142 (15) 0.28390 (8) 0.0291 (3) C5 0.4798 (3) 0.39514 (15) 0.24054 (8) 0.0270 (3) C6 0.5020 (2) 0.52512 (13) 0.21738 (7) 0.0220 (2) C7 0.2896 (2) 0.54128 (14) 0.17189 (7) 0.0239 (2) N1 0.29999 (19) 0.65984 (12) 0.15103 (6) 0.0219 (2) C8 −0.1269 (2) 0.57973 (13) 0.07367 (6) 0.0207 (2) C9 0.0928 (2) 0.81133 (13) 0.10125 (7) 0.0227 (2) C10 0.3012 (3) 0.94216 (14) 0.13560 (7) 0.0248 (3) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Ui /U sup-3 supplementary materials supplementary materials sup-4 C11 0.2842 (2) 0.98403 (13) 0.21717 (7) 0.0210 (2) C12 0.4468 (2) 0.96157 (14) 0.27274 (7) 0.0226 (2) C13 0.4346 (2) 1.00047 (13) 0.34731 (7) 0.0215 (2) C14 0.2607 (2) 1.06384 (13) 0.36891 (7) 0.0198 (2) C15 0.0957 (2) 1.08449 (14) 0.31282 (7) 0.0230 (2) C16 0.1051 (2) 1.04396 (14) 0.23829 (7) 0.0239 (2) C17 0.2509 (2) 1.11216 (14) 0.44918 (7) 0.0232 (2) C18 0.3959 (2) 1.26943 (14) 0.48054 (7) 0.0231 (2) C19 0.3044 (3) 1.06203 (16) 0.09161 (9) 0.0338 (3) C20 0.6693 (3) 1.29519 (17) 0.48410 (9) 0.0302 (3) C21 0.3413 (3) 1.31418 (18) 0.55751 (8) 0.0324 (3) H1A 0.739 (3) 0.725 (2) 0.2216 (10) 0.031 (4) H2A 1.074 (4) 0.684 (2) 0.2993 (11) 0.042 (5)* H4A 0.661 (4) 0.277 (2) 0.3010 (11) 0.042 (5)* H5A 0.325 (4) 0.318 (2) 0.2259 (11) 0.038 (5)* H7A 0.142 (4) 0.462 (2) 0.1596 (11) 0.037 (5)* H10A 0.470 (4) 0.922 (2) 0.1304 (11) 0.035 (5)* H12A 0.570 (3) 0.9154 (18) 0.2584 (9) 0.023 (4)* H13A 0.545 (3) 0.985 (2) 0.3849 (11) 0.035 (5)* H15A −0.026 (3) 1.1270 (19) 0.3265 (10) 0.030 (4)* H16A −0.007 (4) 1.058 (2) 0.2033 (11) 0.040 (5)* H17A 0.314 (3) 1.0534 (19) 0.4791 (10) 0.028 (4)* H17B 0.078 (3) 1.0959 (18) 0.4546 (9) 0.024 (4)* H18A 0.347 (3) 1.331 (2) 0.4477 (11) 0.035 (5)* H19A 0.140 (4) 1.092 (2) 0.0910 (11) 0.044 (5)* H19B 0.452 (4) 1.149 (2) 0.1147 (12) 0.047 (6)* H19C 0.323 (4) 1.027 (2) 0.0392 (11) 0.040 (5)* H20A 0.761 (4) 1.396 (2) 0.5032 (12) 0.047 (6)* H20B 0.714 (4) 1.268 (2) 0.4370 (12) 0.041 (5)* H20C 0.723 (4) 1.237 (2) 0.5162 (12) 0.043 (5)* H21A 0.164 (4) 1.299 (2) 0.5562 (12) 0.048 (6)* H21B 0.421 (4) 1.416 (2) 0.5753 (12) 0.042 (5)* H21C 0.394 (4) 1.258 (2) 0.5923 (11) 0.036 (5)* H1N3 −0.384 (4) 0.641 (2) 0.0196 (11) 0.038 (5)* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 S1 0.02380 (15) 0.01846 (14) 0.01925 (15) −0.00197 (10) 0.00281 (11) −0.00094 (10) F1 0.0338 (5) 0.0443 (5) 0.0371 (5) 0.0151 (4) −0.0013 (4) 0.0066 (4) N2 0.0209 (5) 0.0188 (5) 0.0150 (4) −0.0010 (4) 0.0017 (3) 0.0006 (3) N3 0.0234 (5) 0.0201 (5) 0.0210 (5) −0.0009 (4) −0.0016 (4) −0.0004 (4) N4 0.0311 (6) 0.0190 (5) 0.0210 (5) −0.0011 (4) −0.0011 (4) 0.0012 (4) C1 0.0241 (6) 0.0198 (5) 0.0216 (6) 0.0017 (4) 0.0074 (4) −0.0004 (4) C2 0.0224 (6) 0.0273 (6) 0.0250 (6) 0.0024 (5) 0.0057 (5) −0.0021 (5) C3 0.0287 (6) 0.0307 (7) 0.0225 (6) 0.0101 (5) 0.0038 (5) 0.0011 (5) C4 0.0341 (7) 0.0237 (6) 0.0285 (7) 0.0065 (5) 0.0055 (5) 0.0056 (5) C5 0.0279 (6) 0.0217 (6) 0.0265 (6) 0.0007 (5) 0.0039 (5) 0.0027 (5) Atomic displacement parameters (Å2) sup-4 supplementary materials C6 0.0235 (6) 0.0202 (5) 0.0192 (5) 0.0023 (4) 0.0054 (4) 0.0002 (4) C7 0.0236 (6) 0.0216 (6) 0.0217 (6) −0.0002 (4) 0.0041 (5) 0.0017 (4) N1 0.0207 (5) 0.0236 (5) 0.0171 (5) 0.0009 (4) 0.0028 (4) 0.0015 (4) C8 0.0211 (5) 0.0209 (5) 0.0143 (5) −0.0014 (4) 0.0039 (4) −0.0006 (4) C9 0.0273 (6) 0.0192 (5) 0.0159 (5) −0.0012 (4) 0.0024 (4) 0.0020 (4) C10 0.0281 (6) 0.0208 (6) 0.0174 (5) −0.0035 (5) 0.0018 (5) 0.0013 (4) C11 0.0227 (5) 0.0161 (5) 0.0178 (5) −0.0029 (4) 0.0007 (4) 0.0018 (4) C12 0.0233 (6) 0.0199 (5) 0.0211 (6) 0.0033 (4) 0.0020 (4) 0.0005 (4) C13 0.0232 (6) 0.0198 (5) 0.0189 (5) 0.0049 (4) −0.0007 (4) 0.0024 (4) C14 0.0198 (5) 0.0175 (5) 0.0182 (5) 0.0004 (4) 0.0015 (4) 0.0020 (4) C15 0.0183 (5) 0.0237 (6) 0.0246 (6) 0.0038 (4) 0.0007 (4) 0.0036 (5) C16 0.0209 (6) 0.0245 (6) 0.0215 (6) 0.0016 (4) −0.0035 (4) 0.0052 (5) C17 0.0230 (6) 0.0245 (6) 0.0199 (6) 0.0039 (5) 0.0038 (4) 0.0029 (5) C18 0.0235 (6) 0.0239 (6) 0.0198 (6) 0.0073 (4) 0.0004 (4) 0.0000 (4) C19 0.0446 (9) 0.0252 (7) 0.0250 (7) −0.0006 (6) 0.0034 (6) 0.0071 (5) C20 0.0236 (6) 0.0323 (7) 0.0277 (7) 0.0019 (5) 0.0037 (5) −0.0040 (6) C21 0.0293 (7) 0.0391 (8) 0.0261 (7) 0.0126 (6) 0.0033 (5) −0.0062 (6) Geometric parameters (Å, °) S1—C8 1.6821 (13) C11—C12 1.3904 (18) F1—C3 1.3577 (16) C11—C16 1.3976 (19) N2—C9 1.3824 (16) C12—C13 1.3945 (18) N2—N1 1.3870 (15) C12—H12A 0.989 (16) N2—C8 1.3888 (15) C13—C14 1.3935 (17) N3—C8 1.3389 (17) C13—H13A 0.941 (19) N3—N4 1.3772 (15) C14—C15 1.3980 (17) N3—H1N3 0.85 (2) C14—C17 1.5071 (17) N4—C9 1.3011 (17) C15—C16 1.3923 (19) C1—C2 1.382 (2) C15—H15A 0.958 (18) C1—C6 1.4055 (18) C16—H16A 0.91 (2) C1—H1A 0.977 (18) C17—C18 1.5392 (19) C2—C3 1.387 (2) C17—H17A 0.968 (18) C2—H2A 0.96 (2) C17—H17B 0.988 (17) C3—C4 1.379 (2) C18—C20 1.524 (2) C4—C5 1.385 (2) C18—C21 1.5272 (19) C4—H4A 1.01 (2) C18—H18A 0.989 (19) C5—C6 1.3908 (19) C19—H19A 1.07 (2) C5—H5A 0.98 (2) C19—H19B 1.03 (2) C6—C7 1.4618 (19) C19—H19C 1.01 (2) C7—N1 1.2747 (17) C20—H20A 0.98 (2) C7—H7A 0.97 (2) C20—H20B 0.96 (2) C9—C10 1.5013 (17) C20—H20C 0.97 (2) C10—C19 1.528 (2) C21—H21A 0.99 (2) C10—C11 1.5299 (18) C21—H21B 0.97 (2) C10—H10A 1.059 (19) C21—H21C 0.98 (2) C9—N2—N1 118.18 (10) C11—C12—H12A 118.7 (10) C9—N2—C8 108.16 (11) C13—C12—H12A 120.3 (10) N1—N2—C8 133.59 (11) C14—C13—C12 121.45 (11) sup-5 supplementary materials supplementary materials sup-6 C8—N3—N4 114.43 (11) C14—C13—H13A 117.7 (12) C8—N3—H1N3 127.2 (13) C12—C13—H13A 120.9 (12) N4—N3—H1N3 118.4 (13) C13—C14—C15 117.37 (11) C9—N4—N3 103.92 (11) C13—C14—C17 122.28 (11) C2—C1—C6 120.41 (13) C15—C14—C17 120.33 (11) C2—C1—H1A 121.3 (11) C16—C15—C14 121.37 (12) C6—C1—H1A 118.2 (11) C16—C15—H15A 120.0 (11) C1—C2—C3 118.32 (13) C14—C15—H15A 118.7 (11) C1—C2—H2A 124.2 (12) C15—C16—C11 120.84 (12) C3—C2—H2A 117.5 (12) C15—C16—H16A 119.0 (13) F1—C3—C4 118.41 (13) C11—C16—H16A 120.2 (13) F1—C3—C2 118.51 (13) C14—C17—C18 114.35 (11) C4—C3—C2 123.08 (13) C14—C17—H17A 109.5 (10) C3—C4—C5 117.71 (13) C18—C17—H17A 107.9 (11) C3—C4—H4A 119.6 (12) C14—C17—H17B 108.3 (10) C5—C4—H4A 122.6 (12) C18—C17—H17B 109.7 (10) C4—C5—C6 121.40 (13) H17A—C17—H17B 106.9 (14) C4—C5—H5A 119.0 (11) C20—C18—C21 109.98 (11) C6—C5—H5A 119.6 (11) C20—C18—C17 111.77 (11) C5—C6—C1 119.08 (13) C21—C18—C17 110.13 (12) C5—C6—C7 117.86 (12) C20—C18—H18A 107.0 (11) C1—C6—C7 123.06 (12) C21—C18—H18A 108.4 (11) N1—C7—C6 119.92 (12) C17—C18—H18A 109.4 (12) N1—C7—H7A 121.5 (11) C10—C19—H19A 111.1 (11) C6—C7—H7A 118.5 (11) C10—C19—H19B 109.4 (12) C7—N1—N2 119.06 (11) H19A—C19—H19B 109.8 (16) N3—C8—N2 102.50 (10) C10—C19—H19C 107.9 (11) N3—C8—S1 126.62 (10) H19A—C19—H19C 109.8 (15) N2—C8—S1 130.88 (10) H19B—C19—H19C 108.7 (17) N4—C9—N2 110.96 (11) C18—C20—H20A 111.5 (12) N4—C9—C10 126.38 (12) C18—C20—H20B 113.4 (12) N2—C9—C10 122.59 (12) H20A—C20—H20B 108.1 (18) C9—C10—C19 110.48 (11) C18—C20—H20C 109.1 (13) C9—C10—C11 108.88 (10) H20A—C20—H20C 109.1 (17) C19—C10—C11 113.34 (11) H20B—C20—H20C 105.5 (17) C9—C10—H10A 110.3 (11) C18—C21—H21A 111.1 (13) C19—C10—H10A 103.0 (11) C18—C21—H21B 110.1 (13) C11—C10—H10A 110.7 (11) H21A—C21—H21B 106.5 (17) C12—C11—C16 117.98 (12) C18—C21—H21C 110.7 (11) C12—C11—C10 120.42 (12) H21A—C21—H21C 107.5 (17) C16—C11—C10 121.60 (11) H21B—C21—H21C 110.9 (17) C11—C12—C13 120.95 (12) C8—N3—N4—C9 −0.39 (15) C8—N2—C9—N4 1.56 (14) C6—C1—C2—C3 0.56 (19) N1—N2—C9—C10 −3.85 (17) C1—C2—C3—F1 179.96 (12) C8—N2—C9—C10 178.80 (11) C1—C2—C3—C4 −0.3 (2) N4—C9—C10—C19 −26.92 (19) F1—C3—C4—C5 179.39 (12) N2—C9—C10—C19 156.28 (13) C2—C3—C4—C5 −0.3 (2) N4—C9—C10—C11 98.16 (15) C3—C4—C5—C6 0.8 (2) N2—C9—C10—C11 −78.64 (15) sup-6 supplementary materials supplementary materials C4—C5—C6—C1 −0.5 (2) C9—C10—C11—C12 107.13 (14) C4—C5—C6—C7 −179.87 (13) C19—C10—C11—C12 −129.48 (14) C2—C1—C6—C5 −0.16 (19) C9—C10—C11—C16 −72.11 (15) C2—C1—C6—C7 179.16 (12) C19—C10—C11—C16 51.28 (17) C5—C6—C7—N1 178.23 (12) C16—C11—C12—C13 −1.31 (18) C1—C6—C7—N1 −1.1 (2) C10—C11—C12—C13 179.42 (11) C6—C7—N1—N2 −176.62 (11) C11—C12—C13—C14 −0.41 (19) C9—N2—N1—C7 167.93 (12) C12—C13—C14—C15 1.20 (18) C8—N2—N1—C7 −15.5 (2) C12—C13—C14—C17 −177.44 (12) N4—N3—C8—N2 1.29 (14) C13—C14—C15—C16 −0.28 (18) N4—N3—C8—S1 −178.22 (9) C17—C14—C15—C16 178.39 (12) C9—N2—C8—N3 −1.65 (13) C14—C15—C16—C11 −1.5 (2) N1—N2—C8—N3 −178.43 (12) C12—C11—C16—C15 2.23 (18) C9—N2—C8—S1 177.83 (10) C10—C11—C16—C15 −178.51 (11) N1—N2—C8—S1 1.1 (2) C13—C14—C17—C18 92.42 (15) N3—N4—C9—N2 −0.73 (14) C15—C14—C17—C18 −86.18 (14) N3—N4—C9—C10 −177.84 (12) C14—C17—C18—C20 −66.53 (15) N1—N2—C9—N4 178.91 (11) C14—C17—C18—C21 170.91 (11) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N3—H1N3···S1i 0.85 (2) 2.43 (2) 3.2763 (12) 172.3 (18) C7—H7A···S1 0.96 (2) 2.50 (2) 3.2415 (13) 133.2 (16) C4—H4A···Cg1ii 1.01 (2) 2.85 (2) 3.6276 (16) 133.8 (17) Symmetry codes: (i) −x−1, −y+1, −z; (ii) x, y−1, z. sup-7 supplementary materials supplementary materials supplementary materials sup-8 Fig. 1 1 Fig. 1 sup-8 supplementary materials Fig. 2 Fig. 2 sup-9
https://openalex.org/W4392108972	https://brill.com/downloadpdf/view/journals/ieul/aop/article-10.1163-22125892-bja10030/article-10.1163-22125892-bja10030.pdf	English	null	Vedic evidence for the verbal-governing dā́ti-vāra- ‘type’	Indo-European linguistics	2,024	cc-by	10,775	Indo-European Linguistics (2024) 1–18 brill.com/ieul Vedic evidence for the verbal-governing dā́ti-vāra- ‘type’ A critical reassessment Stephanie W. Jamison \| orcid: 0009-0000-9176-4783 University of California, Los Angeles, CA, USA jamison@humnet.ucla.edu Received 5 August 2023 \| Accepted 21 December 2023 \| Published online 22 February 2024 Abstract For more than a century, a verbal-governing compound type has featured prominently in Indo-Europeanist discourse on compounding, a type exemplified byVedic dā́ti-vāra- ‘granting wishes’, with a -ti-stem first member having a transitive relationship to the nominal second member. However, a critical reexamination of the Vedic evidence for thistyperevealsthatalmostnoneof thestandard,regularlyrepeatedexamplesactually mean what it is claimed they mean. The existence of this “type” is therefore seriously called into question and should not be reconstructed for Indo-European on the basis of the Vedic data. Published with license by Koninklijke Brill nv \| doi:10.1163/22125892-bja10030 © Stephanie W. Jamison, 2024 \| ISSN: 2212-5884 (print) 2212-5892 (online) This is an open access article distributed under the terms of the cc by 4.0 license. Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ Keywords Rig Veda – bahuvrīhi – verbal-governing compound – -ti-stem I am of the opinion that every generation or so we should reexamine the evi- dential support for phenomena that have come to be accepted, indeed cher- ished, items in the Indo-Europeanist repertoire—items about which often the only questions now raised are their prehistory and their formal analysis, with the same examples repeated in publication after publication, their accuracy and correct philological analysis assumed. One of these phenomena is the so- called ‘dāti-vāra type’ in Vedic—that is, verbal governing compounds with a Published with license by Koninklijke Brill nv \| doi:10.1163/22125892-bja10030 © Stephanie W. Jamison, 2024 \| ISSN: 2212-5884 (print) 2212-5892 (online) This is an open access article distributed under the terms of the cc by 4.0 license. Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ Published with license by Koninklijke Brill nv \| doi:10.1163/22125892-bja10030 © Stephanie W. Jamison, 2024 \| ISSN: 2212-5884 (print) 2212-5892 (online) This is an open access article distributed under the terms of the cc by 4.0 license. Downloa via Open Access. This is an o Published with license by Koninklijke Brill nv \| doi:10.1163/22125892-bja10030 © Stephanie W. Jamison, 2024 \| ISSN: 2212-5884 (print) 2212-5892 (online) This is an open access article distributed under the terms of the cc by 4.0 license. Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ jamison 2 first member ending in -ti- supposedly governing the second, as in the stan- dard gloss for dā́ti-vāra- ‘granting wishes’. Although the identification of this type goes back well over a century, it remains a very live item in the perenni- ally popular debate about Indo-European compounding—as witnessed by the posthumous publication of a 1988 handout of Jochem Schindler’s, edited and annotated by Martin Peters, in the 2022 Festschrift for Mark Hale (Schindler & Peters 2022). I will show that almost all of the supposed examples of this type in Vedic not only allow, but require, different interpretations, and the last one standing, dā́ti-vāra- itself, is also seriously tainted and even more mysteri- ous than before. I have been anticipated in some of this by Benedicte Nielsen Whitehead in her 2012 Leiden University dissertation (40–48), summarized by OlgaTribulatoinher2015book, AncientGreekverb-initialcompounds(177–179). Keywords Nonetheless I think I can offer a richer and often more accurate philologi- cal account and rather different analyses of the material. I would also like to emphasize at the outset that I am strictly staying away from both compara- tive and diachronic questions: I will have nothing to say about the type of Gk. βωτι-άνειρα ‘men-feeding,’ nor will I directly confront the issue of the prehis- tory of the compound type—if, indeed, it existed in prehistory. I’ll just muck aroundinmyownlittlepatch,thetextof theRigVeda,whichhasbeencuriously neglected in recent Indo-European treatments of this supposed compound type.1 The dossier of examples in more or less its current form was, to my knowl- edge, first assembled by Wackernagel in the volume on nominal compounds of the Altindische Grammatik (Wackernagel 1905). Though his account occupies only about a page (320–321), he set the parameters and identified most of the class members for all subsequent discussions. His list consists of six items. I give them in his order with his glosses, as well as the number of Rigvedic attes- tations. (No new examples appear after the Rig Veda.2) 1 The literature on this compound type, its possible correspondents elsewhere, and its possi- ble Indo-European source(s) is immense. Since my intent here is to critically reevaluate the Vedic data philologically, I will only fitfully engage with the literature on its presumed pre- history and the various formal and functional proposals made about it. Nor will I attempt to collect the numerous recent publications that simply reproduce Wackernagel’s dossier and Schindler’s explanations for the Vedic forms. 1 The literature on this compound type, its possible correspondents elsewhere, and its possi- ble Indo-European source(s) is immense. Since my intent here is to critically reevaluate the Vedic data philologically, I will only fitfully engage with the literature on its presumed pre- history and the various formal and functional proposals made about it. Nor will I attempt to collect the numerous recent publications that simply reproduce Wackernagel’s dossier and Schindler’s explanations for the Vedic forms. 2 All forms and passages cited are from the Rig Veda and will therefore not be so identified individually. 2 All forms and passages cited are from the Rig Veda and will therefore not be so identified individually. 1 The literature on this compound type, its possible correspondents elsewhere, and its possi- ble Indo-European source(s) is immense. Since my intent here is to critically reevaluate the Vedic data philologically, I will only fitfully engage with the literature on its presumed pre- history and the various formal and functional proposals made about it. Nor will I attempt to collect the numerous recent publications that simply reproduce Wackernagel’s dossier and Schindler’s explanations for the Vedic forms. Keywords One of the anonymous readers for this journal points out that in unpublished (and undated) English and German versions of Schindler’s τερψίμβροτος handout (but not in the handout published as Schindler & Peters 2022), he also listed rantideva-, a personal name in the Mahābhārata, beside the Vedic -ti-stem ránti-, though without glossing the compound. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ the vedic dā́ti-vāra- compound type 3 3 – dā́ti-vāra- (3×) ‘Schätze gebend’ – púṣṭi-gu- (1×) NP, eig. ‘Kühe aufziehend’ – rīty-ā̀p- (2×) ‘Wasser strömend lassend’ – vītí-rādhas- (1×) ‘die Spende geniessend’ – vītí-hotra- (5×) ‘das Opfer geniessend’: – vṛṣṭí-dyu-3 (2×) ‘den Himmel regnen lassend’ The only regular additions4 to this list in later discussions are śrúṣṭi-gu- (1×), rhyming with púṣṭi-gu- and found adjacent to it, and, less commonly, the PN vṛṣṭi-hávya-, which Wackernagel excluded on the grounds of its second- member accent (1905: 320), but Debrunner (1957: 88–89 [Nachtr. to Wack- ernagel 1905: 321 & 12]) adduces, following Renou (1953: 232 with n. 10; see also Schindler & Peters 2022: 333). Debrunner’s glosses for these two addi- tional examples are ‘die Kühe gehorchend machen’ and ‘die Opfergabe reg- nend lassen’, respectively. The same examples (Wackernagel’s plus Debrun- ner’s),withthesameglosses,arecontinuallyrepeatedinthescholarlyliterature (e.g., Pinault 2018: 332–335). To support the transitive interpretation he imposes on the first members, Wackernagel cites parallel Rigvedic syntagms with transitive verbal forms to the appropriate roots and accusative objects, for most of them—but, notably, not for dā́ti-vāra-. In order these are, with my translations: – púṣṭi-gu-: iii.45.3 krátuṃpuṣyasi gā́ iva ‘you foster your resolve like cows’ ̀ – púṣṭi-gu-: iii.45.3 krátuṃpuṣyasi gā́ iva ‘you foster your resolve like cows’ – rīty-ā̀p-: viii.7.28 =x.138.10 (etc.) riṇánn apáḥ‘They let flow the waters’ – púṣṭi-gu-: iii.45.3 krátuṃpuṣyasi gā́ iva ‘you foster your resolve like cows’ – rīty-ā̀p-: viii.7.28 =x.138.10 (etc.) riṇánn apáḥ‘They let flow the waters’ – púṣṭi-gu-: iii.45.3 krátuṃpuṣyasi gā iva you foster your resolve like cows – rīty-ā̀p-: viii.7.28 =x.138.10 (etc.) riṇánn apáḥ‘They let flow the waters’ Given the lateness of the compound and the fact that it is a personal name, I will not pur- sue this form further, but, as the Reader points out, it is worth noting that the -ti-stem is in full grade, like τερψί- and unlike all the other Vedic examples save for dā́tivāra-. Keywords However, the name should probably be assessed with other X-deva- compounds, like Śāntideva (name of a philosopher);Isuspectthatthesearesimplebahuvrīhiswiththemeaning‘havingranti /śānti asone’sdeity.’Wackernagel(1905:320)notesthatBenfeyhadalreadyadducedranti-deva-and other such epic forms as examples of this class. Given the lateness of the compound and the fact that it is a personal name, I will not pur- sue this form further, but, as the Reader points out, it is worth noting that the -ti-stem is in full grade, like τερψί- and unlike all the other Vedic examples save for dā́tivāra-. However, the name should probably be assessed with other X-deva- compounds, like Śāntideva (name of a philosopher);Isuspectthatthesearesimplebahuvrīhiswiththemeaning‘havingranti /śānti asone’sdeity.’Wackernagel(1905:320)notesthatBenfeyhadalreadyadducedranti-deva-and other such epic forms as examples of this class. 3 Wackernagel cites the second member as -dyāv-, but I will use the zero-grade. 4 Nielsen Whitehead (2012: 44–45) adds several other compounds with -ti-stem first members: abhiṣṭí-dyumna- ‘whose glory is protecting or superior’, abhíṣṭi-śavas- ‘rendering powerful assistance’, and ásamāty-ojas- ‘of unequalled strength’ (her glosses); see also Tribulato (2015: 178). These are generally taken as bahuvrīhis without any governing sense. Since Nielsen Whitehead wants to explain all the governing examples as simple bahuvrīhis, these additions were strategic on her part, and I will not comment on them further. 4 Nielsen Whitehead (2012: 44–45) adds several other compounds with -ti-stem first members: abhiṣṭí-dyumna- ‘whose glory is protecting or superior’, abhíṣṭi-śavas- ‘rendering powerful assistance’, and ásamāty-ojas- ‘of unequalled strength’ (her glosses); see also Tribulato (2015: 178). These are generally taken as bahuvrīhis without any governing sense. Since Nielsen Whitehead wants to explain all the governing examples as simple bahuvrīhis, these additions were strategic on her part, and I will not comment on them further. I will also have nothing to say about the Mitanni personal name Šat-ti-ú-az-za, which sup- posedlyhasthesamecompoundelementsasRVvā́ja-sāti-‘winningof prizes’,butreversed(as if sātí-vāja-), which sometimes figures in the recent literature (e.g., Pinault 2018: 332)—on the grounds that interpretation of personal names is hazardous enough even within a single language. 5 It’s worth noting that Grassmann (1872–1875 s.vv.), whoseWörterbuch of course considerably predated Wackernagel’s treatment, gives ‘governing’ interpretations only for the vītí- com- pounds (vītí-rādhas- ‘Genuss gewährend’, vītí-havya- ‘zum Mahle einladend’), but his other, more conventional glosses (‘strömendes Wasser habend’ for rīty-ā̀p-, ‘regnenden Himmel habend’ for vṛṣṭí-dyu-, not to mention ‘das Geben [dāti] liebend [vāra-]’ for dā́ti-vāra-) were silently set aside in favor of Wackernagel’s analysis. By contrast, in Geldner’s (1951) transla- tion, postdating Wackernagel’s treatment and serving as the standard Rig Veda translation for more than a half century, except for the personal names the governing interpretation prevails: ix.106.9 vṛṣṭídyāvo rītyā̀paḥ‘ihr lasset den Himmel regnen, die Gewässer strömen’ (sim. v.68.5); ix.62.29 vītírādhas- ‘der Freigebigkeit liebt’; vītíhotra- ‘opfereifrig’ ii.38.1, iii.24.2, v.26.3, viii.31.9, but ‘der das Hotr̥-Amt besorgt’ i.84.18; dā́ti-vāra- ‘Wünschgewährer’ i.167.8, iii.51.9, v.58.2. Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 Downloaded from Brill.com 10/24/20 via Open Access. This is an open access article distributed of the CC h // i Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 Downloaded from Brill.co via Open Access. This is an open access article jamison 4 – vītí-hotra-: iv.48.1 vihí hótrāḥ‘pursue the oblations’ – vītí-hotra-: iv.48.1 vihí hótrāḥ‘pursue the oblations’ – vṛṣṭí-dyu-: v.63.3, 6; ix.96.3 dyā́m varṣaya- ‘make heaven rain’ – vṛṣṭí-dyu-: v.63.3, 6; ix.96.3 dyā́m varṣaya- ‘make heaven rain’ – vṛṣṭí-dyu-: v.63.3, 6; ix.96.3 dyā́m varṣaya- ‘make heaven rain’ These transitive syntagms are simply requoted in later discussions, including Schindler’s 1988 handout (Schindler & Peters 2022: 332–334). First note that there is no stable relationship between the transitive verb stem and the -ti- compound member—a ya-present, a 9th-class present, a root present, and evenacausative.Further,ImustnowreluctantlyaccuseWackernagelof cherry- picking evidence—indeed, worse, of suppressing evidence. For intransitive syntagms involving the same elements are also found. – púṣṭi-gu-: x.19.3 asmín puṣyantu gópatau ‘Let (the cows) [= the dedicands of the hymn] prosper under this herdsman.’ – púṣṭi-gu-: x.19.3 asmín puṣyantu gópatau ‘Let (the cows) [= the dedicands of the hymn] prosper under this herdsman.’ – rīty-ā̀p-: no ā́po rīyante, but cf. x.40.9 rīyante … síndhavaḥ‘the rivers flow’ – vṛṣṭí-dyu-: no dyaur varṣati, but cf. v.84.3 divó várṣanti vṛṣṭáyaḥ‘The rains rīty āp : no āpo rīyante, but cf. x.40.9 rīyante … síndhavaḥthe rivers flow – vṛṣṭí-dyu-: no dyaur varṣati, but cf. v.84.3 divó várṣanti vṛṣṭáyaḥ‘The rains from/of heaven rain.’ Attheveryleast,Wackernagelshouldhaveacknowledgedthesecompetingsyn- tagms: it would have saved us all a lot of trouble by generating some skepticism about the transitive analysis. As it is, subsequent scholarship seems to have accepted, on the strength of Wackernagel’s prestige, his implicit assertion that atransitiveinterpretationistheonlyonesupportedbythedata,withoutcheck- ing the text independently.5 Attheveryleast,Wackernagelshouldhaveacknowledgedthesecompetingsyn- tagms: it would have saved us all a lot of trouble by generating some skepticism The interpretational problem has become acute as the morphological anal- ysis of the first member has shifted. For Wackernagel (1905: 320–321) the first member must be an old imperative in -ti. Where exactly we find these old -ti imperatives he does not tell us (simply citing Brugmann on this point, but without examples), but such an analysis would easily account for the transitive verb–object function he ascribes to this compound type. Similarly some of the alternative views summarized by Wackernagel—that it is an imperatively used infinitive (Brugmann) or a 3. sg. Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 Downloaded from Brill.com 10/24/20 via Open Access. This is an open access article distributed of the CC h // i finite form (Jacobi)—facilitate a transi- 5 It’s worth noting that Grassmann (1872–1875 s.vv.), whoseWörterbuch of course considerably predated Wackernagel’s treatment, gives ‘governing’ interpretations only for the vītí- com- pounds (vītí-rādhas- ‘Genuss gewährend’, vītí-havya- ‘zum Mahle einladend’), but his other, more conventional glosses (‘strömendes Wasser habend’ for rīty-ā̀p-, ‘regnenden Himmel habend’ for vṛṣṭí-dyu-, not to mention ‘das Geben [dāti] liebend [vāra-]’ for dā́ti-vāra-) were silently set aside in favor of Wackernagel’s analysis. By contrast, in Geldner’s (1951) transla- tion, postdating Wackernagel’s treatment and serving as the standard Rig Veda translation for more than a half century, except for the personal names the governing interpretation prevails: ix.106.9 vṛṣṭídyāvo rītyā̀paḥ‘ihr lasset den Himmel regnen, die Gewässer strömen’ (sim. v.68.5); ix.62.29 vītírādhas- ‘der Freigebigkeit liebt’; vītíhotra- ‘opfereifrig’ ii.38.1, iii.24.2, v.26.3, viii.31.9, but ‘der das Hotr̥-Amt besorgt’ i.84.18; dā́ti-vāra- ‘Wünschgewährer’ i.167.8, iii.51.9, v.58.2. 6 See, e.g., Schindler (1997: 539); Nielsen Whitehead (2012: 44); Pinault (2018: 331–332); Schindler & Peters (2022: 333–334). As is clear from Wackernagel’s summary, this view also has a long history, existing in parallel to the view that the first member is a verb-form. Dunkel (1992) is an exception to the current consensus: he holds to the older view that the first mem- ber is a verb form. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ the vedic dā́ti-vāra- compound type 5 tive interpretation. However, the pendulum has definitely swung: the standard view today is that the first member is a nominal form, a -ti-abstract6—a view that Wackernagel himself explicitly rejects. He does so on eminently reason- able grounds: the function of such nouns as nomina actionis, which makes it difficult to derive additively the transitive sense he ascribes to this compound class. And this is the dilemma that all subsequent attempts to account for these compounds have struggled with: what makes sense morphologically (a nomi- nal -ti-stem first member) makes it difficult to motivate the supposed transitive sense of these compounds; what makes sense functionally (a verbal first mem- ber,imperativevelsim.)cannotbejustifiedmorphologically.Choosingthepath of morphological austerity has led to the fantastically convoluted, multi-step functional derivations associated especially with Schindler, as embodied in Schindler & Peters (2022). But this impasse can be resolved, and rather eas- ily, by asking a simple question: do these compounds really mean what they’re said to mean? Answering this merely requires revisiting the data in context, and in fact on various grounds we can eliminate almost all these forms. If we accept Wacker- nagel’s list as expanded by Debrunner, we have eight items, but in fact several of them are twinned and require only a single explanation: 1. The rhyming hapax personal names púṣṭi-gu- and śrúṣṭi-gu- in a single pāda. 1. The rhyming hapax personal names púṣṭi-gu- and śrúṣṭi-gu- in a single pāda. 2. The two vītí- compounds, of which vītí-rādhas- is only attested once. 3. rīty-ā̀p- and vṛṣṭí-dyu-: both are technically attested twice, but both times they are attested together in the same pāda—and the pādas are essen- tially identical, so we’re actually dealing with hapaxes. 4. If we accept vṛṣṭi-hávya- as a member of this class despite its accent, it presumably pairs with vṛṣṭí-dyu-. 4. If we accept vṛṣṭi-hávya- as a member of this class despite its accent, it presumably pairs with vṛṣṭí-dyu-. In other words, we have only four data points: – púṣṭi-gu-/śrúṣṭi-gu- – vītí-hotra-/vītí-rādhas- – vītí-hotra-/vītí-rādhas- – rīty-ā̀p-/vṛṣṭí-dyu- and, along for the ride, vṛṣṭi-hávya- – dā́ti-vāra- which I will treat in turn in what follows. 6 See, e.g., Schindler (1997: 539); Nielsen Whitehead (2012: 44); Pinault (2018: 331–332); Schindler & Peters (2022: 333–334). 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ As is clear from Wackernagel’s summary, this view also has a long history, existing in parallel to the view that the first member is a verb-form. Dunkel (1992) is an exception to the current consensus: he holds to the older view that the first mem- ber is a verb form. Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 ow oad d f o .co 0/ / 0 0 :05: 6 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons org/licenses/by/4 0/ 6 jamison Before considering the examples individually, we should first note that inde- pendent -ti-stems are attested beside all the first members. This pervasive Before considering the examples individually, we should first note that inde- pendent -ti-stems are attested beside all the first members. This pervasive matching certainly favors the current view that the first members are -ti-stem nominals, against the older view that the first members are verb forms of some sort or other. – -dāti-: only attested as second member of havyá-dāti- (quite common) ‘bestowal of oblations’7 – -dāti-: only attested as second member of havyá-dāti- (quite common) ‘bestowal of oblations’7 – puṣṭí-: very common in a variety of cases and numbers – śruṣṭí-: pretty common, especially in instr. śruṣṭī́ Note the accents of these two, which differ from that of their respective com- pounds púṣṭi-gu / śrúṣṭi-gu-: Note the accents of these two, which differ from that of their respective com- pounds púṣṭi-gu / śrúṣṭi-gu-: – rītí-: 5× – vrṣṭí-: very common, but has been concretized to ‘rain’; no clear examples of abstract ‘raininess, raining’ vel sim. – vītí-: quite common, especially as dative infinitive vītáye – vītí-: quite common, especially as dative infinitive vītáye Now to the actual data. The easiest to dispense with are the rhyming personal names púṣṭi-gu- and śrúṣṭi-gu- found in a single pāda in a Vālakhilya hymn, viii.51.1,8 in a sequence of other names referring to people among whom Indra drank soma. Since these are names, there is no contextual clue to their literal sense,andaswasnotedabove,bothtransitiveandintransitivesyntagms(‘make cows thrive’ / ‘cows thrive’) can be cited as parallels for púṣṭi-gu-. What śrúṣṭi- gu- might mean is even less clear. One might construct ‘obeying cows,’ but this is, alas, pragmatically excluded; ‘making cows obey’ is the preferable governing interpretation, but in the absence of a robust verbal root √śruṣthis remains a hypothetical construction. 7 Also in the zero-grade -tti- in the compounds bhagá-tti- ‘gift of good fortune’, maghá-tti- ‘gift of bounties’. 8 The names are found also in the Anukramaṇī, which ascribes viii.50 to Puṣṭigu Kāṇva and viii.51 to Śruṣṭigu Kāṇva, but these names have simply been extracted from viii.51.1. 7 Also in the zero-grade -tti- in the compounds bhagá-tti- ‘gift of good fortune’, maghá-tti- ‘gift of bounties’. 8 The names are found also in the Anukramaṇī, which ascribes viii.50 to Puṣṭigu Kāṇva and viii.51 to Śruṣṭigu Kāṇva, but these names have simply been extracted from viii.51.1. 9 But see Pinault’s transitive paraphrase (2018: 335) ‘ayant (et produisant) le bétail pourvu de 9 But see Pinault’s transitive paraphrase (2018: 335) ‘ayant (et produisant) le bétail pourvu de 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ (This may be why Wackernagel does not include it.) It should be noted that Schindler (Schindler & Peters 2022: 333) considers both compounds ‘stative’, with púṣṭi- and śrúṣṭi- functionally corresponding to the possessive derivatives puṣṭi-mánt- and śruṣṭi-mánt-. Although he provides no glosses, presumably by his analysis these compounds mean ‘having thriving cattle’ < ‘having cattle that have thriving’ and ‘having obedient cattle’ < ‘having cattle that have obedience’. Alternatively one could construct an interpretation with an instrumental first member: ‘having cows with their thriving / with their obedience’.Eitherseemspreferabletoatransitive,governinginterpretationlike Wackernagel’s.9 q , p y y Now to the actual data. The easiest to dispense with are the rhyming personal names púṣṭi-gu- and śrúṣṭi-gu- found in a single pāda in a Vālakhilya hymn, viii.51.1,8 in a sequence of other names referring to people among whom Indra drank soma. Since these are names, there is no contextual clue to their literal sense,andaswasnotedabove,bothtransitiveandintransitivesyntagms(‘make cows thrive’ / ‘cows thrive’) can be cited as parallels for púṣṭi-gu-. What śrúṣṭi- gu- might mean is even less clear. One might construct ‘obeying cows,’ but this is, alas, pragmatically excluded; ‘making cows obey’ is the preferable governing interpretation, but in the absence of a robust verbal root √śruṣthis remains a hypothetical construction. (This may be why Wackernagel does not include it.) It should be noted that Schindler (Schindler & Peters 2022: 333) considers both compounds ‘stative’, with púṣṭi- and śrúṣṭi- functionally corresponding to the possessive derivatives puṣṭi-mánt- and śruṣṭi-mánt-. Although he provides no glosses, presumably by his analysis these compounds mean ‘having thriving cattle’ < ‘having cattle that have thriving’ and ‘having obedient cattle’ < ‘having cattle that have obedience’. Alternatively one could construct an interpretation with an instrumental first member: ‘having cows with their thriving / with their obedience’.Eitherseemspreferabletoatransitive,governinginterpretationlike Wackernagel’s.9 Now to the actual data. The easiest to dispense with are the rhyming personal names púṣṭi-gu- and śrúṣṭi-gu- found in a single pāda in a Vālakhilya hymn, viii.51.1,8 in a sequence of other names referring to people among whom Indra 9 But see Pinault’s transitive paraphrase (2018: 335) ‘ayant (et produisant) le bétail pourvu de 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. 7 Also in the zero-grade -tti- in the compounds bhagá-tti- gift of good fortune, maghá-tti- gift of bounties’. 8 The names are found also in the Anukramaṇī, which ascribes viii.50 to Puṣṭigu Kāṇva and viii.51 to Śruṣṭigu Kāṇva, but these names have simply been extracted from viii.51.1. 9 But see Pinault’s transitive paraphrase (2018: 335) ‘ayant (et produisant) le bétail pourvu de 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons org/licenses/by/4 0/ the vedic dā́ti-vāra- compound type 7 There is also an alternative possible analysis. In the standard interpreta- tion of these compounds, the second member -gu- is a form of ‘cow’, which is found in other compounds, like bhūri-gu- ‘having many cattle’ (1×, voc.), saptá-gu- ‘having seven cattle’. But there is another -gú-, found in vanar-gú- ‘wandering in the woods’ (2×), built to the root √gā ‘go,’ as analyzed by, e.g., Debrunner (1954: 471–472) and Scarlata (1999: 103, 108). If we assume this -gu- in the two compounds at issue, the meanings would be ‘going to prosperity’, ‘going to obedience/attentive hearing’. For the former see the syntagms puṣ- ṭím … agman (i.122.7), puṣṭíṃyāti (i.77.5) with different synonymous roots ‘go’. True, the accent would be wrong for a pseudo-root-noun compound (expect puṣṭi-gú-, like vanar-gú-), but the accent is wrong anyway: for the governing analysis we should have puṣṭí-gu- to match the -ti-stem puṣṭí- (like vītí-hotra- beside vītí-). Personal name compounds seem often to undergo accent shift (as, in fact, Mayrhofer [2003: 59] claims for púṣṭi-gu- itself); see vṛṣṭi-hávya-, if this form belongs here, as well as the underlying bahuvrīhi vadhry-aśvá- PN < ‘having gelded horses,’ beside vádhri-vāc- ‘having gelded speech’. I don’t partic- ularly champion this alternative analysis, but it adds one more question mark to these two hapaxes, especially since, as names, nothing in their context favors onesemanticinterpretationoveranother.Ithinkitcanbegenerallyagreedthat púṣṭi-gu- and śrúṣṭi-gu-, hapax personal names with unclear semantics in a late and poorly transmitted10 part of the Rig Veda, cannot be used as evidence for the function of this putative compound type. The next pair to be considered are rīty-ā̀p- and vṛṣṭí-dyu-, supposedly mean- ing ‘making waters flow’ and ‘making heaven rain’, respectively. Recall that Wackernagel cites transitive syntagms for the governing meaning(s), but that I also cited matching intransitive syntagms. Recall also that the independent stem vṛṣṭí- has only the concrete sense ‘rain’, not abstract ‘raining, raininess’. prospérité’ →‘faisant prospérer le bétail.’ Pinault implies (334) that his account is based on Schindler’s. 10 Onthenatureandstatusof theVālakhilyahymns,RVviii.49–59,see,e.g.,Oldenberg(1912: 116–117); Geldner (1951: ii.370); Jamison & Brereton (2014: 1019 & 1130). prospérité’ →‘faisant prospérer le bétail.’ Pinault implies (334) that his account is based on Schindler’s. 10 Onthenatureandstatusof theVālakhilyahymns,RVviii.49–59,see,e.g.,Oldenberg(1912 116–117); Geldner (1951: ii.370); Jamison & Brereton (2014: 1019 & 1130). 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ Thus, if vṛṣṭí-dyu- really means ‘causing heaven to rain’, the -ti-stem in the com- pound will have to have preserved the abstract verbal action-noun sense only there, not in the abundant attestations of the stem elsewhere, which only refer to the concrete substance (rainwater, raindrops) and could not have a verbal relationship, whether intransitive (‘heaven raindrops’) or transitive (‘causes heaven to raindrop’), with the second member—thus adding yet another dis- tancing step to the analysis. Recall even further that these two stems, each found twice in the Rig Veda, are only found together, adjacent to each other in Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. htt // ti g/li /b /4 0/ Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 via Open Access. This is an open access article jamison 8 almost identical pādas: v.68.5 vṛṣṭídyāvā rītyā̀pā [dual, of Mitra and Varuṇa], ix.106.9 vṛṣṭídyāvo rītyāpaḥ[voc. pl., of soma drops], so that the two occur- rences are really only one.Whatever these compounds are doing, they’re doing it together. And there’s more. Another coincidence of pairing: as noted above, the -tí-stem rītí- occurs independently 5×; two of those occurrences are con- struedwiththegen.apā́m,withthesameelementsasourcompound,andthose two occurrences are found in the same pāda as another, more common, geni- tival syntagm, divó vṛṣṭí-: vi.13.1 divó vṛṣṭíḥ… rītír apā́m; ix.108.10 vṛṣṭíṃdiváḥ … rītím apā́m both meaning ‘the rain of heaven, the streaming of waters’. This to me is the smoking gun. The two compounds appear together; the two syn- tagms appear together. And the independent phrases are not transitive, as the compounds are supposed to be—they mean ‘the rain of heaven … the flowing of waters’—not ‘the causing of heaven to rain, the causing of waters to flow’. In vi.13.1 these are good things that Agni arranges for us to have; in ix.108.10 (very near the compounds in ix.106.9, notice) they either are the good liquids that Soma purifies himself into or that he attracts through his purification. The two compounds and the two syntagms make a formulaic magic square: vṛṣṭí- diváḥ rītí- apā́m vṛṣṭí-dyu- rīty-ā̀p- The two pairs have to be interpreted together because they pattern together, and this to me excludes a transitive / governing interpretation of those com- pounds. 11 In the published Jamison & Brereton Rig Veda translation (2014), in both passages, v.68.5 (JPB) and ix.106.9 (SWJ), the two compounds are translated with governing value—e.g., ix.106.9 ‘bringing the heavens to rain and the waters to streaming’. Both translators too credulously accepted the standard view, but see now the discussion and correction in my on-line commentary (Jamison 2015–, ad ix.106.9). Scarlata (1999: 526) produces a first-pass analysis of vṛṣṭí-dyu- as ‘der Himmel mit Regen hat’ but then reconfigures it into the governing type ‘den Himmel regnen lassend’. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ vṛṣṭí- diváḥ rītí- apā́m vṛṣṭí-dyu- rīty-ā̀p- The two pairs have to be interpreted together because they pattern together, and this to me excludes a transitive / governing interpretation of those com- pounds. What then do they mean and how should their first members be con- strued? Here a scenario rather like Schindler’s for púṣṭi-gu-—a ‘stative’ double- possessive bahuvrīhi—comes to mind, perhaps mediated through an instru- mental first member: ‘having waters that have flowing’ →‘having waters with their flowing’; ‘having heaven that has rain’ →‘having heaven with its rain’.11 In the case of Mitra and Varuṇa the latter would be pretty literal: as gods they do in some sense possess heaven; for the soma drops, metaphorical, but soma has 11 In the published Jamison & Brereton Rig Veda translation (2014), in both passages, v.68.5 (JPB) and ix.106.9 (SWJ), the two compounds are translated with governing value—e.g., ix.106.9 ‘bringing the heavens to rain and the waters to streaming’. Both translators too credulously accepted the standard view, but see now the discussion and correction in my on-line commentary (Jamison 2015–, ad ix.106.9). 11 In the published Jamison & Brereton Rig Veda translation (2014), in both passages, v.68.5 (JPB) and ix.106.9 (SWJ), the two compounds are translated with governing value—e.g., ix.106.9 ‘bringing the heavens to rain and the waters to streaming’. Both translators too credulously accepted the standard view, but see now the discussion and correction in my on-line commentary (Jamison 2015–, ad ix.106.9). Scarlata (1999: 526) produces a first-pass analysis of vṛṣṭí-dyu- as ‘der Himmel mit Regen hat’ but then reconfigures it into the governing type ‘den Himmel regnen lassend’. It’s this reconfiguration that seems unnecessary and counterindicated by the formulaic evidence. 12 Unfortunately the re-evaluation of the semantics of √vī has not penetrated the Indo- Europeanist literature on this compound type: the older glosses of vītí-X prevail. See, e.g., Tribulato (2015: 178) vītí-rādhas- ‘enjoying the gift,’vītí-hotra- ‘enjoying the offering’. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ the vedic dā́ti-vāra- compound type 9 cosmic properties in the Rig Veda. For a possible mediating instrumental, see v.53.5 vṛṣṭī́dyā́vo yatī́r iva ‘like the heavens coming with their rain’. Let us briefly treat the personal name vṛṣṭi-hávya-, which, as we saw, Wack- ernagel excluded from his list on the grounds of accent, but which figures in subsequent literature, including Schindler’s handout (Schindler & Peters 2022: 333). It is found only once in a late hymn (x.115.9) and as a name has no contextual semantics; the referent is the father of the poet(s) of the hymn in question. PaceWackernagel, its accent doesn’t disqualify it from consideration, since, as we saw above, personal names can undergo accent shift. Although Schindler groups it with his ‘stative’ type (like púṣṭi-gu-)—hence, ‘having obla- tions that have rain’? (Schindler & Peters 2022: 333)—others have attributed transitive value to it: Mayrhofer (2003: 88) ‘die Opfertränke regnen lassend’ or ‘dessen Opferguss Regen bewirkt’, Scarlata (1999: 526) ‘dessen Opferguss Regen hat/bewirkt’. Once again I would point out that vṛṣṭí- is only concrete, so that an interpretation based on verbal transitivity has to assume a usage of vṛṣṭí- that predates this functional shift. The simplest interpretation, and the one that best conforms to the Rigvedic usage of vṛṣṭí-, is to take the compound as an equational metaphorical bahuvrīhi: ‘whose X is (like) Y’—hence ‘whose oblations are (like) rain’. In the ixth Maṇḍala soma is regularly described as flowing like the rain of/from heaven: e.g., ix.89.1 divó ná vṛṣṭíḥpávamāno akṣāḥ ‘Like the rain from heaven, the self-purifying (soma) has flowed’. The image is of gushing liquid abundance. Wenowturntothelastsetof paireditemsonourlist: vītí-hotra-(5×)and vītí- rādhas-(1×).Oneof theissuesrelevanttothesecompoundsisthatthemeaning of the root √vī has been reassessed since the compound type was identified, and so the compounds and the root from which they’re derived have been glossed in numerous, not-very-principled ways. For the now-standard view of the root’s semantics, namely ‘pursue, follow’, see EWAia s.v. vayi with further literature. Older assessments of the root’s meaning often contain the notion ‘enjoy’;Grassmann(1872–1875)glossesvītí-as‘Geniessen/Genuss’(enjoyment) and concretely ‘Opfermahl’. Wackernagel (see above) glosses the compound vītí-hotra-as‘dasOpfergeniessend’,with vītí-astransitive‘enjoying’.12However, re-evaluation of the uses of the root √vī and a stricter imposition of seman- tic boundaries have unified the various uses of √vī as ‘pursue, follow’, with the further implication that what one pursues is something one desires. The -tí-stem vītí- ‘pursuit, pursuing’ is quite common, especially in the dat. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ vītáye, 12 Unfortunately the re-evaluation of the semantics of √vī has not penetrated the Indo- Europeanist literature on this compound type: the older glosses of vītí-X prevail. See, e.g., Tribulato (2015: 178) vītí-rādhas- ‘enjoying the gift,’vītí-hotra- ‘enjoying the offering’. Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons org/licenses/by/4 0/ Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 via Open Access. This is an open access article d 10 jamison which as a purpose infinitive means ‘for pursuing, to pursue’, often with an accusative object. As noted above, Wackernagel cites a transitive syntagm vihí hótrāḥ(iv.48.1) as support for his compound gloss ‘das Opfer geniessend’. I freely admit that both finite and non-finite forms of √vī, especially including theinfinitive vītáye,takesynonymsof hótrā-13asobject,especiallyhavyá-‘obla- tion’: see hávyāni (…) vītáye (i.74.6, 135.3–4, 142.13; ii.2.6; viii.20.10, 16, 101.7). But it is absolutely crucial to note who is the subject of these forms of √vī. Both gods and mortals can serve as subject of √vī, but their objects differ.When a mortal is the subject, a god is usually the object, as in viii.4.17 vémi tvā pūṣan ‘I pursue you, Pūṣan’. The mortals are actively seeking the gods to bring them to the sacrifice. But in the far more numerous cases in which gods are the sub- jects, they are pursuing what we mortals and ritualists have to offer them, often oblations—as in iii.53.1 vītáṃhavyā́ni adhvaréṣu devā ‘Pursue the oblations at (our) ceremonies, o you two gods [=Indra and Parvata].’ But—crucially—the compound that has the same apparent structure as these verb phrases, vītí- hotra-, which should mean, by the ‘governing’ hypothesis, ‘pursuing the obla- tions’, does not modify the gods who are doing the pursuing, but generally the mortals who are providing the objects being pursued.14 Cf. i.84.18, whose sub- ject is the energetic ritualist: (1) i.84.18 kó agním īṭṭe havíṣā ghṛténa, srucā́ yajatā ṛtúbhir dhruvébhiḥ …, kó maṃsate vītíhotraḥsudeváḥ Who reverently invokesAgni with oblation and ghee? (Who) will perform sacrifice with a ladle according to the fixed ritual sequences? … Who might think himself (a man) whose oblations are worth pursu- ing, who has gods well on his side? (1) i.84.18 kó agním īṭṭe havíṣā ghṛténa, srucā́ yajatā ṛtúbhir dhruvébhiḥ …, kó maṃsate vītíhotraḥsudeváḥ Who reverently invokesAgni with oblation and ghee? 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ (Who) will perform sacrifice with a ladle according to the fixed ritual sequences? … Who might think himself (a man) whose oblations are worth pursu- ing, who has gods well on his side? 13 The Rig Veda contains two different but related stems, neuter hótra- and feminine hótrā-. The former often means ‘Hotarship, office of Hotar’ (a priestly title), the latter generally ‘oblation, offering’. (In addition there’s a homonymous hótrā- ‘ritual call’.) Since vītí-hotra- is a bahuvrīhi, i.e., an adjective, with the inherent gender of the noun final member neu- tralized, its second member could be either one, and in fact it is surely hótrā-. See the syntagmquotedbyWackernagel,iv.48.1 vihíhótrāḥ‘pursuetheoblations’,whoseindepen- dent object is the acc. pl. fem. of hótrā-. A second member ‘Hotarship’ would be difficult to construe. 13 The Rig Veda contains two different but related stems, neuter hótra- and feminine hótrā-. The former often means ‘Hotarship, office of Hotar’ (a priestly title), the latter generally ‘oblation, offering’. (In addition there’s a homonymous hótrā- ‘ritual call’.) Since vītí-hotra- is a bahuvrīhi, i.e., an adjective, with the inherent gender of the noun final member neu- tralized, its second member could be either one, and in fact it is surely hótrā-. See the syntagmquotedbyWackernagel,iv.48.1 vihíhótrāḥ‘pursuetheoblations’,whoseindepen- dent object is the acc. pl. fem. of hótrā-. A second member ‘Hotarship’ would be difficult to construe. 14 Of the five occurrences of vītí-hotra-, three modify mortals in a ritual situation (i.84.18, ii.38.1, viii.31.9). The other two (iii.24.2, v.26.3) modify Agni, but Agni is of course the havya-vā́h-, the ‘oblation-conveyor’, who takes to the gods what mortals have offered. He is therefore in possession of oblations that the gods pursue. Similarly, the single occur- rence of vītí-rādhas- (ix.62.29) ‘having largesse worth pursuing’ modifies the soma drop, which offers bounty to Indra. 13 The Rig Veda contains two different but related stems, neuter hótra- and feminine hótrā-. The former often means ‘Hotarship, office of Hotar’ (a priestly title), the latter generally ‘oblation, offering’. (In addition there’s a homonymous hótrā- ‘ritual call’.) Since vītí-hotra- is a bahuvrīhi, i.e., an adjective, with the inherent gender of the noun final member neu- tralized, its second member could be either one, and in fact it is surely hótrā-. See the syntagmquotedbyWackernagel,iv.48.1 vihíhótrāḥ‘pursuetheoblations’,whoseindepen- dent object is the acc. pl. fem. of hótrā-. A second member ‘Hotarship’ would be difficult to construe. 15 Nielsen Whitehead (2012: 45–48) also considers these compounds to be bahuvrīhis (save for dā́ti-vāra- [pp. 45, 48]) and treats each of them in turn, summarizing their various translations and treatments in the secondary literature. I concur with her overall interpretation (p. 48), but the discussions of the individual items are somewhat inconclusive and insufficiently grounded philologically—understandably, since the treatment is a very small part of a dissertation focused primarily on late Latin and Romance. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ the vedic dā́ti-vāra- compound type 11 In other words, the pragmatic ritual context excludes a straight transitive-type reading,‘pursuingtheoblations’,suchaswasassumedbyWackernagel,because that should be appropriate only to gods. The referent of the compound is not pursuing the oblations, but possesses the oblations for others to pursue. I take the first member, vītí-, as standing functionally for the dative infinitive vītáye, in an otherwise standard bahuvrīhi. In the relationship between its parts vītí- hotra- is no different from its etymological and structural close kin, vītá-havya- (3×), with a past participle as first member. In all cases this latter compound qualifies a mortal and should mean ‘whose oblations are pursued’. The differ- ence between the -tí-stem and the -tá- stem in these two compounds may be that of ‘potential’ (to be X-ed) versus ‘achieved’ (X-ed). Unfortunately I do not know of any other pairs of this type. I have now demonstrated that none of the compounds just discussed shows a transitive governing relationship between first member in -ti- and second member, though they do all have reliable relationships with independently attested -ti-stems. Leaving aside the two names púṣti-gu- and śrúṣṭi-gu-, whose meaning and structure cannot properly be assessed, I consider all the others just treated to be bahuvrīhis of a more or less standard sort: ‘whose Y is (with) X’ or, in the case of the vītí- compound, ‘whose Y is for Xing’.15 Crucially, how- ever, none of these needs to undergo a second step to produce a Schindlerian factitive double bahuvrīhi. I do not see why it is necessary, or how it is even possible, to get from rīty-ā̀p- ‘having waters provided with flowing’ to ‘having waters flowing’ to ‘making waters flow’, as in the—to me, highly implausible— semantic derivation provided by Peters (Schindler & Peters 2022: 335; see also Pinault 2018: 335) for the hapax OAv. jə̄nar- (= jan-nar-): ‘having the men pro- videdwithkilling’→‘havingthemenkilled’→‘killingthemen’.16Inthisexample I don’t see why his interpretation, especially of the middle step, would not lead to the hatá-putra- (‘having slain/dead sons’) bahuvrīhi type, with no agentive governing sense, before moving on to the verbal-governing stage. 16 For several such chains of paraphrases, see Pinault (2018: 335), including, for vṛṣṭí-dyu-, ‘ayant (et produisant) le ciel pourvu de pluie’ →‘faisant pleuvoir le ciel.’ The parenthetic addition of ‘(et produisant)’ seems a bit of trickery to me. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ Since this is 16 For several such chains of paraphrases, see Pinault (2018: 335), including, for vṛṣṭí-dyu-, ‘ayant (et produisant) le ciel pourvu de pluie’ →‘faisant pleuvoir le ciel.’ The parenthetic addition of ‘(et produisant)’ seems a bit of trickery to me. Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons org/licenses/by/4 0/ 12 jamison clearly not the sense, I am quite dubious about the sequence of derivational steps; I think it easier to get to ‘killing men’ directly.17 It is the tyranny of the bahuvrīhi that complicates the analysis and requires the positing of a function- ally implausible derivational chain. This leaves us with dā́ti-vāra-,18 which seems to present us with an entirely different situation. But before delving into it, I’d like to point out that what the previous discussion has done is to show that there’s no ‘dā́ti-vāra- type’; the other standard examples have been eliminated, and there is just dā̇ti-vāra-. Whatever historical and comparative conclusions one wishes to draw will have to depend solely on this form. This compound occurs three times in the Rig Veda (i.167.8, iii.51.9, v.58.2). It always occurs at the end of a Triṣṭubh line, and in all three occurrences it modifies the Maruts.19 I do not see any way out of taking it as a transitive con- struction ‘granting wishes, giving choice things,’20 and so I concur with the usual interpretation. E.g., (2) v.58.2 tveṣáṃgaṇáṃtávasaṃkhā́dihastaṃ, dhúnivratam māyínaṃdā́ti- vāram … the turbulent (Marut) flock, powerful, with bangles on their hands, of boisterous commandment, masters of artifice, granting wishes … 17 In fact, this hapax is a bit of a weak reed, since its grammatical analysis is disputed. I think the analysis of jə̄nǝrąm as a gen. pl. of jan-nar- ‘killing men,’ directly adjacent to and conjoined with xrū-nǝrąm, probably meaning ‘injuring men,’ in Y. 53.8 is almost surely correct—an analysis that goes back to Humbach and is already reflected in his first Gāthā translation (1959: i.159 and ii.97) and accepted by most subsequent translators and com- mentators. However, it should be noted that Bartholomae (1904 s.vv.) takes both forms as accusatives of long-ā feminines, jə̄nǝrā- ‘Töten, Mord’ and xrūnǝrā- ‘blutige Misshand- lung, Blutbad’ respectively, and that Kellens & Pirart (1991: 273) interpret both forms as middle 3. pl. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ imperatives (on what seem to me dubious grounds) (though see Kellens 1974: 387, which accepts Humbach’s analysis). 18 Scarlata (1999: 219) also tackles dā́ti-vāra- in the course of a discussion of havyá-dāti-. For dā́ti- in dā́ti-vāra- he considers both nominal and verbal possibilities, but reaches no firm conclusions. 19 In my opinion. In i.167.8 others think that it modifies a mortal worshiper. See Jamison (2015–, ad loc.). 20 One could try an interpretation parallel to that of vītí-hotra-, namely ‘having wishes for granting’. Although the verb-phrase formulaic evidence that is about to be presented might seem to exclude that, Elizabeth Tucker suggests to me that, assuming the com- pound →verb phrase scenario (see below), it could have had this meaning to begin with, but the first member was synchronically reinterpreted as a third singular verb, spawning the finite form dāti. 20 One could try an interpretation parallel to that of vītí-hotra-, namely ‘having wishes for granting’. Although the verb-phrase formulaic evidence that is about to be presented might seem to exclude that, Elizabeth Tucker suggests to me that, assuming the com- pound →verb phrase scenario (see below), it could have had this meaning to begin with, but the first member was synchronically reinterpreted as a third singular verb, spawning the finite form dāti. 21 We might also adduce the phrase dhā́ti rátnam with a similarly anomalous primary form to the root aor. adhāt, to the phonologically and semantically parallel root √dhā ‘place’, in ii.38.1 nūnáṃdevébhyo ví hí dhā́ti rátnam# ‘for now he distributes treasure to the gods’. The object rátnam fills the same semantic slot as vā́ryam, vā́jam, vásu.There are two other attestations of dhāti (iv.55.1, vii.90.3); although these do not participate in the formulaic web, they do occupy the same metrical slot: syllables 8 and 9 in a Triṣṭubh cadence. 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ the vedic dā́ti-vāra- compound type 13 Like most of the other putative compound examples we examined above, it participatesinaformulaicweb—but,significantly,notwitha-ti-stemnominal, as they all did. Recall that though -dāti- is found in the well-attested compound havyá-dāti- ‘bestowal of oblations,’ it is not attested independently. Instead dā́ti-vāra- exists parallel to a number of syntagms containing almost the same elements, but with a finite verb. The verb is 3. sg. dāti, seemingly belonging to the root aorist, but with a primary ending—making it, of course, strikingly anomalous (though see below). As object we find vā́riya-, a variant of vā́ra- whose suffix allows distraction and therefore a Jagatī cadence (or a dimeter iambic cadence). Like most of the other putative compound examples we examined above, it participatesinaformulaicweb—but,significantly,notwitha-ti-stemnominal, as they all did. Recall that though -dāti- is found in the well-attested compound havyá-dāti- ‘bestowal of oblations,’ it is not attested independently. Instead dā́ti-vāra- exists parallel to a number of syntagms containing almost the same elements, but with a finite verb. The verb is 3. sg. dāti, seemingly belonging to the root aorist, but with a primary ending—making it, of course, strikingly anomalous (though see below). As object we find vā́riya-, a variant of vā́ra- whose suffix allows distraction and therefore a Jagatī cadence (or a dimeter iambic cadence). (3) vii.15.12 dítiś ca dāti vā́riyam# And Diti gives something to be desired. (4) v.48.5 yáto bhágaḥsavitā́ dā́ti vā́riyam# … from which Bhaga (and) Savitar give something to be desired. (4) v.48.5 yáto bhágaḥsavitā́ dā́ti vā́riyam# … from which Bhaga (and) Savitar give something to be desired. The phrase is also found pāda-internal: The phrase is also found pāda-internal: (5) vii.42.4 sá … dāti vā́riyam … He gives something to be desired. And instead of dāti, the imperative is once substituted—but it’s entirely para- sitic on dāti, which follows in the next verse (see vii.15.12 cited above). And instead of dāti, the imperative is once substituted—but it’s entirely para- sitic on dāti, which follows in the next verse (see vii.15.12 cited above). (7) vi.24.2 vājī́… dā́ti vā́jam# The prize-winner gives a prize. 22 Three examples among the extremely numerous forms of √dā: x.17.7 … vā́riyaṃdāt#; viii.71.11 #dānā́ya vā́riyāṇām#; and in a compound viii.71.3 rayím dehi viśvávāram. 23 Scarlata (1999: 219) also floats this possibility, and it is implicit in Lubotsky’s location of the forms in his listings (1997: 669 & 746). 23 Scarlata (1999: 219) also floats this possibility, and it is implicit in Lubotsky’s location of the forms in his listings (1997: 669 & 746). 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ And instead of dāti, the imperative is once substituted—but it’s entirely para- sitic on dāti, which follows in the next verse (see vii.15.12 cited above). (6) vii.15.11 bhágaś ca dātu vā́riyam# And let Bhaga give something to be desired. We also find dāti with semantically similar objects:21 (7) vi.24.2 vājī́… dā́ti vā́jam# The prize-winner gives a prize. 21 We might also adduce the phrase dhā́ti rátnam with a similarly anomalous primary form to the root aor. adhāt, to the phonologically and semantically parallel root √dhā ‘place’, in ii.38.1 nūnáṃdevébhyo ví hí dhā́ti rátnam# ‘for now he distributes treasure to the gods’. The object rátnam fills the same semantic slot as vā́ryam, vā́jam, vásu.There are two other attestations of dhāti (iv.55.1, vii.90.3); although these do not participate in the formulaic web, they do occupy the same metrical slot: syllables 8 and 9 in a Triṣṭubh cadence. Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. 22 Three examples among the extremely numerous forms of √dā: x.17.7 … vā́riyaṃdāt#; viii.71.11 #dānā́ya vā́riyāṇām#; and in a compound viii.71.3 rayím dehi viśvávāram. jamison jamison 14 Once non-adjacent: (8) iv.8.3 dā́ti priyā́ṇi cid vásu He gives especially dear goods. (8) iv.8.3 dā́ti priyā́ṇi cid vásu He gives especially dear goods. All of these have Verb Object order and most are adjacent and pāda-final, just like the compound dā́ti-vāra-. Outside of this little clutch of passages, vā́ra- / vā́rya- are seldom objects of √dā,22 so this phenomenon is not a byproduct of a larger formulaic system, but localized in the dā́ti-vāra- realm. I find these data baffling—and I also find it baffling that with all the ink spilled on the compound dā́ti-vāra- these syntagms barely figure. Among other things these are the only five occurrences of the anomalous finite dā́ti with pri- mary ending, and the fact that it’s only attested in this formulaic sphere, closely tied with the anomalous compound dā́ti-vāra-, should give us pause. But to keep our focus on the compound, one thing we can say for certain is that synchronically, to Vedic poets, dā́ti-vāra- contains a finite verb, not a -ti-stem noun, and in this it is quite distinct from the other forms supposedly belonging to this ‘type’, whose formulaic affinities to -ti-stems were explored above. I would like to emphasize the word ‘synchronically’. As for their history, twodiametricallyopposeddiachronicscenariospresentthemselvestoaccount for the relationship between dā́ti-vāra- and dāti vā́riyam (etc.)—scenarios that are almost equally balanced—and therefore almost equally flawed—in a clas- sic chicken-or-egg dilemma. On the one hand, the compound dā́ti-vāra-, the lone example of the precious PIE verbal-governing type in -ti- to be found in Vedic, was inherited, but, quite possibly because of its isolation, lost any trans- parency it once had. It was then reanalyzed as containing a finite verb, and a limited set of formulaic verb phrases were then generated from this misanaly- sis: compound →verb phrase. Alternatively, the finite verb dāti in the formulaic phrase dāti vā́ram could have lost its transparency, since it should belong to a root aorist stem, where indicative forms should not have primary endings. The best way to account for such a finite form is as a root aorist subjunctive (dā-a-ti).23 There is no posi- tive evidence for this analysis unfortunately; a distracted root syllable would be highly desirable but is not found. However, distraction is not a universal requirement in these forms, and in fact all of the finite occurrences of dā́ti / 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. 24 Most of them are so rendered in Jamison & Brereton (2014: dā́ti iv.8.3, v.48.5, dāti vii.52.4, dhāti iv.55.1, vii.90.3). Adjustments to the others (dāti vi.24.2, vii.15.12, dhā́ti ii.38.1) have been registered ad locc. in Jamison (2015–). 25 For a rich, though incomplete, collection, see Wackernagel (1905: 323–329, esp. 326–328). In addition to the selection of early Vedic examples cited in the text and by Wacker- nagel, consider well-known later forms like nāstika- ‘atheist’, nāstitā- ‘non-existence’, both < na+asti ‘(it) does not exist’. 24 Most of them are so rendered in Jamison & Brereton (2014: dā́ti iv.8.3, v.48.5, dāti vii.52.4, dhāti iv.55.1, vii.90.3). Adjustments to the others (dāti vi.24.2, vii.15.12, dhā́ti ii.38.1) have been registered ad locc. in Jamison (2015–). (8) iv.8.3 dā́ti priyā́ṇi cid vásu He gives especially dear goods. https://creativecommons.org/licenses/by/4.0/ 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. the vedic dā́ti-vāra- compound type 15 dāti and dhā́ti / dhāti in the RV are compatible with a subjunctive interpreta- tion.24Nonetheless,sincenon-distracteddātiappearstobeapresentindicative because its root vowel and mood sign coalesced, it sits uncomfortably in the well-attestedrootaoristof √dā;unlikeother rootaoristsubjunctiveslikekarati, the root syllable and the mood sign cannot be kept separate and so the mor- phological analysis is not clear: the default analysis would be as root present indicative 3. sg. active. According to this scenario, because of this apparent anomaly the verb phrase became univerbated to the dā́ti-vāra- nominal com- pound that we have: verb phrase →compound. For parallels to this phrasal univerbation I can point to other examples of compounds based on phrases:25 already in the RV we have forms like mama- sat-yá- (x.42.4) ‘[struggle for] what’s mine’ (< ‘[this] being mine’), khada-prī́- (i.38.1, viii.7.31) / kadha-priya (voc. i.30.20) ‘when?-friend’ (= fair-weather friend), in the AV (Ś+P) aham-uttará- / aham-uttara-tvá- ‘(contest for) preem- inence’ (lit. ‘I (am) higher’); verbal syntagms are found in forms like AV (Ś+P) mām-paśya-‘lookatme’(nameof aplantusedinalovecharm)andthenameof the literary genre itihāsá- < íti ha+āsa ‘thus indeed it was; it was just so’ already AV(Ś+P) and well attested throughout the epic and Classical periods. However, thesephrasalcompoundsgenerallyrefertoanentityexternaltothephrase,e.g., theliterarygenreItihāsa,whereasthecompounddā́ti-vāra-issemanticallyand functionally indistinguishable from the free syntagm. Still, on the whole, this latter scenario, verb phrase →compound, seems somewhat more likely to me, primarily because under the compound →verb phrase scenario it is hard to envision how this archaic inherited compound made its way from PIE to Vedic: a single precious relic surviving the perilous centuries, indeed millennia, leaving no trace in Iranian (a significant absence), to fetch up three times in the Rig Veda and then die. But this does not mean that the alternative scenario, verb phrase →com- pound, is without problems. First, even if the aorist subjunctive dāti isn’t a particularly favored form, what could possibly motivate the univerbation to an unprecedented type of compound—since, as I have just shown, the scat- tered -ti-stem compounds usually grouped with dā́ti-vāra- are functionally and Indo-European Linguistics (2024) 1–18 \| 10.1163/22125892-bja10030 via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons org/licenses/by/4 0/ 16 jamison formally distinct and can’t provide a model? 26 One possible motivation could be that the wish-granting referent is not a nominative sin- gular subject, and so the 3. sg. verb dāti would not be appropriate. Indeed, only one of the three occurrences of the compound is nom. sg. dā́tivāraḥ(i.167.8, modifying [in my view] the Marut flock); the other two are acc. sg. masc. dā́tivāram (v.58.2) and nom. pl. masc. dā́tivārāḥ. However, this strikes me as an after-the-fact justification, which presupposes that the compound already existed. In other words, I can’t imagine a Vedic bard thinking, ‘I want to say that the Maruts grant wishes, but I can’t use dāti in the plural. Hanta, I’ll make it into a compound!’ And in any case, why would the same argument not hold for dāti vā́riyam? (8) iv.8.3 dā́ti priyā́ṇi cid vásu He gives especially dear goods. Moreover, the syntagms involv- ing dāti vā́riyam (etc.) show that dāti was capable of remaining an indepen- dent finite verb. Why did the vā́ram forms univerbate, but not vā́riyam?26 As I pointed out above, those formulae are mere metrical variants, with -vāram suiting a Triṣṭubh cadence and vā́riyam a Jagatī cadence. What could trigger such an extreme syntactic cleavage? I do not have answers for these serious questions (and do not want to force them), but I still weakly favor the verb phrase →compound trajectory over compound →verb phrase, simply because the time depth required to bring the isolated compound safely from PIE to Vedic seems excessive and its absence from Iranian concerning. But, either way, the fact that dā́ti-vāra- ‘presents’ as a verb phrase precludes the production of new such compounds within Vedic on its model, with nomi- nal -ti-stems and transitive semantics: dā́ti-vāra- stands apart. To sum up briefly, for well over a century a small group of Vedic com- pounds with first members ending in -ti- have been interpreted as transi- tive verbal-governing compounds comparable to supposedly similar forma- tions in other IE branches and supposedly bolstered by transitive syntagms in Vedic itself. Both the roster of examples and their interpretation have become frozen, deployed as an unexamined class in publication after publication, with the accuracy of the interpretations assumed and theoretical superstructures erected upon them. By reexamining the evidence, I have shown that all but one of the canonical Vedic examples have to be interpreted in other ways—leaving only one standing, dā́ti-vāra- ‘giving choice things’. But since this compound is closely linked to verbal syntagms with the same or similar elements, contain- ing dāti as a finite verb with accusative object, the history and status of this compound are muddied: is it a recent univerbation of a verb phrase, or were the associated verb phrases generated from the compound? If the former is the case, Vedic has no evidence for the supposed PIE verbal governing com- pounds to -ti- stems. If the latter, dā́ti-vāra- may be inherited, even from PIE, but as an isolate, since, as I’ve just demonstrated, none of the other compounds that have been associated with dā́ti-vāra- since Wackernagel actually belongs 26 One possible motivation could be that the wish-granting referent is not a nominative sin- gular subject, and so the 3. sg. verb dāti would not be appropriate. (8) iv.8.3 dā́ti priyā́ṇi cid vásu He gives especially dear goods. Indeed, only one of the three occurrences of the compound is nom. sg. dā́tivāraḥ(i.167.8, modifying [in my view] the Marut flock); the other two are acc. sg. masc. dā́tivāram (v.58.2) and nom. pl. masc. dā́tivārāḥ. However, this strikes me as an after-the-fact justification, which presupposes that the compound already existed. In other words, I can’t imagine a Vedic bard thinking, ‘I want to say that the Maruts grant wishes, but I can’t use dāti in the plural. Hanta, I’ll make it into a compound!’ And in any case, why would the same argument not hold for dāti vā́riyam? 10.1163/22125892-bja10030 \| Indo-European Linguistics (2024) 1–18 Downloaded from Brill.com 10/24/2024 04:05:46AM via Open Access. This is an open access article distributed under the terms of the CC BY 4.0 license. https://creativecommons.org/licenses/by/4.0/ the vedic dā́ti-vāra- compound type 17 to such a class. In this case those who wish to claim deep antiquity for the sup- posed type have only a single Vedic form, not a class, to play with. Acknowledgments I am grateful to the two anonymous Readers for this journal, and to Joel Brere- ton, José Luis García Ramón, Jesse Lundquist, and Elizabeth Tucker, for care- ful reading and helpful comments on earlier drafts of the paper, as well as to the audience of its first public presentation, at the East Coast Indo-European Conference, Harvard University, June 2022, and to the members of the UCLA Greco-Indo-Iranian seminar in Spring 2022, where it first took shape. References Bartholomae, Christian. 1904. Altiranisches Wörterbuch. Strassburg: Trübner. Bartholomae, Christian. 1904. Altiranisches Wörterbuch. Strassburg: Trübner. Bartholomae, Christian. 1904. Altiranisches Wörterbuch. Strassburg: Trübner. Debrunner, Albert. 1954. Altindische Grammatik ii/2, Die Nominalsuffixe. Göttingen: Debrunner, Albert. 1954. Altindische Grammatik ii/2, Die Nominalsuffixe. Göttingen: Vandenhoeck & Ruprecht. ff g Vandenhoeck & Ruprecht. Vandenhoeck & Ruprecht. Debrunner, Albert. 1957. 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https://openalex.org/W2136063721	https://europepmc.org/articles/pmc2695808?pdf=render	English	null	Cytoplasmic Continuity Revisited: Closure of Septa of the Filamentous Fungus Schizophyllum commune in Response to Environmental Conditions	PloS one	2,009	cc-by	3,901	Abstract Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: h.a.b.wosten@uu.nl The major groups of fungi within the Basidiomycota contain different types of septa. The Pucciniomycotina and the Ustilagi- nomycotina have relatively simple septa [1,11,12]. In contrast, septa of the Agaricomycotina are relatively complex. They consist of a barrel-shaped swelling around the pore, the dolipore, which is associated with a septal pore cap (SPC) [6]. This septal pore cap, which restricts organelle translocation, can be of the vesiculate type, the perforate type or the imperforate type [13] and is assumed to be derived from the endoplasmic reticulum [14–16]. The SPC of Schizophyllum commune is of the perforate type. Its base, i.e. the part closest to the septum, has a diameter of 450–600 nm and the whole structure is regularly perforated by openings of approximately 100 nm [15,17]. Cytoplasmic Continuity Revisited: Closure of Septa of the Filamentous Fungus Schizophyllum commune in Response to Environmental Conditions Arend F. van Peer1, Wally H. Mu¨ ller2, Teun Boekhout3, Luis G. Lugones1, Han A. B. Wo¨ sten1* 1 Department of Microbiology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands, 2 Department of Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands, 3 CBS Fungal Biodiversity Centre, Utrecht, The Netherlands Abstract Background: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. Methodology/Principal Findings: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. Conclusions/Significance: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous perse. Citation: van Peer AF, Mu¨ller WH, Boekhout T, Lugones LG, Wo¨sten HAB (2009) Cytoplasmic Continuity Revisited: Closure of Septa of the Filamentous Fungus Schizophyllum commune in Response to Environmental Conditions. PLoS ONE 4(6): e5977. doi:10.1371/journal.pone.0005977 Editor: Alexander Idnurm, University of Missouri-Kansas City, United States of America Editor: Alexander Idnurm, University of Missouri-Kansas City, United States of America Received April 10, 2009; Accepted May 27, 2009; Published June 22, 2009 Copyright: 2009 van Peer 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: 2009 van Peer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by Utrecht University. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. June 2009 \| Volume 4 \| Issue 6 \| e5977 PLoS ONE \| www.plosone.org Results and Discussion S. commune was grown in a glass bottom culture dish in a thin layer of solidified minimal medium (MM) containing glucose as a carbon source. The solidified medium was overlaid with liquid MM (Figure 1A). Extension of selected hyphae was followed during a 3 min period using the light microscope of the PALM CombiSys- tem. Compartments of growing hyphae were disrupted with the laser of the PALM Combi-system within 30 mm from the septum. Cytoplasm of disrupted compartments spilled into the surrounding medium (Figure 2). Cytoplasmic flow from the adjacent compart- ment into the medium depended on the state of the septum (Figure 2; Movies S1, S2, S3, S4, S5, S6). Loss of cytoplasm was considerable (septum was open and was slowly closed; Movies S5- S6), minor (septum was open but was quickly closed; Movies S3-S4) or not detected (septum was already closed; Movies S1-S2). By The fact that the second and third septa were often closed came to a surprise considering the phenomenon of streaming of cytosol in a fungal mycelium [26]. We argued that in the presence of a surplus of glucose in the medium cytosolic streaming is not essential and that therefore septal pores can be closed. To test this hypothesis, hyphae were grown from a glucose-containing medium into a medium without this carbon source (Figure 1B). Indeed, in MM without glucose many more septa of growing hyphae were open (i.e. 11 out of 12 apical septa, 22 out of 29 second septa and 26 out of 41 third septa; Table 1). Taken together, these findings show that the continuity of the cytoplasm of S. commune depends on the presence of carbon source in the medium. Figure 1. Glass bottom culture dish used to assess plugging of septa in S. commune. In most cases S.commune was grown in a thin layer of agar medium overlaid with minimal medium (A). However, liquid medium was not added when it was assessed whether septa were open or closed in the absence of glucose in the medium. In this case, the culture dish contained distinct patches of minimal medium with or without glucose, which were separated by a gap of 5 mm (B). (C) Magnification of boxed area in (A) and (B) showing a hypha with the first and second septum as referred to in the text. Introduction A fungal mycelium is the result of fusing hyphae that grow at their apices and that branch subapically. In general, hyphae of the lower fungi, i.e. the Glomeromycota, Zygomycota, and Chytri- diomycota are sparsely, if at all, septated [1–3]. Therefore, the cytoplasm within mycelia of these fungi is continuous. Hyphae of the higher fungi, i.e. the Ascomycota and Basidiomycota, are compartmentalized by septa. These septa contain central pores of up to 500 nm that allow streaming of cytoplasm and translocation of organelles like mitochondria and nuclei [1–3]. Therefore, the cytoplasm within these mycelia is also considered to be continuous. This discriminates the filamentous fungi from plants and animals. In these latter two kingdoms there are also intercellular cytoplasmic connections but they are much smaller. Gap junctions in animals and plasmodesmata in plants have pores with a diameter of about 1.5 to 3.0 nm. These pores allow streaming of inorganic ions and small water-soluble organic molecules [8–10]. It should be noted that the diameter of the pores of plasmodesmata and gap junctions is dynamic. For instance, the channels in plasmodesmata can be closed or their width increased to 5 to 9 nm. Septa of Ascomycota and the Basidiomycetes become plugged in response to hyphal damage to prevent loss of cytoplasm [18– 20]. Peroxisome-like organelles, called Woronin bodies, plug the septa of the ascomycetes [21–23], whereas in basidiomycetes septa are closed by electron dense, plugging material [19]. It has been proposed that the SPC is involved in the plugging process [15,20,24,25]. Here, it is shown by laser dissection that septa of growing hyphae of S. commune not only plug in response to hyphal damage but that this is also caused by environmental conditions PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org June 2009 \| Volume 4 \| Issue 6 \| e5977 1 PLoS ONE \| www.plosone.org June 2009 \| Volume 4 \| Issue 6 \| e5977 Fungal Cytoplasmic Continuity cutting the second compartment and following cytoplasmic streaming from the apical compartment into the medium it was shown that 45 out of 45 apical septa were open (Table 1). In contrast, only 25 out of 53 of the second septum (separating compartment 2 and 3; see Figure 1C) and 1 out of 14 of the third septum (separating compartment 3 and 4) were open. This was shown to occur in growing hyphae throughout the mycelium. Introduction The second and third septa closed quickly in the case they were open. In contrast, closure of the most apical septum was generally slower. such as availability of carbon source, exposure to high tempera- ture, osmotic shock or toxic agents. The results thus imply that the cytoplasmic continuity of this fungus depends on the environmen- tal conditions. Results and Discussion doi:10.1371/journal.pone.0005977.g001 PLoS ONE \| www.plosone.org 2 June 2009 \| Volume 4 \| Issue 6 \| e5977 Figure 1. Glass bottom culture dish used to assess plugging of septa in S. commune. In most cases S.commune was grown in a thin layer of agar medium overlaid with minimal medium (A). However, liquid medium was not added when it was assessed whether septa were open or closed in the absence of glucose in the medium. In this case, the culture dish contained distinct patches of minimal medium with or without glucose, which were separated by a gap of 5 mm (B). (C) Magnification of boxed area in (A) and (B) showing a hypha with the first and second septum as referred to in the text. doi:10.1371/journal.pone.0005977.g001 June 2009 \| Volume 4 \| Issue 6 \| e5977 PLoS ONE \| www.plosone.org 2 Fungal Cytoplasmic Continuity Figure 2. A septum is open when cytoplasm moves through the septum after laser dissection of a neighbouring compartment. Compartments before (A, E) and after (B–D, F–H) dissection of a hypha with a closed (A–D) and an open (E–H) septum. Cytoplasmic flow in E–H is indicated by the movement of a vacuole (arrows) through the septum (). Dots represent the position of the laser. doi:10.1371/journal.pone.0005977.g002 Figure 2. A septum is open when cytoplasm moves through the septum after laser dissection of a neighbouring compartment. Compartments before (A, E) and after (B–D, F–H) dissection of a hypha with a closed (A–D) and an open (E–H) septum. Cytoplasmic flow in E–H is indicated by the movement of a vacuole (arrows) through the septum (). Dots represent the position of the laser. doi:10.1371/journal.pone.0005977.g002 We reasoned that another environmental condition that could affect the septal pore is exposure to stress. To test this, the liquid medium overlaying the solid minimal medium (Figure 1A) was replaced by deionized water. This neither affected growth nor closure of the apical septum in a 60 minutes interval (Table 2). In contrast, addition of 1 M MgSO4 to the liquid medium did have an effect on the state of the septal pore. Addition of MgSO4 initially resulted in the accumulation of vacuoles and after 15 minutes most hyphae had stopped growing. At this point, all apical septa were still open. However, 5 out of 5 apical septa had closed after an additional 30 minutes of exposure to 1M MgSO4. Results and Discussion Absence of streaming of cytoplasm into the medium from the adjacent compartment was not Table 1. Plugging of a septal pore depends on its position in the hypha and the presence of glucose in the medium. Treatment Septum* Total number of Septa Open Septa Closed Septa % Open Septa MM + glucose 1 45 45 0 100 2 53 25 28 47 3 14 1 13 7 MM - glucose 1 12 11 1 92 2 29 22 7 76 3 41 26 15 63 Septum 1 separates the apical compartment from the second compartment; septum 2 separates compartment 2 and 3, and septum 3 compartment 3 and 4. doi:10.1371/journal.pone.0005977.t001 Table 1. Plugging of a septal pore depends on its position in the hypha and the presence of glucose in the medium. Septum 1 separates the apical compartment from the second compartment; septum 2 separates compartment 2 and 3, and septum 3 compartment 3 and 4. doi:10.1371/journal.pone.0005977.t001 l compartment from the second compartment; septum 2 separates compartment 2 and 3, and septum 3 compartment 3 and 4. 977.t001 Septum 1 separates the apical compartment from the second compartment; septum 2 separates compartment 2 and 3, and septum 3 compartment 3 and 4. doi:10.1371/journal.pone.0005977.t001 June 2009 \| Volume 4 \| Issue 6 \| e5977 PLoS ONE \| www.plosone.org 3 Fungal Cytoplasmic Continuity Table 2. Plugging of the apical septum depends on environmental stress. Treatment Total number of Septa** Open Septa Closed Septa % Open Septa Hypotonic, 45 min 5 5 0 100 Hypertonic, 45 min 5 0 5 0 20 mg ml21 nourseothricin 5 1 4 20 45uC, 30 min 5 0 5 0 45uC, 30 min; 25uC, 15 min 5 4 1 80 0uC, 30 min 5 4 1 80 220uC, 30 min 5 5 0 100 Hyphae were grown in MM medium with glucose. Hypotonic or hypertonic conditions were created by overlaying the agar medium with water and 1 M MgSO4, respectively. Nourseothricin was added to the liquid medium overlaying the agar medium. Hyphae were analysed in two independent experiments. doi:10.1371/journal.pone.0005977.t002 Hyphae were grown in MM medium with glucose. Hypotonic or hypertonic conditions were created by overlaying the agar medium with water and 1 M MgSO4, respectively. Nourseothricin was added to the liquid medium overlaying the agar medium. **Hyphae were analysed in two independent experiments. doi:10.1371/journal.pone.0005977.t002 source. Analysis of plugging Glass bottom culture dishes were mounted on a PALM CombiSystem (Carl Zeiss MicroImaging GmbH, Munich, Ger- many). Disruption of compartments was performed with laser pulses (laser setting ‘‘dots’’, laser power 65%). Movies were captured to assess whether septa were open or closed. To this end, spilling of cytoplasm from compartments adjacent to the disrupted compartment was monitored. Supporting Information Movie S1 This septum is closed. There is no spilling of cytoplasm from the compartment that is adjacent to the one that is damaged. Our data show that hyphae initially accumulate vacuoles upon exposure to stress, after which their growth halts. This is followed by septal closure. Closure of the septum may thus be a final rescue system that is activated to have individual compartments survive the stress condition. Notably, plugging was shown to be reversible, at least when induced by heat. This indicates that septal pores are dynamic structures that can open and close depending on the environmental conditions. Taken together, it is concluded that the cytoplasm of a mycelium of a higher fungus is not continuous per se, as is generally assumed. The cytoplasmic connections within a mycelium of S. commune thus resemble those in plants and animals. Like the septal pore of this basidiomycete, plasmodesmata and gap junctions can reversibly open and shut [8,27,28]. Found at: doi:10.1371/journal.pone.0005977.s001 (6.14 MB MP3) Movie S2 This septum is closed. There is no spilling of cytoplasm from the compartment that is adjacent to the one that is damaged. Found at: doi:10.1371/journal.pone.0005977.s002 (2.40 MB MP3) Found at: doi:10.1371/journal.pone.0005977.s002 (2.40 MB MP3) Movie S3 This septum is open but it closes quickly after the compartment is damaged. There is minor spilling of cytoplasm from the compartment that is adjacent to the one that is damaged. Found at: doi:10.1371/journal.pone.0005977.s003 (6.65 MB MP3) Results and Discussion In this case the agar media were not topped with liquid medium after inoculation with a plug of mycelium. due to the presence of vacuoles near the septum. Exposure of S. commune to 20 mg ml21 nourseothricin, which inhibits protein synthesis, triggered a similar response as 1M MgSO4 (Table 2). Within 30 minutes, the hyphal tips stopped growing and mild vacuolization was observed. Yet, septa were still open. After another 30 minutes, all hyphae were heavily vacuolized and almost all apical septa had closed. Transfer of the mycelium from 25uC to 0 or 220uC did not cause plugging. Hyphae continued their original growth rate when they were placed back at 25uC. Exposing the mycelium to 45uC for 30 minutes stopped growth. Hyphae vacuolized and all apical septa had closed (Table 2). Interestingly, most septa opened again 15 minutes after colonies were placed back at 25uC. During this time, hyphae restored normal growth and vacuolization was decreased to normal levels. Materials and Methods Strains and growth conditions References 16. Moore RT, Patton AM (1975) Parenthesome fine structure in Pleurotus cystidiosus and Schizophyllum commune. Mycologia 67: 1200–1205. 1. Bauer R, Begerow D, Sampaio JP, Weiss M, Oberwinkler F (2006) The simple septate basidiomycetes: a synopsis. Mycol Prog 5: 41–66. 1. Bauer R, Begerow D, Sampaio JP, Weiss M, Oberwinkler F (2006) The simple septate basidiomycetes: a synopsis. Mycol Prog 5: 41–66. septate basidiomycetes: a synopsis. Mycol Prog 5: 41–66. p y y g 17. Mu¨ller WH, van Aelst AC, van der Krift TP, Boekhout T (1994) Scanning 2. Barr DJS (2001) Chytridiomycota. In: McLaughlin DJ, McLaughlin EG, Lemke PA, eds. The Mycota VII, Systematics and evolution, Part A;Berlin: Springer-Verlag, 93–112. 17. Mu¨ller WH, van Aelst AC, van der Krift TP, Boekhout T (1994) Scanning electron microscopy of the septal pore cap of the basidiomycete Schizophyllum commune. Can J Microbiol 40: 879–883. electron microscopy of the septal pore cap of the basidiomycete Schizophyllum commune. Can J Microbiol 40: 879–883. 3. Benny GL, Humber RA, Morton JB (2001) Zygomycota: Zygomycetes. In: McLaughlin DJ, McLaughlin EG, Lemke PA, eds. The Mycota VII, Systematics and evolution, Part A. Berlin: Springer-Verlag, pp. 113–146. 18. Trinci APJ, Collinge AJ (1974) Occlusion of the septal pores of damaged hyphae of Neurospora crassa by hexagonal crystals. Protoplasma 80: 57–67. of Neurospora crassa by hexagonal crystals. Protoplasma 80: 57–67. 19. Aylmore RC, Wakley GE, Todd NK (1984) Septal sealing in the basidiomycete Coriolus versicolor. J Gen Microbiol 130: 2975–2982. 4. Shatkin AJ, Tatum EL (1959) Electron microscopy of Neurospora crassa mycelia. J Biophys Biochem Cytol 6: 423–426. J Biophys Biochem Cytol 6: 423–426. 20. Markham P (1994) Occlusions of septal pores in filamentous fungi. Mycol Res 98: 1089–1106. 5. Moore RT, McAlear JH (1962) Fine structures of mycota. Observations on septa of ascomycetes and basidiomycetes. Am J Bot 49: 86–94. of ascomycetes and basidiomycetes. Am J Bot 49: 86–94. 21. 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Perkins G, Goodenough D, Sosinsky G (1997) Three-dimensional structure of the gap junction connexon. Biophys J 72: 533–544. 24. Mu¨ller WH, Koster AJ, Humbel BM, Ziese U, Verkleij AJ, et al. (2000) Automated electron tomography of the septal pore cap in Rhizoctonia solani. J Struct Biol 131: 10–18. 10. Ghoshroy S, Lartey R, Sheng J, Citovsky V (1997) Transport of proteins and nucleic acids through plasmodesmata. Annu Rev Plant Physiol Plant Mol Biol 48: 27–50. 25. van Driel KGA, van Peer AF, Grijpstra J, Wo¨sten HAB, Verkleij AJ, et al. (2008) Septal pore cap protein SPC18, isolated from the basidiomycetous fungus Rhizoctonia solani, also resides in pore plugs. Eukaryot Cell 7: 1865–1873. 11. Oberwinkler F, Bandoni RJ (1982) A taxonomic survey of the gasteroid, auricularioid Heterobasidiomycetes. Can J Bot 60: 1726–1750. 26. Jennings DH (1987) Translocation of solutes in fungi. Biol Rev 62: 215–243. 12. Bauer R, Oberwinkler F, Vanky K (1997) Ultrastructural markers and systematics in smut fungi and allied taxa. Can J Bot 75: 1273–1314. 27. Oparka KJ, Roberts AG (2001) Plasmodesmata. A not so open-and-shut case. Plant Physiol 125: 123–126. systematics in smut fungi and allied taxa. Can J Bot 75: 1273–13 13. McLaughlin DJ, Frieders EM, Lu¨ H (1995) A microscopist’s view of hetero- basidiomycete phylogeny. Stud Mycol 38: 91–109. 28. Ruan Y-L, Llewellyn DJ, Furbank RT (2001) The control of single-celled cotton fiber elongation by developmentally reversible gating of plasmodesmata and coordinated expression of sucrose and K+ transporters and expansin. Plant Cell 13: 47–63. 14. Girbardt M (1961) Licht- und Elektronenmikroskopische Untersuchungen an Polystictus versicolor. Arch Mikrobiol 39: 351–359. y 15. Mu¨ller WH, Montijn RC, Humbel BM, van Aelst AC, Boon EJM, et al. (1998) Structural differences between two types of basidiomycete septal pore caps. Microbiology 144: 1721–1730. 29. Dons JJM, de Vries OMH, Wessels JGH (1979) Characterization of the genome of the basidiomycete Schizophyllum commune. Author Contributions Conceived and designed the experiments: AFvP LGL HAW. Performed the experiments: AFvP LGL. Analyzed the data: AFvP LGL HAW. Conceived and designed the experiments: AFvP LGL HAW. Performed the experiments: AFvP LGL. Analyzed the data: AFvP LGL HAW. Strains and growth conditions Movie S4 This septum is open but it closes quickly after the compartment is damaged. There is minor spilling of cytoplasm from the compartment that is adjacent to the one that is damaged. Found at: doi:10.1371/journal.pone.0005977.s004 (3.62 MB MP3) g S. commune strain 4–8 (FGSC # 9210 VT # H4–8) was grown in the light at 25uC on minimal medium with 2% glucose (MM; 29). Plugging experiments were performed in glass bottom culture dishes (P35G-0-20-C, MatTek Corporation, Ashland, MA, USA). To this end, wells in the dishes (20 mm in diameter, 1 mm in height) were filled with 400 ml MM containing 1% agarose. Cultures were inoculated with a plug of S. commune mycelium that was gently pushed in the agar medium containing glucose. Dishes were filled with 2 ml liquid MM (Figure 1A) and transferred to a water vapour saturated chamber at 25uC for 2–3 days. Heat stress was applied by floating the glass bottom culture dish in a water bath at 45uC. Alternatively, wells were filled with MM that contained glucose on one side but no carbon source on the other side (Figure 1B). These media were separated by a 5 mm gap preventing glucose to diffuse into the medium without carbon Movie S5 This septum is open and it closes slowly after the compartment is damaged. There is major spilling of cytoplasm from the compartment that is adjacent to the one that is damaged. Found at: doi:10.1371/journal.pone.0005977.s005 (1.80 MB MP3) Movie S6 This septum is open and it closes slowly after the compartment is damaged. There is major spilling of cytoplasm from the compartment that is adjacent to the one that is damaged. Found at: doi:10.1371/journal.pone.0005977.s006 (0.15 MB MP3) PLoS ONE \| www.plosone.org June 2009 \| Volume 4 \| Issue 6 \| e5977 4 Fungal Cytoplasmic Continuity Contributed reagents/materials/analysis tools: WHM. Wrote the paper: AFvP WHM TB LGL HAW. PLoS ONE \| www.plosone.org References Biochim Biophys Acta 563: 100–112. PLoS ONE \| www.plosone.org June 2009 \| Volume 4 \| Issue 6 \| e5977 5
https://openalex.org/W2899316164	https://europepmc.org/articles/pmc6247474?pdf=render	English	null	Biological Effects of Tetrahydroxystilbene Glucoside: An Active Component of a Rhizome Extracted from <i>Polygonum multiflorum</i>	Oxidative medicine and cellular longevity	2,018	cc-by	13,323	Hindawi Oxidative Medicine and Cellular Longevity Volume 2018, Article ID 3641960, 15 pages https://doi.org/10.1155/2018/3641960 Hindawi Oxidative Medicine and Cellular Longevity Volume 2018, Article ID 3641960, 15 pages https://doi.org/10.1155/2018/3641960 Lingling Zhang 1 and Jianzong Chen 2 1Translational Medicine Center, Honghui Hospital, Xi’an Jiaotong University, Xi’an 710054, China 2 1Translational Medicine Center, Honghui Hospital, Xi’an Jiaotong University, Xi’an 710054, China 2T di i l Chi M di i D Xiji H i l F h Mili M di l U i i Xi’ 710032 Chi 1Translational Medicine Center, Honghui Hospital, Xi an Jiaotong University, Xi an 710054, China 2Traditional Chinese Medicine Department, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, g p J g y Traditional Chinese Medicine Department, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Ch Correspondence should be addressed to Lingling Zhang; sxjxzhangll2018@126.com and Jianzong Chen; chenjz2016@126.com Correspondence should be addressed to Lingling Zhang; sxjxzhangll2018@126.com and Jianzo Received 27 July 2018; Revised 8 October 2018; Accepted 23 October 2018; Published 4 November 2018 Academic Editor: Daniele Vergara Academic Editor: Daniele Vergara Copyright © 2018 Lingling Zhang and Jianzong Chen. 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. Polygonum multiﬂorum Thunb. (PM), a traditional Chinese medicinal herb, has been widely used in the Orient as a tonic and antiaging agent. 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside (TSG, C20H22O9, FW = 406.38928) is one of the active components extracted from PM. TSG is an antioxidant agent, which exhibits remarkable antioxidative activities in vivo and in vitro. The antioxidant eﬀect of TSG is achieved by its radical-scavenging eﬀects. TSG can inhibit apoptosis and protect neuronal cells against injury through multifunctional cytoprotective pathways. TSG performs prophylactic and therapeutic activities against Alzheimer’s disease, Parkinson’s disease, and cerebral ischemia/reperfusion injury. It is also antiatherosclerotic and anti-inﬂammatory. However, the mechanisms underlying these pharmacological activities are unclear. This study aimed at reviewing experimental studies and describing the eﬀectiveness and possible mechanisms of TSG. 2. Antioxidative Effect 3.1. Protective Eﬀect against Alzheimer’s Disease. Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive decline in cognitive abilities and associated with neuropathological features, including extensive loss of neurons (particularly cholinergic neurons), intracellular neuroﬁbrillary tangles composed of the tau protein, and extracellular deposition of plaques composed of β-amyloid (Aβ), which is a cleavage product of amyloid precursor pro- tein (APP). These two insoluble protein aggregates are accompanied by chronic inﬂammatory responses and exten- sive oxidative damage [31]. Learning and memory abilities are important cognition aspects that reﬂect the advanced integrative functions of the brain. Modern research has shown that an impaired memory function may be associated with cholinergic system synthases, such as acetylcholine transferase (ChAT) and hydrolytic enzymes, such as acetyl- cholinesterase (AChE). The levels of ChAT and acetylcholine (Ach) decrease in patients with AD, whereas the concentra- tion of AChE increases [32]. Learning and memory abilities are closely related to central nervous system function, and their regulation involves monoamine neurotransmission; NE, DA, and 5-HT levels change with age [33]. Senescence- accelerated-prone 8 (SAMP8) mice exhibit early onset impaired learning and memory [34]. TSG signiﬁcantly improves memory ability (P < 0 01) compared with that of the control group and prolongs the life span of SAMP8 mice by 17% (P < 0 01) compared with that of the control group [3]. TSG increases the protein level of neural klotho and reduces the levels of neural insulin, insulin receptor, insulin-like growth factor-1 (IGF-1), and IGF-1 receptor in Oxidative stress is a phenomenon that induces various health disturbances and diseases by enhancing the oxidation of bio- logically important molecules in vivo. Oxidation reactions by reactive oxygen species (ROS) are regarded as factors that trigger oxidative stress [19]. They are a group of very reactive short-lived chemicals produced during normal metabolism or after an oxidative reaction. ROS include free radicals, such as superoxide anion (O2 ⋅−) and hydroxyl radical (⋅OH), and nonradical molecules, such as hydrogen peroxide (H2O2) and singlet oxygen (1O2) [20]. Under physiological condi- tions, an appropriate level of intracellular ROS should be maintained to achieve redox balance and cell proliferation [21]. However, excessive ROS accumulation is highly cyto- toxic because these chemicals induce DNA damage, lipid peroxidation, and protein degradation [20, 22]. Oxidative damages caused by ROS may cause various neurodegenera- tive and chronic diseases, such as coronary heart diseases, atherosclerosis, cancer, and aging [23]. 1. Introduction of PM have been widely explored in academic medicine. The pharmacological characteristics of the compound have also been investigated. TSG possesses special biological actions and considerable values in scientiﬁc research and clinical medicine. The structure of TSG is similar to that of resveratrol (3,4′,5-trihydroxy-trans-stilbene), which is well known for its numerous biological activities, especially in cardiovascular protection and neuroprotection. TSG pos- sesses strong antioxidant and free radical-scavenging activi- ties, which are much stronger than those of resveratrol in superoxide anion radical scavenging, hydroxyl radical scavenging, and DPPH radical scavenging [8]. Parkinson’s disease (PD) is among the most common age-related neuro- degenerative disease. Current pharmacological treatments of PD remain largely symptomatic, and the development of new therapeutic strategies may provide eﬀective alternative treat- ment options. Vergara et al. [9] provided experimental evidence supporting the beneﬁcial eﬀects of resveratrol in preserving cellular homeostasis in parkin-mutant ﬁbroblasts, which may be relevant for PD treatment. The polyphenolic The root of Polygonum multiﬂorum Thunb. (PM), which is also known as heshouwu, is a famous traditional Chinese medicinal herb that has been used as a tonic and antiaging agent in the Orient. PM and its extract can be used to treat age-related diseases [1–3]. The medic- inal eﬀects of PM in the treatment of these age-related dis- eases are possibly mediated by the antioxidant capacity of this plant [4], because free radical-induced oxidative stress has been implicated in the aging process. PM consists of anthraquinone, stilbene, phospholipid, and other com- pounds, and modern chromatographic separation studies have demonstrated that many bioactive compounds (e.g., stilbene glycosides) in PM are responsible for its medicinal activities [5]. One of the main components that can be extracted from the root of PM is 2,3,5,4′-tetrahydroxystil- bene-2-O-β-D-glucoside (TSG), which is a stilbene mono- mer [6]. Since the discovery of TSG by Hata et al. [7], who were Japanese scientists, the chemical components Oxidative Medicine and Cellular Longevity 2 structure of TSG is similar to that of resveratrol. TSG also elicits neuroprotective eﬀects in various neurodegenerative diseases and cerebral ischemia. In aged mice, TSG treatment rescues synapses and suppresses α-synuclein overexpression in the brain, subsequently improving their memory and movement functions [10]. TSG protects dopamine (DA) neurons against lipopolysaccharide- (LPS-) induced neuro- toxicity through dual modulation on glial cells by attenuating microglial-mediated neuroinﬂammation and enhancing astroglial-derived neurotrophic eﬀects [11]. 1. Introduction In an ischemia- reperfusion-injured rat model, TSG promotes the postopera- tive recovery of rats by minimizing the volume of cerebral infarcts and improving neurological dysfunction, thereby upregulating the expression levels of CD31, angiopoietin 1, and angiopoietin receptor-2 [12]. TSG possesses antiathero- sclerosis, anti-inﬂammatory, and anticardiac ﬁbrotic eﬀects. Although these eﬀects are widely known, the mechanisms of action have yet to be established. Numerous mecha- nisms, such as activation of adenosine 5′-monophosphate- activated protein kinase [13, 14], protein kinase B (Akt) [15, 16], peroxisome proliferator-activated receptor gamma (PPAR-γ) [17], and silent mating type information regula- tion 2 homolog 1 [18] and inhibition of the classical nuclear factor-κB (NF-κB) signal [13], mediate the thera- peutic eﬀects of TSG. This study aimed at reviewing vari- ous experimental studies and describing the eﬀectiveness and possible mechanisms of TSG. brain microvascular endothelial cells, TSG exhibits cytopro- tective eﬀects against H2O2-induced oxidative stress by inhi- biting malondialdehyde (MDA) and ROS and upregulating SOD and GSH [25]. To further evaluate the in vivo anti- oxidative eﬀects of TSG on rats, Lv et al. [26] administered D-galactose (100 mg/kg/day body weight; single hypodermic injection) to senile rats and applied TSG (20 and 40mg/kg/ day body weight). They found an increase in the activities of SOD and GSH-Px and a decrease in the content of thiobarbituric acid reactive species in the serum and heart, brain, and liver tissues of rats. TSG also reduces serum MDA and tissue 8-OHDG levels and increases serum GSH-Px activity in hypertensive rats aged spontaneously [27]. Hemeoxygenase-1 (HO-1) is a highly inducible and stress-responsive protein (also called heat shock protein 32) that catalyzes the ﬁrst and rate-limiting step in the degradation of heme, which is a potent oxidant [28]. NADPH-quinone oxidoreductase 1 (NQO1) is a predomi- nantly cytosolic enzyme that provides cells with multiple layers of protection against oxidative stress, including the direct detoxiﬁcation of highly reactive quinones [29]. The pretreatment of cells with TSG (5 and 10μM) for 24 h can attenuate H2O2-induced ROS production and upregulate the expression of antioxidant enzymes HO-1 and NQO1 in a dose-dependent manner [30]. These results suggest that TSG is a potent antioxidant. 2. Antioxidative Effect In the same man- ner, TSG induces a dose-dependent enhancement of the activity of ChAT, decreases the activity of AChE, promotes the synthesis of monoamine neurotransmitter (NE, DA, and 5-HT), downregulates glutamic acid and aspartic acid, and retrieves the metabolic disorder of amino acid neuro- transmitters in the brain tissues of SAMP8 mice compared with those of the SAMP8 group (P < 0 05 or 0.01) [36, 37]. with those of the SAMP8 group (P < 0 05 or 0.01) [36, 37]. Aβ, a major protein component of senile plaques, is considered as a critical cause of the pathogenesis of AD [31]. The alternative splicing of APP exon 7 generates iso- forms containing a Kunitz protease inhibitor (KPI) domain. APP–KPI levels in the brain are correlated with Aβ pro- duction [38]. TSG protects nerve cells against Aβ-induced cell damage and improves learning–memory deﬁcit in an AD model of APP transgenic mice and in a rat model of cholinergic damage induced by injecting ibotenic acid into the basal forebrain [39, 40]. Moreover, intragastric TSG (100 mg/kg/day) administration for 1 month can signiﬁ- cantly improve learning, memory, spatial orientation, and other behavioral functions in transgenic mice and attenu- ate Aβ neurotoxicity-induced injury to endoplasmic retic- ulum functions [41]. In cell lines and APPSWE/PSENdE9 (APP/PS1) transgenic mice, TSG suppresses APP–KPI+ and amyloid plaque formation. The mechanism of the neuro- protective eﬀect of TSG may involve the activation of the AKT–GSK3β signaling pathway, the attenuation of the splic- ing activity of ASF, and the decrease in APP–KPI+ levels, leading to the decline in Aβ deposition [15]. In addition, the intragastrical administration of 60 and 120 mg/kg TSG increases mitochondrial COX activities; decreases Aβ1–42 contents; reduces APP, beta-site APP cleaving enzyme 1, and presenilin-1 expression; and enhances nerve growth factor, brain-derived neurotrophic factor, and tropomyosin- related kinase B expression in the hippocampus of NaN3- infused rats [42]. Aβ deposition-induced microglial activation is a crucial event in the pathology of AD [43]. Jiao et al. [44] found that TSG attenuates Aβ-induced microglial activation and inhibits the production of inﬂammatory molecules, such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2 (COX-2), and prostaglandin E2. Further- more, Aβ exposure increases the levels of microglial M1 markers, interleukin- (IL-) 1β, IL-6, and tumor necrosis factor α (TNF-α). 2. Antioxidative Effect DPPH is a free radical widely used to test the free radical-scavenging ability of various chemicals. TSG possesses a strong DPPH radical- scavenging activity in vitro with an IC50 of 33.24 μΜ [24]. TSG also exhibits a strong capacity to scavenge ⋅OH and superoxide anion radical with IC50 of 2.75 and 0.57 μg/ml, respectively [8]. The crucial components of an antioxidant defense system in the body are cellular antioxidant enzymes (e.g., superoxide dismutase (SOD) and glutathione peroxi- dase (GSH-Px)), which are involved in the reduction of ROS and peroxides produced in living organisms and in the detoxiﬁcation of certain exogenous compounds. In human Oxidative Medicine and Cellular Longevity 3 LDH release and MDA content, and increases SOD activity and Bcl-2 expression [47]. In vivo, the intragastric adminis- tration of TSG (100 mg/kg/day) in drinking water for 8 weeks provides protection against memory impairment induced by intracerebroventricular treatment with Aβ1–40 (3 μg/mouse, i.c.v.) in mice. In addition, TSG reduces interleukin-6 (IL-6) content (P < 0 05), MDA content (P < 0 01), and MAO-B activity (P < 0 05) and increases T- AOC activity (P < 0 01) compared with those of the model group [48, 49]. Other studies have shown that TSG poten- tially reverses alterations in cognitive behavioral, biochemi- cal changes, and oxidative damage induced by Aβ1–42 in mice. These beneﬁcial eﬀects of TSG can be attributed partly by inhibiting the expression of the Keap1/Nrf2 pathway in hippocampus and cerebral cortex tissues [50]. Some investi- gations have also suggested that TSG protects neuronal HT- 22 cells against Aβ-induced neurocytotoxicity by ameliorat- ing mitochondrial-dependent oxidative stress and apoptotic pathway via the activation of Nrf2-HO-1 signaling. These data may elucidate a new mechanism about how TSG attenuates the pathologic process of AD by repairing Aβ- induced hippocampal neuron impairment [51]. the brain of SAMP8 mice (P < 0 01) compared with that of the control group [3]. In a hidden platform test and a spatial probe test, age-related learning and memory impairment in SAMP8 mice is prevented by daily treatment with TSG (33, 100, and 300 mg/kg/day) for 50 days [35]. Oxidative Medicine and Cellular Longevity TSG remarkably enhances the antioxidant enzyme activities of SOD, catalase (CAT), and GSH-Px and eﬃciently reduces the MDA content in PC12 cells [71]. MPP+ transports into DA neurons and accumulates in the mitochondria, resulting in ATP depletion and mitochondrial membrane potential alteration [72]. TSG inhibits the increase in intracellular reactive oxy- gen species levels and the MPP+-induced disruption of mito- chondrial membrane potential. TSG markedly upregulates the Bcl-2/Bax ratio, reverses the cytochrome c release, and inhibits the caspase-3 activation in MPP+-induced PC12 cells or SH-SY5Y cells [71, 73]. In vitro studies have shown that MPP+ increases α-Syn expression [74], and the overexpres- sion of mutant human α-Syn aggravates MPP+-induced neu- rotoxicity [75]. TSG (3.125–50 μM) protects A53T AS cells against MPP+-induced cell damage, and the mechanisms of TSG’s neuroprotective eﬀects are mediated by inhibiting α- Syn overexpression and aggregation, enhancing mitochon- drial function, reducing ROS levels, and inhibiting apoptosis in A53T AS cells exposed to MPP+ [76]. NO is involved in the pathogenesis of PD [77]. NOS has four known isoforms: nNOS, iNOS, eNOS, and mitochondrial NO synthase. nNOS and iNOS are closely related to the pathogenesis of PD [78]. Our study showed that the exposure of PC12 cells to 75mM 6-OHDA for 24 h signiﬁcantly increases the level of intracel- lular ROS and NO, induces the overexpression of iNOS and nNOS, and elevates the level of 3-NT. However, these changes are markedly reversed in a dose-dependent manner after PC12 cells are pretreated with diﬀerent TSG concentra- tions for 24h. These results suggest that TSG may protect PC12 cells from 6-OHDA-induced apoptosis by regulating the ROS–NO pathway [79]. p Neuroinﬂammation is an important contributor to PD pathogenesis with the hallmark of microglial activation [82]. Daily intraperitoneal injection of TSG for 14 consec- utive days signiﬁcantly protects DA neurons from 6- OHDA-induced neurotoxicity and suppresses microglial activation. A similar neuroprotection is shown in primary neuron–glial cocultures. In vitro studies have further dem- onstrated that TSG inhibits the activation of microglia and the subsequent release of proinﬂammatory factors. More- over, TSG-mediated neuroprotection is closely related to the inactivation of mitogen-activated protein kinase signal- ing pathway [83]. Astroglia also plays an important role in PD development and becomes the prime target of PD treatment [84]. TSG protects DA neurons against LPS- induced neurotoxicity through dual modulation on glial cells by attenuating microglial-mediated neuroinﬂamma- tion and enhancing astroglial-derived neurotrophic eﬀects. Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity TSG (20 or 40mg/kg, i.g.) for 14 days, conducted pole and open ﬁeld tests, and found that this treatment signiﬁcantly reverses the MPTP-induced behavioral deﬁcits compared with those of the MPTP treatment group (P < 0 05). Behav- ioral disorders occur when the number of TH-positive neurons in the SNpc of MPTP-treated mice decreases to 57.04% of the normal amount [81]. In another study, mice injected with MPTP show an approximately 63% decrease in TH neurons and manifest typical symptoms and behav- ioral disorders, such as tremor, piloerection, and bradykine- sia. Conversely, mice treated with TSG exhibit a signiﬁcant improvement in behavior and TH-positive neurons, which recover to 66% of that in the control group [80]. DA and its metabolites (DOPAC and HVA) decrease after MPTP is injected in striatal neurons. TSG dose-dependently counter- acts the MPTP-induced loss of striatal DA [80], prevents the loss of striatal DA transporter protein [16], and provides protection against MPTP lesions partly by controlling ROS- mediated JNK, P38, and mitochondrial pathways and PI3K/ Akt-mediated signaling mechanism. These in vivo eﬀects extend previous in vitro observations [70, 71]. 3.2. Protective Eﬀect against Parkinson’s Disease. Parkinson’s disease (PD) is the second-most common neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the pres- ence of ubiquitinated alpha-synuclein- (α-syn-) containing cytoplasmic inclusions called Lewy bodies in surviving SNpc neurons [62]. Several factors, including oxidative stress, mitochondrial dysfunction, neuroinﬂammation, and dysreg- ulated kinase signaling, likely operate in the mechanism of death of nigrostriatal DA neurons in PD [63–65]. Many stud- ies have used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its active metabolite 1-methyl-4-phenylpyridi- nium (MPP+), 6-hydroxydopamine (6-OHDA) and paraquat (PQ) as neurotoxins or Parkinsonism mimetics in cell cultures or animal PD models. These toxins induce oxidative stress and lead to cell death of DA neurons to mimic the situation in PD [66]. Oxidative stress is an important factor that can modulate intracellular signaling and lead to neuronal death by apopto- sis or necrosis [67, 68]. These include JNK signaling, p38 acti- vation, PI3K/Akt signaling inactivation, and signaling through bcl-2 family proteins [65]. MTT assay, ﬂow cytome- try, and DNA fragmentation have conﬁrmed that TSG elicits protective eﬀects against MPP+-induced PC12 cell apoptosis by inhibiting ROS generation and modulating the activation of JNK and the PI3K/Akt pathway [69, 70]. 2. Antioxidative Effect TSG pretreatment suppresses the increase in M1 markers and enhances the levels of M2 markers, such as IL-10, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and arginase-1, in N9 and BV2 cells. PU.1 overexpression partly eliminates the anti-inﬂammatory eﬀects of TSG, suggesting that the roles of TSG in Aβ-induced microglial cells are mediated by PU.1 expression. Aβ enhances the oligomerization, accumulation, and toxicity of α-synuclein [52]. α-Synuclein is a highly abundant protein at presynaptic terminals, is associated with the distal reserve pool of synaptic vesicles, and has a role in the regula- tion of neurotransmitter release, synaptic function, and plasticity [53]. Synaptic plasticity in the hippocampus has been considered the key phenomenon of learning and mem- ory processes [54]. A previous study demonstrated that 120 and 240μmol/kg/day TSG improves learning–memory impairment, decreases Aβ content, and inhibits α-synuclein overexpression and aggregation in the hippocampus of APPV717I transgenic mice in an AD model [40, 55]. The intragastric administration of 60 mg/kg/day TSG to 21-month-old rats for 3 months remarkably improves their learning–memory abilities in water maze tests, increases the number of synapses and synaptic vesicles, and increases the expression of synaptophysin in the hippocampal CA1 region of aged rats [56]. Another study has demonstrated that the oral administration of TSG for 3 months enhances memory and movement functions, protects the synaptic ultrastruc- ture, and increases the synaptic connections and the levels of synapse-related proteins and p-CaMKII in the hippocam- pus, striatum, and cerebral cortex of aged mice [10]. The long-term potentiation (LTP) of synaptic transmission trig- gered by high-frequency stimulation (HFS) in the hippocam- pal CA1 area requires postsynaptic molecular mechanisms, such as the activation of N-methyl-D-aspartate (NMDA) receptors, calcium–calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinases (ERKs) of the mitogen-activated protein family [57–60]. In vitro, 1 and 5μM TSG induce neurite outgrowth, promote PC12 cell diﬀerentiation, and increase intracellular calcium levels in hippocampal neurons in a concentration-dependent manner. Moreover, TSG facilitates HFS-induced hippocampal LTP in a bell-shaped manner. The facilitation of LTP induction by TSG requires CaMKII and ERK activation [61]. Therefore, TSG can be used to treat Alzheimer’s disease. Oxidative damage in macromolecules is associated with the accumulation of Aβ in the progressive development of AD [45] and is a critical event in Aβ-induced neuronal cell death [46]. In vitro models, TSG prevents Aβ1–42-induced PC12 cell injury by improving the cell survival rate, reduces 4 Oxidative Medicine and Cellular Longevity The cytokines erythropoietin (EPO) and insulin-like growth factor I (IGF-I) are individually found to be eﬀective [90]. The administration of TSG (60 mg/kg/d) can improve the neurological function of rats after reperfusion and increase the protein expression of HIF-1α and EPO after reperfusion compared with those of the model group [91]. In a related paper by Zhao et al. [92], TSG (30, 60, and 120 mg/kg/d) taken orally for 7 days can inhibit cell apoptosis caused by focal cerebral ischemia in rats, and its mechanism may be involved in the increase in the expression of Bcl-2 protein, which can inhibit cell apoptosis, and in the decrease in the expression of Bax protein, which can induce cell apoptosis and decrease the ratio of Bcl-2/Bax. TSG inhibits atherosclerosis by regulating blood fat, anti- oxidant and anti-inﬂammatory eﬀects, suppressing matrix metalloproteinase expression, and relaxing blood vessels [100–103]. Gao et al. [100] reported that the administration of TSG (90 and 180 mg/kg/day) to hyperlipidemic rats for 1 week signiﬁcantly reduces atherosclerosis index (AI, LDL- C/HDL-C) and serum total cholesterol (TC) and low- density lipoprotein cholesterol (LDL-C) levels (P < 0 01) but increases the mRNA expression of LDL receptor (LDLR) (P < 0 05) in liver cells. In another study, after rats are orally administered with 12 and 24 g/kg of TSG and a high-fat diet for 4 weeks, the contents of TC, TG, LDL-c, apoB, and MDA decrease signiﬁcantly. The energy of serum SOD, CAT, GSH- Px, and T-AOC and the ratios of HDL-c/TC and apoAI/apoB also signiﬁcantly increase in TSG groups, suggesting that TSG can regulate lipid metabolism and elicit an antioxidant eﬀect [101]. ApoE−/−mouse is the ﬁrst mouse model to develop lesions similar to those of humans and mimic the ini- tiation and progression of human atherosclerosis. After 8 weeks of treatment, TSG ameliorates the serum levels of TC, TG, and LDL-C and increases the serum level of high- density lipoprotein cholesterol in ApoE−/−mice. TSG suppresses hepatic steatosis, atherosclerotic lesion formation, and macrophage foam cell formation in ApoE−/−mice. Moreover, TSG improves the expression levels of hepatic scavenger receptor class B type I (SR-BI), ABCG5, and CYP7A1 and upregulates the protein expression levels of aor- tic ATP-binding cassette transporters G1 and A1 (ABCG1 and ABCA1). An in vitro study has shown that TSG pro- motes the cholesterol eﬄux of macrophages and increases the protein expression levels of ABCA1 and ABCG1 [104]. Oxidative Medicine and Cellular Longevity Neuronal death that follows 10 min of ischemia is associ- ated with a late increase in AChE activity [93]. Microtubule- associated protein-2 (MAP-2) is depleted in the early hours after an in vivo ischemia insult in a rat hippocampal slice [94]. Experiments involving rats with chronic cerebral ische- mia have demonstrated that the administration of TSG (30, 60, and 120 mg/kg) for 11 weeks causes a dose-dependent decrease in the AChE activity and increases the expression of protein phosphatase-2A (PP-2A) and MAP-2 in the hippocampus [95]. Angiogenesis is a prognostic marker of the survival and functional improvement of patients with cerebral stroke. Intraperitoneal injection of TSG (30, 60, and 120 mg/kg) promotes postoperative recovery in rats by minimizing the volume of cerebral infarcts and improving neurological dysfunction in a dose- and time-dependent manner. Additionally, TSG signiﬁcantly increases the micro- vessel density in the brain and upregulates the CD31 expres- sion in the ischemic penumbra relative to that in the control. Finally, treatment with TSG signiﬁcantly upregulates the relative levels of vascular endothelial growth factor, angiopoietin 1, and angiopoietin receptor-2 expression in the brain lesions of rats [12]. Thus, TSG elicits neuroprotec- tive eﬀects against cerebral ischemia injury by modulating various pathways. Endothelial dysfunction is a key early event in atheroscle- rotic plaque formation and characterized by inﬂammatory processes, reduced NO bioavailability, and increased oxida- tive stress [105, 106]. TSG can prevent the development of atherosclerosis by inﬂuencing endothelial function in athero- genic diet-fed rats [103]. The administration of TSG (30, 60, and 120 mg/kg/day) after atherosclerosis is induced for 6 weeks results in a dose-dependent increase in the NO levels in the serum and aorta, the NOS content in the aorta, and the expression of eNOS but reduces the expression of iNOS in the aorta of atherosclerotic rats [107]. In the same manner, TSG improves ACh-induced endothelium-dependent relaxa- tion, prevents intimal remodeling, and inhibits the decreased NOx content in the serum and aorta of atherogenic diet-fed Oxidative Medicine and Cellular Longevity These ﬁndings may serve as a basis for developing new alternative PD treatments [11]. 3.3. Protective Eﬀects against Cerebral Ischemia Injury. Multiple pathogenic mechanisms are involved in ischemia/ reperfusion- (I/R-) related injury, including oxidative stress, inﬂammation, excitotoxicity, calcium overload, and apopto- sis [85, 86]. ROS have been considered important mediators of brain damage after I/R injury [86, 87]. In vitro, various TSG concentrations (25, 50, and 100 μM) have been reported to protect the primary culture of cortical neurons against cytotoxicity induced by oxygen–glucose deprivation followed by reperfusion (OGD-R). Moreover, TSG (25μM) reverses OGD-R-induced neuronal injury, intracellular ROS genera- tion, and mitochondrial membrane potential dissipation and attenuates H2O2-induced increase in [Ca2+]i [18]. Xu et al. [88] showed an ameliorating eﬀect of TSG against focal cerebral I/R injury-induced apoptosis that can be attributed to its antioxidative actions. Doses of 0.038, 0.114, and 0.342 g/kg/day of TSG administered intragastrically at the onset of I/R in rats for 7 days result in a signiﬁcant increase To further evaluate the in vivo neuroprotective eﬀect of TSG, Zhang et al. and He et al. [16, 80] treated mice with Oxidative Medicine and Cellular Longevity 5 calcium, and large inﬂammatory cells called macrophages build up in arterial walls. Although the pathophysiological mechanisms underlying atherosclerosis are poorly under- stood, inﬂammation and oxidative stress play important roles in all the phases of atherosclerosis evolution [96]. ROS can promote inﬂammation, alter vasomotion, induce cell death, cause platelet aggregation, and stimulate vascular smooth muscle cell (VSMC) proliferation [97]. One of its main risk factors is low-density lipoprotein (LDL) cholesterol [98]. When LDL penetrates endothelial cells, more ROS are generated. In turn, ROS oxidize LDL to oxidative low- density lipoprotein (OX-LDL), which can cause endothelial injury and inﬂammatory reaction [99]. All these events contribute to cardiovascular lesion formation. in GSH-Px and SOD activities (P < 0 05) but a decrease in the MDA content (P < 0 01) [88]. In another study that involves TUNEL staining, the intraperitoneal administration of TSG at dosages of 15 and 40 mg/kg at the onset of reperfusion after 90 min of middle cerebral artery occlusion (MCAO) in mice causes signiﬁcant reductions in the brain infarct volume and the number of positive cells in the cerebral cortex compared with those of the MCAO group [18]. Oxidative Medicine and Cellular Longevity In addition, the contin- uous intragastric administration of TSG to mice for 6 days before I/R relieves the increase in the binding force of NMDA receptor (P < 0 05). Optical section results have shown that the calcium concentration in the group treated with TSG is remarkably lower than that in the ischemia model group (P < 0 05 or 0.01) [89]. A decrease in cellular oxygen tension during ischemic stroke also promotes an endogenous adaptive response accompanied by the upregulation of vari- ous cytoprotective factors to attenuate ischemic injury. The cytokines erythropoietin (EPO) and insulin-like growth factor I (IGF-I) are individually found to be eﬀective [90]. The administration of TSG (60 mg/kg/d) can improve the neurological function of rats after reperfusion and increase the protein expression of HIF-1α and EPO after reperfusion compared with those of the model group [91]. In a related paper by Zhao et al. [92], TSG (30, 60, and 120 mg/kg/d) taken orally for 7 days can inhibit cell apoptosis caused by focal cerebral ischemia in rats, and its mechanism may be involved in the increase in the expression of Bcl-2 protein, which can inhibit cell apoptosis, and in the decrease in the expression of Bax protein, which can induce cell apoptosis and decrease the ratio of Bcl-2/Bax. in GSH-Px and SOD activities (P < 0 05) but a decrease in the MDA content (P < 0 01) [88]. In another study that involves TUNEL staining, the intraperitoneal administration of TSG at dosages of 15 and 40 mg/kg at the onset of reperfusion after 90 min of middle cerebral artery occlusion (MCAO) in mice causes signiﬁcant reductions in the brain infarct volume and the number of positive cells in the cerebral cortex compared with those of the MCAO group [18]. In addition, the contin- uous intragastric administration of TSG to mice for 6 days before I/R relieves the increase in the binding force of NMDA receptor (P < 0 05). Optical section results have shown that the calcium concentration in the group treated with TSG is remarkably lower than that in the ischemia model group (P < 0 05 or 0.01) [89]. A decrease in cellular oxygen tension during ischemic stroke also promotes an endogenous adaptive response accompanied by the upregulation of vari- ous cytoprotective factors to attenuate ischemic injury. 5. Effects on Inflammatory Disease NF-κB is a transcription factor that promotes the transcrip- tion of genes involved in proinﬂammatory responses [120]. The activation of NF-κB leads to the formation of NF-κB dimers (p65 and p50) that translocate to the nucleus to promote the transcription of the proinﬂammatory mediators TNF-α, IL-6, and COX-2, resulting in a series of inﬂamma- tory cascade responses [121]. PPAR-γ, a member of the nuclear hormone receptor superfamily, can inhibit the activation of NF-κB through several mechanisms and repress the NF-κB-mediated transcription of proinﬂammatory cytokines [122]. The intragastric administration of TSG (10, 30, and 60mg/kg/day) for 7 days after colitis is induced by acetic acid irrigation dramatically attenuates acetic acid- induced colon lesions in mice, reverses body weight loss, and improves histopathological changes. TSG apparently decreases the content of MDA, which is a marker of lipid peroxidation. TSG appears to exert its beneﬁcial eﬀects on acetic acid-induced experimental colitis through the upregu- lation of the mRNA and protein levels of PPAR-γ and the inhibition of the NF-κB pathway, which in turn decreases the protein overexpression of the downstream inﬂammatory mediators TNF-α, IL-6, and COX-2 [123]. Neuroinﬂammation is closely implicated in the pathogenesis of neurological diseases [124]. The hallmark of neuroinﬂammation is microglial activation. The activation of microglial cells and the consequent release of proinﬂam- matory and cytotoxic factors, such as TNF-α, IL-1β, NO, and prostaglandin E synthase 2, possibly contribute to the onset of neurodegenerative diseases [125]. TSG suppresses matrix metalloproteinase expression and inﬂammation in rats with diet-induced atherosclerosis [102] and inhibits cyclooxygenase-2 activity and expression in RAW264.7 mac- rophage cells [126]. As a member of the intracellular phase II enzyme family, HO-1 is necessary to maintain cellular redox homeostasis. The overexpression of HO-1 markedly sup- presses TNF-α, thereby inducing airway inﬂammation by the inhibition of oxidative stress [127]. TSG treatment strongly induces the expression of HO-1 in an NRF2- dependent manner. Furthermore, TSG attenuates the LPS- mediated activation of RAW264.7 cells and the secretion of proinﬂammatory cytokines, including IL-6 and TNF-α [128]. NADPH oxidase is recognized as a key ROS- producing enzyme during inﬂammation and widely expressed in various immune cells, such as macrophages, eosinophils, microglia, and neutrophils [129]. TSG attenu- ates LPS-induced NADPH oxidase activation and subse- quent ROS production [130]. 4. Antiatherosclerosis Effect Atherosclerosis is a chronic and progressive disease in which plaques consisting of deposits of cholesterol and other lipids, Oxidative Medicine and Cellular Longevity 6 rats after 12 weeks of treatment. The decreased mRNA and protein expression of eNOS and the increased mRNA and protein expression of iNOS in atherogenic diet-fed rats are attenuated by TSG treatment. These results suggest that TSG can restore vascular endothelial function, which may be related to its ability to prevent changes in eNOS and iNOS expression, leading to the preservation of NO bioactivity [103]. rats after 12 weeks of treatment. The decreased mRNA and protein expression of eNOS and the increased mRNA and protein expression of iNOS in atherogenic diet-fed rats are attenuated by TSG treatment. These results suggest that TSG can restore vascular endothelial function, which may be related to its ability to prevent changes in eNOS and iNOS expression, leading to the preservation of NO bioactivity [103]. rats, and TSG attenuates vimentin mRNA and protein levels in oxLDL-induced HUVECs. This protective eﬀect may be mediated partly by TSG-induced inhibition of vimentin expression via the interruption of the TGFβ/Smad signaling pathway and caspase-3 activation. These ﬁndings help eluci- date the molecular mechanism underlying the beneﬁcial eﬀects of TSG on atherosclerosis and suggest that TSG may be an eﬀective agent for cardiovascular disease. y Inﬂammatory processes also play important roles in the onset, development, and remodeling of atherosclerotic lesions [108]. C-reactive protein (CRP) is probably a media- tor of atherosclerosis and may increase the vulnerability of an atherosclerotic plaque to rupture [109]. After adhering to and migrating into the vascular wall at atherosclerotic sites, leu- kocytes secrete proinﬂammatory cytokines, such as IL-6, which likely play a major role in the pathogenesis of athero- sclerosis [110]. Tumor necrosis factor- (TNF-) α, another cytokine with proinﬂammatory eﬀects, is upregulated in ath- erosclerotic plaques and may contribute to the pathogenesis of atherosclerosis [111]. Diﬀerent doses of TSG (120, 60, or 30 mg/kg/day) administered to Sprague–Dawley rats for 12 weeks signiﬁcantly and dose-dependently attenuate hyperlipidemic diet-induced alterations in serum lipid proﬁle and increase CRP, IL-6, and TNF-α levels [102]. Zhao et al. 4. Antiatherosclerosis Effect [112, 113] showed that TSG inhibits lysopho- sphatidylcholine- (LPC-) induced apoptosis of human umbilical vein endothelial cells (HUVECs) by blocking the mitochondrial apoptotic pathway, and this process is accompanied with the activation of superoxide dismutase and glutathione peroxidase, the clearance of intracellular reactive oxygen species, and the reduction of lipid peroxi- dation. In addition, TSG provides protection against LPC- induced endothelial inﬂammatory damage. This protective eﬀect is indicated by improved cell viability, adhesive ability, and migratory ability. Moreover, TSG reduces the expression and prevents the LPC-enhanced expression of Notch1, Hes1, and MCP-1. Therefore, the protective eﬀects of TSG against inﬂammatory damage partly depends on Notch1 inhibition. 5. Effects on Inflammatory Disease Therefore, TSG may be used VSMCs are the main cellular components in the blood vessel wall, and their excessive proliferation plays an important role in the pathogenesis of atherosclerosis [114]. Although several growth factors and cytokines are involved in the development of atherosclerotic lesions, one of the principal regulators of mitogenesis in VSMCs is platelet- derived growth factor- (PDGF-) BB whose expression is increased in atherosclerotic lesions. PDGF-BB has been shown to activate key extracellular signaling transducers, including ERK 1/2, which are associated with cell growth and movement and critical for the initiation and progression of vascular lesions [115]. Xu et al. [116] demonstrated that TSG (10 and 100 μmol/l) signiﬁcantly inhibits VSMC prolif- eration induced in the serum, cell cycle transition from the G0/G1 phase to the S phase, and proliferating cell nuclear antigen expression in the nucleus of VSMCs. Vimentin is a cytoskeletal protein involved in VSMC proliferation, mono- cyte migration across the endothelium walls, and foam cell formation [117]. Yao et al. [118, 119] reported that vimentin is a key protein in the TSG treatment for atherosclerosis in 7 Oxidative Medicine and Cellular Longevity Table 1: Antioxidative eﬀect. Experimental model IC50/dose Eﬀects and possible mechanism Reference number 33.24 μΜ Scavenge DPPH radical [24] 2.75 and 0.57 μg/ ml Scavenge ⋅OH and superoxide anion radical [8] HBMECs 50 and 100 μM ↓MDA and ROS; ↑SOD and GSH [25] Rats 20 and 40 mg/kg ↑SOD, GSH-Px activities; ↓thiobarbituric acid reactive species content [26] Hypertensive rats 50 mg/kg ↓Serum MDA and tissue 8-OHDG levels; ↑serum GSH-Px activity [27] GES-1 cells and SGC-7901 cells 5 and 10 μM ↓ROS production; ↑the expression of HO-1 and NQO1 [30] Table 2: Protective eﬀect against Alzheimer’s disease. 5. Effects on Inflammatory Disease Experimental model Dose Eﬀects and possible mechanism Reference number MPP+-induced PC12 cells 1–10 μM ↑Cell viability; ↓cell apoptosis; ↓intracellular ROS and the phosphorylated JNK; ↑SOD, CAT, and GSH-Px activities, ↑Bcl-2/Bax ratio, MMP; ↓MDA content, cytochrome c, caspase-3; activation of PI3K/Akt pathway [69–71] MPP+-induced A53T AS cells 3.125–50 μM ↓α-Syn overexpression and aggregation; enhancing mitochondrial function; ↓ROS level; ↓cell apoptosis [76] 6-OHDA-induced PC12 cells 10–50 μM ↓Intracellular ROS and NO; ↓overexpression of iNOS, nNOS; ↓3-NT level [79] MPTP-induced mice 20 or 40 mg/ kg Reverses the MPTP-induced behavioral deﬁcits; ↑TH-positive neurons in SNpc ↑DA and its metabolites contents, and DAT protein in striatum; activation of PI3K/Akt pathway; inhibition of the ROS-mediated JNK, P38, and mitochondrial pathways [16, 80] 6-OHDA-induced rats; primary rat midbrain neuron-glia cocultures 10 and 50 mg/ kg; 20–80 μM ↓Neurotoxicity; suppressed microglia activation and proinﬂammatory factors; inactivation of MAPK signaling pathway [83] Primary rat microglia- and astroglia- enriched cultures; LPS-induced rats 20–80 μM; 10 and 50 mg/ kg ↓Microglia-mediated neuroinﬂammation; enhancing astroglia-derived neurotrophic eﬀects [11] Table 4: Protective eﬀects against cerebral ischemia injury. Experimental model Dose Eﬀects and possible mechanism Reference number OGD-R-induced cell; MCAO mice 25 μM; 15 and 40 mg/kg ↓Neuronal injury; ↓intracellular ROS; ↓[Ca2+]i, ↑MMP; ↓brain infarct volume; ↓ cell apoptosis [18] Rat 0.038, 0.114, and 0.342 g/kg ↑GSH-PX and SOD activities; ↓MDA content [88] Gerbils 0.038, 0.114, and 0.342 g/kg ↓Binding force of NMDA receptor; ↓intracellular [Ca2+]i [89] Rats 60 mg/kg ↑The protein expression of HIF-1α and EPO [91] Rats 30, 60, and 120 mg/kg ↓Cell apoptosis; ↑Bcl-2; ↓Bax [92] Rats 30, 60, and 120 mg/kg ↓AChE activity; ↑the expression of protein PP-2A and MAP-2 [95] Rats 30, 60, and 120 mg/kg Promoted postoperative recovery in rats; ↓volume of cerebral infarcts; improving neurological dysfunction; ↑microvessel density in the brain; ↑CD31 expression; ↑levels of VEGF, Ang 1, and Tie-2 [12] to treat inﬂammatory diseases. However, further research should be performed. to treat inﬂammatory diseases. However, further research should be performed. protecting vascular endothelial cells [131]. Klotho and insulin/IGF-1 signaling pathways are closely associated with ROS accumulation, and the protein levels of klotho and insulin signaling pathway are critical for antiaging [131]. The long-term administration of TSG improves the memory ability and regulates the body weight of mice with D-galactose-induced aging, reduces the levels of IGF- 1, and increases the levels of klotho in serum. 5. Effects on Inflammatory Disease Klotho and insulin/IGF-1 signaling pathways are closely associated with ROS accumulation, and the protein levels of klotho and insulin signaling pathway are critical for antiaging [131]. The long-term administration of TSG improves the memory ability and regulates the body weight of mice with D-galactose-induced aging, reduces the levels of IGF- Table 3: Protective eﬀect against Parkinson’s disease. Experimental model Dose Eﬀects and possible mechanism Reference number MPP+-induced PC12 cells 1–10 μM ↑Cell viability; ↓cell apoptosis; ↓intracellular ROS and the phosphorylated JNK; ↑SOD, CAT, and GSH-Px activities, ↑Bcl-2/Bax ratio, MMP; ↓MDA content, cytochrome c, caspase-3; activation of PI3K/Akt pathway [69–71] MPP+-induced A53T AS cells 3.125–50 μM ↓α-Syn overexpression and aggregation; enhancing mitochondrial function; ↓ROS level; ↓cell apoptosis [76] 6-OHDA-induced PC12 cells 10–50 μM ↓Intracellular ROS and NO; ↓overexpression of iNOS, nNOS; ↓3-NT level [79] MPTP-induced mice 20 or 40 mg/ kg Reverses the MPTP-induced behavioral deﬁcits; ↑TH-positive neurons in SNpc ↑DA and its metabolites contents, and DAT protein in striatum; activation of PI3K/Akt pathway; inhibition of the ROS-mediated JNK, P38, and mitochondrial pathways [16, 80] 6-OHDA-induced rats; primary rat midbrain neuron-glia cocultures 10 and 50 mg/ kg; 20–80 μM ↓Neurotoxicity; suppressed microglia activation and proinﬂammatory factors; inactivation of MAPK signaling pathway [83] Primary rat microglia- and astroglia- enriched cultures; LPS-induced rats 20–80 μM; 10 and 50 mg/ kg ↓Microglia-mediated neuroinﬂammation; enhancing astroglia-derived neurotrophic eﬀects [11] Table 4: Protective eﬀects against cerebral ischemia injury. Experimental model Dose Eﬀects and possible mechanism Reference number OGD-R-induced cell; MCAO mice 25 μM; 15 and 40 mg/kg ↓Neuronal injury; ↓intracellular ROS; ↓[Ca2+]i, ↑MMP; ↓brain infarct volume; ↓ cell apoptosis [18] Rat 0.038, 0.114, and 0.342 g/kg ↑GSH-PX and SOD activities; ↓MDA content [88] Gerbils 0.038, 0.114, and 0.342 g/kg ↓Binding force of NMDA receptor; ↓intracellular [Ca2+]i [89] Rats 60 mg/kg ↑The protein expression of HIF-1α and EPO [91] Rats 30, 60, and 120 mg/kg ↓Cell apoptosis; ↑Bcl-2; ↓Bax [92] Rats 30, 60, and 120 mg/kg ↓AChE activity; ↑the expression of protein PP-2A and MAP-2 [95] Rats 30, 60, and 120 mg/kg Promoted postoperative recovery in rats; ↓volume of cerebral infarcts; improving neurological dysfunction; ↑microvessel density in the brain; ↑CD31 expression; ↑levels of VEGF, Ang 1, and Tie-2 [12] 8 Oxidative Medicine and Cellular Longevity Table 3: Protective eﬀect against Parkinson’s disease. Table 3: Protective eﬀect against Parkinson’s disease. Table 3: Protective eﬀect against Parkinson’s disease. 5. Effects on Inflammatory Disease TSG upregulates the expression of klotho in the cerebrum, heart, kidney, testis, and epididymis tissues of mice with D-galactose-induced aging [132]. Further studies have shown that TSG extends the life span of mice by upregu- lating neural klotho and downregulating neural insulin or 5. Effects on Inflammatory Disease Experimental model Dose Eﬀects and possible mechanism Reference number SAMP8 mice 2, 20, and 50 μM Improves the memory ability and prolonged the life span of mice; ↑the protein level of neural klotho; ↓the levels of neural insulin, insulin-receptor, IGF-1, and IGF-1 receptor [3] SAMP8 mice 33, 100, and 300 mg/kg Prevention of age-related learning and memory impairment; ↑ChAT activity; ↓AChE activity; ↑the synthesis of NE, DA, and 5-HT; ↓ glutamic acid and aspartic acid contents [35–37] APP695V717I transgenic mice 100 mg/kg Improve learning, memory, and spatial orientation behavioral functions in mice; ↓Aβ neurotoxicity-induced injury to endoplasmic reticulum functions [41] HEK-293FT cells; SH-SY5Y cells; APP/ PS1 transgenic mice NaN3-induced rats 10–200 μM; 50 mg/kg Activation of AKT-GSK3β signaling pathway; ↓APP–KPI+ and amyloid plaque formation; ↓the splicing activity of ASF, APP–KPI+ levels, Aβ deposition [15] 60 and 120 mg/kg ↑Mitochondrial COX activity; NGF, BDNF, TrkB expression; ↓Aβ1–42 content; APP, BACE1, and PS1 expression [42] Aβ-induced N9 and BV2 cells 90 μM ↓iNOS, NO, COX-2, and PGE2, IL-1β, IL-6, and TNF-α; ↑IL-10, BDNF, and GDNF, Arg-1; ↓PU.1 overexpression [44] Aβ1–42-induced PC12 cells 0.1, 1, and 10 μmol/l ↑Cell survival rate; ↓LDH release, MDA content; ↑SOD activity and Bcl-2 expression [47] Aβ1–40-induced mice 100 mg/kg Prevention of learning and memory impairment; ↓IL-6 content, MDA content, and MAO-B activity; ↑T-AOC activity [48, 49] Aβ1–42-induced mice 30, 60, and 120 mg/kg ↓Oxidative damage; inhibited the expression of Keap1/Nrf2 pathway [50] Aβ-induced HT-22 cells 60 μmol/l ↓Oxidative stress; activation of Nrf2-HO-1 signaling [51] APPV717I transgenic mice 120 and 240 μmol/ kg Improves learning–memory impairment; ↓Aβ content; ↓α-synuclein overexpression and aggregation [40, 55] Aged rats 30 and 60 mg/kg ↑Synapses and synaptic vesicles, SYP expression in the hippocampal CA1 region [56] Aged mice 50,100, and 200 mg/kg Enhances the memory and movement functions, protected the synaptic ultrastructure; ↑synapse-related proteins and p-CaMKII [10] PC12 cells 1 μM and 5 μM Induces neurite outgrowth, promotes PC12 cell diﬀerentiation; ↑ intracellular calcium levels, facilitates HFS-induced hippocampal LTP, activates CaMKII and extracellular ERK [61] Table 2: Protective eﬀect against Alzheimer’s disease. Oxidative Medicine and Cellular Longevity 8 8 to treat inﬂammatory diseases. However, further research should be performed. 6. Other Effects Oxidative stress is believed to play a role in physiological and pathological aging processes, such as age-related protecting vascular endothelial cells [131]. 6. Other Effects Oxidative stress is believed to play a role in physiological and pathological aging processes, such as age-related neurodegenerative diseases. Various studies have been performed about the antiaging eﬀects of TSG [2]. Klotho, a serum secretory protein, is closely related to age. Klotho has many physiological functions, such as regulating calcium and phosphorus levels in vivo, delaying senes- cence, improving cognition, reducing oxidative stress, and Oxidative Medicine and Cellular Longevity 9 Table 5: Eﬀects on inﬂammatory disease. Experimental model Dose Eﬀects and possible mechanism Reference number Mice 10, 30, and 60 mg/kg Attenuates acetic acid-induced colon lesions; reverses body weight loss, and improves histopathological changes; ↓the content of MDA; ↑the mRNA and protein levels of PPAR-γ; inhibition of the NF-κB pathway; ↓the protein overexpression of the TNF-α, IL-6, and COX-2 [123] RAW264.7 macrophage cells 1, 10, and 100 μmol/l ↓COX-2 enzyme activity and expression [126] Microglia BV2 cell lines 20–80 μM ↓NADPH oxidase activation; ↓ROS production [130] Table 6: Antiatherosclerosis eﬀect. Experimental model Dose Eﬀects and possible mechanism Reference number Hyperlipidemic rats 90 and 180 mg/kg ↓Serum TC, LDL-C, and AI levels; ↑the mRNA expression of LDLR in the liver cells [100] Hyperlipidemia rats 12 and 24 g/kg ↓Serum TC, TG, LDL-c, apoB, and MDA; ↑serum SOD, CAT, GSH-PX, and T-AOC; ↑the ratios of HDL-c/TC and apoAI/apoB [101] ApoE−/−mice; RAW264.7 cells 50 and 100 mg/kg; 10 and 100 μM ↓TC, TG, and LDL-C; ↑HDL-c, SR-BI, ABCG5, and CYP7A1 expression; ↑the protein expressions of ABCA1 and ABCG1 [104] Atherosclerotic rats 30, 60, and 120 mg/kg ↑NO levels in the serum and aorta; ↑NOS content and the expression of eNOS, eNOS mRNA; ↓the expression of iNOS, iNOS mRNA [103, 107] Atherosclerotic rats 120, 60, or 30 mg/kg ↓CRP, IL-6, and TNF-α levels [102] HUVECs 0.1, 1, or 10 μmol/l ↓Mitochondrial apoptotic pathway, lipid peroxidation, ROS, and MDA; ↑SOD and GSH-Px; ↓the expression of Notch1, Hes1, and MCP-1 [112, 113] VSMCs 10 and 100 μmol/l ↓VSMC proliferation; ↓cell cycle transition from G0/G1 phase to S phase; ↓PCNA expression in the nucleus of VSMCs [116] HUVECs 50 and 100 μM ↓Vimentin mRNA and protein levels; ↓TGFβ/Smad signaling pathway and caspase-3 activation [119] Table 7: Other eﬀects. Experimental model Dose Eﬀects and possible mechanism Reference number D-galactose- induced aging mice 42, 84, and 168 mg/kg Improves the memory ability and regulates the body weight of mice; ↓the levels of IGF-1; ↑the expression of klotho [132] C. Abbreviations PM: Polygonum multiﬂorum Thunb TSG: 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D- glucoside ROS: Reactive oxygen species H2O2: Hydrogen peroxide SOD: Superoxide dismutase GSH-Px: Glutathione peroxidase TBARS: Thiobarbituric acid reactive species HUVECs: Human umbilical vein endothelial cells HO-1: Hemeoxygenase-1 NQO1: NADPH-quinone oxidoreductase 1 Aβ: Beta-amyloid peptide APP: Amyloid precursor protein ChAT: Acetylcholine transferase ACH: Acetylcholine SAMP8: Senescence-accelerated-prone 8 IGF-1: Insulin-like growth factor-1 KPI: Kunitz protease inhibitor BACE1: Beta-site APP cleaving enzyme 1 PS1: Presenilin 1 TrkB: Tropomyosin-related kinase B SYP: Synaptophysin NMDA: N-Methyl-D-aspartate CaMKII: Calcium–calmodulin-dependent protein kinase II ERK: Extracellular signaling-regulated kinase IL-6: Interleukin-6 MPTP: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPP+: 1-Methyl-4-phenylpyridinium MDA: Malondialdehyde Bax: Bcl2-associated protein Bcl-2: B-cell lymphoma protein-2 MCAO: Middle cerebral artery occlusion MAP-2: Microtubule-associated protein-2 PP-2A: Protein phosphatase-2A VSMC: Vascular smooth muscle cell LDL: Low-density lipoprotein LDL-C: Low-density lipoprotein cholesterol NO: Nitric oxide iNOS: Inducible NO synthase TNF-α: Tumor necrosis factor CRP: C-reactive protein PCNA: Proliferating cell nuclear antigen COX: Cyclooxygenase. ROS can enhance bone resorption by directly or indirectly promoting osteoclast formation and activity [135]. TSG protects MC3T3-E1 cells from H2O2-induced cell damage and inhibition of osteoblastic diﬀerentiation. The protective eﬀect of TSG on osteoblastic MC3T3-E1 cells may be mediated partly by its antioxidant ability [136]. In addition, TSG increases the density, content, and size of minerals in bone tissues and enhances the resistance to exo- genic action, structural toughness, and strength of bone tis- sues [137]. These results suggest that TSG can be used as a good candidate for the protection of osteoblasts against oxi- dative stress-induced dysfunction and may have a potential therapeutic value for osteoporosis. Conflicts of Interest The authors declare that there are no conﬂicts of interest. 6. Other Effects elegans 50 and 100 μM Enhances the stress resistance; ↑the life span of the nematode C. elegans [1] Rat cardiac ﬁbroblasts 3–100 μmol/l ↓ERK1/2 activation; ↓overall production of ECM components [133] Pressure overload rats 60 and 120 mg/ kg ↓Angiotensin II level; ↓transforming growth factor-β1 expression; ↓ERK1/2 and p38 MAPK activation [134] Pressure overload rats 120 mg/kg ↑Endogenous PPAR-γ expression [17] MC3T3-E1 cells 0.1–10 μM ↓Osteoblastic diﬀerentiation; ↓oxidative damage [136] SD rats 150, 300, and 600 mg/kg ↑The density, content, and size of minerals in bone tissues; enhances the resistance to exogenic action, structural toughness, and strength of bone tissues [137] Cardiac ﬁbroblasts play an important role in regulating normal myocardial function and adverse myocardial remod- eling. Angiotensin (Ang) II, the eﬀector peptide of the renin- angiotensin system, is a key pathogenic factor in the insulin-like growth factor 1 [3]. TSG enhances the stress resistance and increases the life span of the nematode Cae- norhabditis elegans [1]. These results strongly conﬁrm the potential of TSG as a pharmaceutical antiaging drug. Oxidative Medicine and Cellular Longevity 10 development of hypertension and heart failure. TSG can inhibit Ang II-induced cardiac ﬁbroblast proliferation by suppressing the ERK1/2 pathway and reducing the overall production of extracellular matrix components [133]. TSG can prevent cardiac remodeling induced by pressure over- load in rats. The underlying mechanisms may be related to a decreasing angiotensin II level, an antioxidant eﬀect of the tested compound, the suppression of transforming growth factor-β1 expression, and the inhibition of ERK1/ 2 and p38 mitogen-activated protein kinase activation [134]. Further studies have suggested that the upregulation of endogenous PPAR-γ expression by TSG may be involved in the beneﬁcial eﬀect of TSG on pressure overload-induced cardiac ﬁbrosis [17]. foundation for further studies to assess the mechanisms underlying the eﬀects and clinical applications of TSG. 7. Conclusion TSG has broad biological actions, including radical- scavenging eﬀects and beneﬁcial eﬀects, for the treatment of various conditions, such as neuronal disease, cardiovas- cular disease, inﬂammatory disease, and osteoporosis. Several studies have elucidated the underlying mechanism of TSG action. The eﬀects of TSG against oxidative stress have helped elucidate many aspects of its mechanism of action. TSG is a strong free radical scavenger and potent antioxidant [24] (Table 1). TSG can enhance cognitive performance and prevent or treat AD in multistages and multitargets (Table 2). TSG may also act as an eﬀective neuroprotective agent against PD through the modulation of the PI3K/Akt signaling pathway and ROS-mediated JNK, p38, and mitochondrial pathways [69–71, 80] (Table 3). TSG elicits anti-inﬂammatory eﬀects and provides protection against cerebral I/R injury through multifunctional cytoprotective pathways (Tables 4 and 5). Previous studies demonstrated that TSG reduces the blood lipid content and inhibits the atherosclerotic process [102] (Table 6). TSG can prevent pressure overload-induced cardiac remodeling and Ang II- induced cardiac ﬁbrosis in vitro [17] and may function against H2O2-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells (Table 7), but these abilities should be further investigated. References [19] M. Kuwabara, T. Asanuma, K. Niwa, and O. Inanami, “Reg- ulation of cell survival and death signals induced by oxidative stress,” Journal of Clinical Biochemistry and Nutrition, vol. 43, no. 2, pp. 51–57, 2008. [6] X. Qiu, J. 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https://openalex.org/W2980636360	https://europepmc.org/articles/pmc6982648?pdf=render	English	null	Need vs. Financing Capability: Human Papillomavirus Vaccinations among Adolescents	Asian Pacific journal of cancer prevention	2,019	cc-by	5,228	DOI:10.31557/APJCP.2019.20.10.2959 DOI:10.31557/APJCP.2019.20.10.2959 Need vs. Financing Capability: Human Papillomavirus Vaccinations among Adolescents RESEARCH ARTICLE Editorial Process: Submission:03/27/2019 Acceptance:09/28/2019 Abstract Background:The incidence of Human Papillomavirus (HPV) infection and cervical cancer in adulthood may be prevented by HPV vaccination in adolescence. Currently, the HPV vaccination coverage rate in developing countries is about 15%. The reason for this low vaccination coverage is most likely due to a lack of information among adolescents and adults. Purpose: To explore adolescents, parents and teachers’ needs, obstacles, and expectations around the HPV vaccination. Methods: This research used a qualitative method with a focus group discussion. The research participants were divided into three groups: 21 female students, 17 parents, and 20 teachers. This research was conducted in junior high schools that have programs run by their adolescent reproductive health counseling information centers. The data were analyzed by employing content analysis. Results: HPV vaccination has not been made a priority for adolescents because: 1) There is a lack of available education about HPV and HPV vaccinations for adolescents, parents, and teachers. 2) The high cost for parents to vaccinate their children. 3) Adolescents, parents and teachers believe that the HPV vaccine needs to be administered to adolescents, but they feel that the vaccine is not affordable. Conclusion: It is important to consider a program which will provide accurate information about the HPV vaccination to the community, especially adolescents. Financial management, such as insurance or vaccination savings schemes, may be one way to overcome the problem of the HPV vaccination’s cost. Keywords: HPV- cervical cancer- HPV vaccine- knowledge- financing Asian Pac J Cancer Prev, 20 (10), 2959-2964 Asian Pac J Cancer Prev, 20 (10), 2959-2964 Asian Pac J Cancer Prev, 20 (10), 2959-2964 However, at the national level, the administration of the HPV vaccination is still in its initiation process. The HPV vaccination is administered to female students in grades five (first dosage) and six (second dosage) at elementary schools through the School Children’s Immunization Month (BIAS) program (Arifah et al., 2017). Asian Pacific Journal of Cancer Prevention, Vol 20 2959 1Department of Pediatric and Maternity Nursing, 2Department of Medical Education, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Indonesia. For Correspondence: wiwien_lismidati@ugm.ac.id Need vs. Financing Capability: Human Papillomavirus Vaccinations among Adolescents Wiwin Lismidiati1, Ova Emilia2, Widyawati Widyawati1 1Department of Pediatric and Maternity Nursing, 2Department of Medical Education, Faculty of Medicine, Publ Nursing, Universitas Gadjah Mada, Indonesia. *For Correspondence: wiwien_lismidati@ugm.ac.id Results The teachers’ group included teachers who teach school subjects and counseling teachers who have the important role of helping female students at school on a daily basis. Ten teachers were invited to the discussion from each school, and all of them were permitted to attend the discussions. Ten to 12 female students were invited from each school to the Focus Group Discussion (FGD); in all, 10 female students were present from Junior High School A and 11 female students were present from Junior High School B. The parents’ group was sampled using the criteria of those with female children in grades seven and eight at the same schools. 10 to 12 parents were invited from each school; six parents were present during the FGD activity from Junior High School A and 11 parents were present from Junior High School B. There were 58 participants of the FGDs: 21 female students, 17 parents, and 20 teachers. The participants’ characteristics can be seen in Table 1. There were five themes namely: (1) Lack of understanding about human papillomavirus vaccine; (2) Difficult to explain to children and make embarrassment are the constrain arise in the health education; (3) Parents’limited funds for children’s human papilloma virus vaccination; (4) Insurance covering, discount and savings is the effort that parent’s thought to solve financial problems; (5) The constrain in female vaccine are expensive, low economy, not priority in household budget. Theme 1: Lack of understanding about human papillomavirus and the human papillomavirus vaccine Theme 1: Lack of understanding about human papillomavirus and the human papillomavirus vaccine Fifty of participants expected that adolescents would receive the HPV vaccinations, considering the future effects of exposure to HPV infection. However, most of the participants lacked knowledge about HPV, cervical cancer, and the HPV vaccine. Some participants identified the importance of health education and health promotion with regard to HPV and the HPV vaccine. They stated that they wanted adequate information about HPV, cervical cancer, and the HPV vaccine. Data analysis This qualitative research took place in junior high schools in Bantul and Sleman under the Adolescent Reproductive Health Counseling Information program in Yogyakarta Special Region. The recording of each discussion was transcribed by the researcher. The written notes made by the research assistant were added to the record. The research data were analyzed manually with the following qualitative research measures: 1) Transcribing the data collected from the FGDs and making notes during the data’s collection about data related to the informants. 2) Reconfirming the accuracy of the data with the participants and triangulating the data with the community health center officer in charge of the local junior high schools health units. 3) Reading all the data or transcripts for general ideas presented by the informants and any other necessary information. 4) Starting coding. 5) Using the coding process’s results to develop the theme for further analysis. 6) Presenting the description and theme in a qualitative narration. 7) Interpreting the data. Study design and sample This focus group discussion was carried out from December 2017 to January 2018. The research design was qualitative using focus group discussion approach. This research sample consists of three groups: female junior high school students, parents, and teachers. The inclusion and exclusion criteria for the female students’ group included female students in grades seven and eight who were willing to be participants and who signed the informed consent form; the inclusion and exclusion criteria for the parents’ group included the parents of female junior high school students in grades seven and eight who were willing to be participants, and the inclusion and exclusion criteria of the teachers’group included female teachers who were actively teaching subjects during the research period and willing to be respondents. The themes were analyzed for each question during the FGD sessions with the three participant groups. al., 2017). research studies. The FGD instructions contained some open-ended questions that were adapted to each discussion group. This research was approved by the Medical and Health Research Ethical Committee under number KE/ FK/1100/EC/2017. The research question is what are the needs, challenges and expectations of female students, parents and teachers for the HPV vaccination program? Introduction The World Health Organization (WHO) states that the Human Papillomavirus (HPV) prophylactic vaccine is highly effective when administered to 16-20 year-old females. The HPV vaccine is expected to prevent up to 70% of HPV incidence in vaccinated females (WHO, 2006). The HPV vaccination is most effective when administered to 9-13 year-old adolescents, and to 14-26 year-old non-vaccinated people before they have had sexual intercourse. Research shows that if administered in this way, the HPV vaccination may achieve a nearly 90% protection rate (Cunningham et al., 2015; Gallagher et al., 2018). Low- to middle-income countries face some constraints in introducing the HPV vaccination to their people. These challenges include: historical/structural factors; programmed vaccinations; and delivery constraints (Gallagher et al., 2018). Other constraints found include socio-cultural, health system, political, and financial factors (Wigle et al., 2013). In Indonesia, constraints include the parents’ acceptance of the HPV vaccinations, the vaccine’s cost, the fear of side effects, the preferred location for HPV vaccination (Jaspers et al., 2011), the lack of access to service centers with adequate laboratory and health workers, and the requirement for repeated visits to the service center (Karneli et al., 2013). In addition, a lack of knowledge about the HPV vaccination, the vaccine’s safety, side effect considerations, and not being recommended by physicians or health workers are some of the other challenges found around the HPV vaccination’s administration (Arifah et In 34 low- and low- to middle-income countries, the vaccination program is supported by the Global Alliance for Vaccines and Immunizations (GAVI). From 2013 to 2016, GAVI provided support to more than 20 countries eligible for a 2-year HPV vaccine demonstration project. In 2014, 1.2% of 10-14 year-old adolescents were expected to have had at least one dose of the vaccine administered (Bruni et al., 2014; Gallagher et al., 2018). In Indonesia, the HPV vaccine is available and is recommended by the Indonesian Pediatric Society. Wiwin Lismidiati et al Asian Pacific Journal of Cancer Prevention, Vol 20 2960 Data collection The FGD groups met in the same place as their respective schools. The FGDs were conducted in classrooms and laboratory rooms in the respective schools, and lasted for 60-90 minutes. Before starting the group discussions, the purpose of the FGD was explained to the participants and they were asked to provide their written consent through an informed consent form. The participants were told that any data would be kept confidential and that answer anonymity would be maintained. Each discussion group was led by one research facilitator, and an assistant was assigned to take notes and help manage the group. The group discussions were digitally recorded with the permission of the participants. Parent Participant (PP12): “I agree on socialization, but parents must be involved.” Adolescent Participant (AP7): “Socialization at school to provide information of reproductive health and teenager relationship” The research instruments included prepared FGD instructions. The FGD instructions were arranged by the researcher pursuant to previous theories and related Teacher Participant (TP1): “Maybe the knowledge should be prioritized, since it must be a process, not something instant. Children’s source of funding is from Asian Pacific Journal of Cancer Prevention, Vol 20 2960 DOI:10.31557/APJCP.2019.20.10.2959 Need vs. Financing Capability: Human Papillomavirus Vaccinations among Adolescents their parents. When the parents have no understanding or knowledge of its benefits, it will be hard.” Teacher Participant (TP13): It (socialization) is also needed, but through the biology subject, during the study of reproduction.” Theme 2: Difficult to explain to children and make embarrassment are the constrain arise in the health education Parent Participant (PP9): “How to communicate, how to talk about it? Data collection Will they understand?” Adolescent Partisipant Age Characteristic AP 1 13 years old Eight grade junior high female students in Srandakan Bantul AP 2 12 years old Seventh grade junior high female students in Srandakan Bantul AP 3 13 years old Eight grade junior high female students in Srandakan Bantul AP 4 12 years old Seventh grade junior high female students in Srandakan Bantul AP 5 13 years old Eight grade junior high female students in Srandakan Bantul AP 6 13 years old Eight grade junior high female students in Srandakan Bantul AP 7 14 years old Eight grade junior high female students in Srandakan Bantul AP 8 12 years old Eight grade junior high female students in Srandakan Bantul AP 9 12 years old Seventh grade junior high female students in Srandakan Bantul AP 10 13 years old Eight grade junior high female students in Srandakan Bantul AP 11 14 years old Eight grade junior high female students in Ngaglik Sleman AP 12 13 years old Eight grade junior high female students in Ngaglik Sleman AP 13 14 years old Eight grade junior high female students in Ngaglik Sleman AP 14 14 years old Eight grade junior high female students in Ngaglik Sleman AP 15 13 years old Eight grade junior high female students in Ngaglik Sleman AP 16 13 years old Eight grade junior high female students in Ngaglik Sleman AP 17 13 years old Eight grade junior high female students in Ngaglik Sleman AP 18 14 years old Eight grade junior high female students in Ngaglik Sleman AP 19 14 years old Eight grade junior high female students in Ngaglik Sleman AP 20 13 years old Eight grade junior high female students in Ngaglik Sleman PS 21 13 years old Eight grade junior high female students in Ngaglik Sleman Table 1. Characteristic of Adolescent’Participants Parents Participant Age Keterangan PP1 42 years Mothers of seventh grade junior high female students in Srandakan Bantul. She is an employed. Educational background was senior high school. PP 2 37 years Mothers of eight grade junior high female students in Srandakan Bantul. She is self employed. Educational background was senior high school. PP 3 37 years Mothers of seventh grade junior high female students in Srandakan Bantul. She is an employed. Educational background was junior high school. PP 4 45 years Mothers of eight grade junior high female students in Srandakan Bantul. Data collection She is self employed. Educational background was bachelor. PP 5 41 years Mothers of seventh grade junior high female students in Srandakan Bantul. She is in private sector. Educational background was senior high school. PP 6 47 years Mothers of eight grade junior high female students in Srandakan Bantul. She is self employed. Educational background was senior high school. PP 7 38 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is in private sector. Educational background was senior high school. PP 8 45 years Mothers of eight grade junior high female students in Srandakan Bantul. She is an employed. Educational background was senior high school. PP 9 39 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is an employed. Educational background was senior high school. PP 10 40 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is an employed. . Educational background was senior high school. PP 11 38 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is self employed. Educational background was Diploma. PP 12 37 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is an employed. Educational background was Diploma. PP 13 46 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is self employed. Educational background was senior high school. PP 14 39 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is an employed. Educational background was vocational school. PP 15 39 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is an employed. Educational background was vocational school. PP 16 44 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is in private sector. Educational background was vocational school PP 17 40 years Mothers of eight grade junior high female students in Ngaglik Sleman. She is an employed. Educational background was Diploma. Table 2. Characteristic of Parents’ Participants Need vs. Financing Capability: Human Papillomavirus Vaccinations among Adolescents Table 2. Characteristic of Parents’ Participants Table 1. Characteristic of Adolescent’Participants Theme 2: Difficult to explain to children and make embarrassment are the constrain arise in the health education Parent Participant (PP9): “How to communicate, how to talk about it? Data collection Characteristic of Teachers‘Participant (Regional Health Insurance) or Jamkesmas (Social Health Insurance)?” (Regional Health Insurance) or Jamkesmas (Social Health Insurance)?” Table 3. Characteristic of Teachers‘Participant Parent Participant (PP12): “There is normally a discount, for example, buy two get one free.” Adolescent Participant (AP11):”Yes, may be after saving for it.” Teacher Participant (TP9): “Create a program at school which may be budgeted next year.” Teacher Participant (TP9): “Create a program at school which may be budgeted next year.” Theme 5: The constrain in female adolescent vaccine are expensive, low economy, not priority in house hold budget Theme 5: The constrain in female adolescent vaccine are expensive, low economy, not priority in house hold budget Parent Participant (PP1/PP6): “The objection is about the high cost. I think it is too much for us in the village. We are in the village, and thus it is too expensive” Parent Participant (PP1/PP6): “The objection is about the high cost. I think it is too much for us in the village. We are in the village, and thus it is too expensive” Teacher Participant (TP10) :“Parents are of a low economic status, from the perspective of their education and employment, 50% have a weak economy.” Teacher Participant (TP10) :“Parents are of a low economic status, from the perspective of their education and employment, 50% have a weak economy.” Adolescent Participant (AP7): “There are still many necessities for school tasks.” Discussion Adolescents, parents, and teachers have limited knowledge about HPV, cervical cancer, and the HPV vaccine; thus, the need for health education and the socialization of HPV, cervical cancer, and the HPV vaccine is an important factor in changing behavior toward the HPV vaccination. Not much progress has been made by Indonesian health workers with the socialization of HPV, and getting people to have the HPV vaccine to prevent cervical cancer. This is confirmed by the results of an interview with a community health center worker, who stated that the HPV vaccine is not a priority program and that it is not the community health center’s policy to socialize it in the adolescent reproductive health concern (PKPR) program. Currently, the PKPR program at community health centers only socializes the issues of older marital age, an early introduction to sex, and adolescent anemia. The lack of recommendations by health workers when socializing the HPV vaccine was also found by Cartmell et al., 2018, which stated that factors contributing to the lack of information provided about the HPV vaccine include: 1) The lack of awareness among some pediatricians and general practitioners about the HPV vaccine directive. 2) Service providers are uncomfortable discussing the topic. Other researchers state that the reason for the lack of parents’ knowledge about HPV and the HPV vaccination, as well as for the parents’ concern about the side effects, is that there is no recommendation for it by health workers (Morales-Campos et al., 2013; Fernández et al., 2014; Masika et al., 2015). This is confirmed by previous research studies (Reiter et al., 2011; Bartolini et al.,2012) proposing that health workers and teachers are credible sources of information who could provide significant support for an HPV vaccination campaign. Recommendations from physicians is the consistent key predictor of the HPV vaccine’s acceptance (Rosenthal et al., 2011). Data collection Will they understand?” y Parent Participant (PP10): “No courage to explain it, since the children cannot understand what we tell them.” Parent Participant (PP10): “No courage to explain it, since the children cannot understand what we tell them.” Teacher Participant (TP12): “Lack of awareness and thinking that everything is alright.” Teacher Participant (TP12): “Lack of awareness and thinking that everything is alright.” Teacher Participant (TP11): “Anything related to female matters is an embarrassment, even if it is a free check-up. The children will also get embarrassed.” Asian Pacific Journal of Cancer Prevention, Vol 20 2961 Theme 3: Parents’ limited funds for children’s human Wiwin Lismidiati et al Wiwin Lismidiati et al Teacher Participants Usia Keterangan TP 1 43 years Math at junior high school teacher in Srandakan Bantul TP 2 44 years Civilization Education at junior high school teacher in Srandakan, Bantul TP 3 42 years Biology at junior high school teacher in Srandakan, Bantul TP 4 47 years Math at junior high school teacher in Srandakan Bantul TP 5 45 years Social science at junior high school teacher in Srandakan Bantul TP 6 46 years Guidance counselor at junior high school teachers in Srandakan Bantul TP 7 46 years Social science at junior high school teacher in Srandakan Bantul TP 8 43 years Civilization Education at junior high school teacher in Srandakan, Bantul TP 9 46 years Math at junior high school teacher in Srandakan Bantul TP 10 46 years Guidance counselor at junior high school teachers in Srandakan Bantul TP 11 44 years Math at junior high school teacher in Ngaglik Sleman TP 12 45 years Gym at junior high scholl teacher in Ngaglik Sleman TP 13 46 years Civilization Education at junior high school teacher in Ngaglik Sleman TP 14 43 years Physics at junior high school teacher in Ngaglik Sleman TP 15 44 years Biology at junior high school teacher in Ngaglik Sleman TP 16 43 years Physics at junior high scholl teacher in Ngaglik Sleman TP 17 44 years Gym at junior high scholl teacher in Ngaglik Sleman TP 18 45 years Biology at junior high school teacher in Ngaglik Sleman TP 19 44 years Social science at junior high school teacher in Ngaglik Sleman TP 20 46 years Physics at junior high school teacher in Ngaglik Sleman Table 3. Asian Pacific Journal of Cancer Prevention, Vol 20 2962 papillomavirus vaccination This is also confirmed by research (Dempsey and Zimet, 2015; Degarege et al., 2018) stating that health education programs should target the parents and extended family members, so as to increase the coverage rate for future vaccination programs. Parental and school involvement strategies are also an important factor for improving the school vaccination program for adolescents (Whelan et al., 2014). One main constraint causing parents not to accept the administration of the HPV vaccine in Indonesia is the cost. The HPV vaccination’s cost is not affordable for most Indonesians. The people believe that the government should either fund the vaccination or there should be joint funding between the government and parents for the HPV vaccinations (Arifah et al., 2017; Jaspers et al., 2011; Karneli et al., 2013). ( , ) In this research, both the parents’ and teachers’ groups stated that they face constraints in providing their daughters with education about sexual matters. The cultural/taboo factor prevents them from providing adolescents with sexual information. Reproductive and sexual health education is a sensitive topic, which needs advocacy to allow it to be provided to young people and the general public. So far, sexual and reproductive health education in schools is incomprehensive and not relevant to the actual sexual behavior and risks adolescents face. This implies the students have limited knowledge about reproductive health in general. Sexual and reproductive health education provided in schools tends to see adolescent reproductive and sexual health issues as merely biological phenomena, and often considers adolescent sexuality as something taboo and dangerous, which should be controlled through moral and religious discourse instead of education (Pakasi and Kartikawati, 2013). The results of the research conducted by Francis et al., (2011) also state that cultural norms and gender form the clearest response with regard to sexual health issues in communications about sexuality between mothers and children. Education about HPV, cervical cancer, and the HPV vaccine is recommended as necessary for adolescents, parents, and teachers. The results of research into the parent and teacher groups states that HPV, cervical cancer and HPV vaccine education should also be provided by involving parents. Parental involvement, through meetings at school, is required since parents make the decisions about their children’s vaccinations. Parental involvement in school meetings is an important strategy to educate about HPV, cervical cancer, and the HPV vaccination (Remes et al., 2012). papillomavirus vaccination Almost all the participants of the three groups disclosed constraints to buying the human papillomavirus vaccine. Most of them stated that even if many people wanted to buy the HPV vaccine for their daughters, they were unable to buy it because it is expensive. The parents’ group disclosed that the vaccine could be included in health insurance plans like the Social Insurance Administration Body (BPJS), which is an important factor in increasing the HPV vaccine’s acceptance. Some parent participants stated that the vaccine should not be expensive or there should be a discount for the HPV vaccine’s purchase. Theme 4: Insurance covering, discount and saving is the effort that parent needed to solve financial problems were identified, as follows: In Indonesia, parents have a low level of knowledge about HPV, the HPV vaccination, and cervical cancer topics in general (Jaspers et al., 2011). Only about Parent Participant (PP18): “Is it covered by the government’s BPJS? Or is it covered by Jamkesda Asian Pacific Journal of Cancer Prevention, Vol 20 2962 DOI:10.31557/APJCP.2019.20.10.2959 Need vs. Financing Capability: Human Papillomavirus Vaccinations among Adolescents HPV; about 15 8% regional regulations Need vs. Financing Capability: Human Papillomavirus Vaccinations among Adolescents Financing Capability: Human Papillomavirus Vaccinations among Adolescents bout 15.8% regional regulations. 16.6% of parents have heard about HPV; about 15.8% have heard of the HPV vaccine; and more than 40% lack understanding about HPV, the HPV vaccination, and cervical cancer. This percentage is nearly equal to that found in Malaysia and Singapore. There, 12.2% and 20.0% (respectively) have heard about HPV, and 10.5% and 15.8% (respectively) have heard about the HPV vaccine (Jaspers et al., 2011; Sam et al., 2009). According to these research results, we can conclude that greater information is needed about HPV, cervical cancer, and HPV vaccine topics, considering that the knowledge levels are still low.i The Minister of Health (2016) stated that the HPV vaccine has just been initiated as part of the government’s national program, but it is not a national program for vaccination, so the HPV vaccination’s cost is still high. The government’s currently prioritized measure is to mobilize resources to strengthen the health system and buy the HPV vaccine. Indonesia has not taken innovative measures, in terms of financing, for introducing the HPV vaccination. Meanwhile, Malaysia has included the HPV vaccine as a priority in its adolescent program, through its school health program (Ezat and Syed, 2011). papillomavirus vaccination It is important to focus on improving the overall level of knowledge. Similarly, consideration should be made for an alternative to the HPV vaccination’s financing, such as an adolescent reproductive health savings scheme, in order to maximize the HPV vaccination’s coverage rate. Funding Statement: Funding for this publication was provided by the Ministry of Research, Technology and Higher Education. The funder had no role in the study’s design, data collection, analysis, interpretation or publication of the report. Acknowledgements We would like to acknowledge the staff of the Nursing Department, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada. We thank the study participants for taking time to complete the interview and the school staff for their assistance in conducting this study. Last but not least, we would like to give our highest appreciation to Ibu Vania as the editor of this manuscript. The results of this research also identify constraints in financing for the HPV vaccination. The high price of the vaccination, combined with no recommendation for it from physicians, is the most influential factor in the HPV vaccination’s acceptance. Most of the respondents are from the middle to lower socio-economic groups. Reproductive health savings for the HPV vaccinations’ financing is an alternative method for increasing the HPV vaccination’s coverage in schools. The research results also show that the three groups of female students, parents, and teachers expect the HPV vaccination program to be free. If the program was free, the HPV vaccination coverage rate would be higher. The triangulation result with health workers states that the community health centers’ current program for cervical cancer prevention is still in its early stages, with Visual Inspection with Acetic Acid (VIA) examinations, and there is no socialization of the HPV vaccine as yet. The reason for this is that the program and activity have not yet been included in the Asian Pacific Journal of Cancer Prevention, Vol 20 2963 References Arifah K, Damayanti W, Sitaresmi MN (2017). Human papillomavirus acceptability among female adolescents in Yogyakarta. Sari Pediatri, 18, 430-5. Bartolini RM, Winkler JL, Penny ME, Lamontagne DS (2012). Parental acceptance of human papillomavirus in Peru: A Bartolini RM, Winkler JL, Penny ME, Lamontagne DS (2012). Parental acceptance of human papillomavirus in Peru: A Asian Pacific Journal of Cancer Prevention, Vol 20 2963 Wiwin Lismidiati et al Engaging parents and schools improves uptake of the human papillomavirus vaccine: examining the role of the public health nurse. Vaccine, 32, 4665-71.i decision framework. PLoS One, 7, 1–8. decision framework. PLoS One, 7, 1–8. Bruni L, Albero G, Serrano B, et al (2014). Human papillomavirus and related diseases report in the world.ICO/IARCHPV Information Centre, pp 8-23. WHO (2006). 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This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. Dempsey AF, Zimet GD (2015). Interventions to improve adolescent vaccination. Am J Prev Med, 49, 445–54. Fernández ME, Le YL, Fernández-espada N, et al (2014). Knowledge, attitudes, and beliefs about human papillomavirus vaccination among Puerto Rican mothers and daughters: a qualitative study. Prev Chronic Dis, 11, 1–8.i Francis SA, Battle-fisher M, Liverpool, J, Hipple L, Mosavel M (2011). A qualitative analysis of South African women’s knowledge, attitudes, and beliefs about human papillomavirus and cervical cancer prevention, vaccine awareness and acceptance, and maternal-child communication about sexual health. Vaccine, 29, 8760–5. Gallagher KE, Lamontagne D, Watson-jones D (2018). Asian Pacific Journal of Cancer Prevention, Vol 20 2964 This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. References Status of human papillomavirus vaccine introduction and barriers to country uptake. Vaccine, 36, 4761–7. Jaspers L, Budiningsih S, Wolterbeek R, Henderson FC, Peters AA (2011). Parental acceptance of human papillomavirus vaccination in Indonesia: a cross-sectional study. Vaccine, 29, 7785–93. Karneli NK, Suwiyoga K, Sudibya A (2013). Parental willingness to pay the cervical cancer vaccination cost of senior high school aged students in Badung Regency. PHPM, 1, 70–7. Masika MM, Ogembo JG, Chabeda SV, Wamai RG, Mugo N (2015). Knowledge on human papillomavirus vaccine and cervical cancer facilitates vaccine acceptability among school teachers in Kitui County, Kenya. PLoS One, 10, 1–14. Morales-Campos DY, Markham CM, Peskin M (2013). Hispanic mothers and high school girls perceptions of cervical cancer, human papillomavirus, and the human papillomavirus vaccine. J Adolesc Health, 52, 69–75. Pakasi DT, Kartikawati R (2013). Between needs and taboos: sexuality and reproductive health education for high school students. Makara Seri Kesehatan, 17, 79–87. Reiter PL, Stubbs B, Panozzo CA, Whitesell D, Brewer NT (2011). Human papillomavirus and human papillomavirus vaccine education intervention: effects on parents, healthcare staff , and school staff. Cancer Epidemiol Biomarkers Prev, 20, 2354–62. Remes P, Selestine V, Changalucha J, et al (2012). A qualitative study of human papillomavirus vaccine acceptability among health workers, teachers, parents, female pupils, and religious leaders in Northwest Tanzania. Vaccine, 30, 5363–7. Rosenthal SL, Weiss TW, Zimet GD, et al (2011). Predictors of human papillomaviru vaccine uptake among women aged 19-26: Importance of a physician’s recommendation. Vaccine, 29, 890–5. Sam IC, Wong LP, Rampal S, et al (2009). Maternal acceptance of human papillomavirus vaccine in Malaysia. J Adolesc Health, 44, 610–2. Whelan NW, Steenbeek A, Martin-Misener R, et al (2014). Asian Pacific Journal of Cancer Prevention, Vol 20 2964
W2894047459.txt	https://revistas.ufrj.br/index.php/aigeo/article/download/28615/15753	en		Tool for Prospecting of Remaining Hydro-Energetic Potential	Anuário do Instituto de Geociências	2,018	cc-by	4,212	Anuário do Instituto de Geociências - UFRJ www.anuario.igeo.ufrj.br Tool for Prospecting of Remaining Hydro-Energetic Potential Ferramenta para Prospecção de Potencial Hidroenergético Remanescente Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad Escola de Engenharia de São Carlos (EESC USP), Centro de Recursos Hídricos e Estudos Ambientais, Avenida dos Trabalhores São-Carlense, 400, Parque Arnold Schimidt, 13566-590, São Carlos – SP E-mails: claudio@ana.gov.br; dalva@sc.usp.br; bbernardo@sc.usp.br; mauadfm@sc.ups.br Recebido em: 15/05/2018 Aprovado em: 18/07/2018 DOI: http://dx.doi.org/10.11137/2018_2_427_437 Abstract In the context of renewable energy sources, small hydropower plants are a good cost-benefit alternative and they can be implemented in remote communities that have waterfalls. The objective of this study was to develop and apply a tool in a GIS environment capable of pointing out sites for the remaining hydro-energy utilization in three hydrographic basins based on altimetric data and regionalization of flow rates. The results were verified by comparing hydro-energetic potentials identified with previously inventoried sites. From the comparative analysis, it was possible to identify the remaining potentials efficiently and quickly, being the tool an important instrument to assist in the prospecting of hydro-energetic potential. Keywords: GIS; Hidropower; Water Resources Resumo No contexto de fontes de energia renováveis as pequenas centrais hidrelétricas apresentam-se como uma boa alternativa em relação ao custo benefício podendo ser implantadas em comunidades remotas que disponham de queda d’água. Este estudo teve por objetivos desenvolver e aplicar uma ferramenta em ambiente SIG capaz de apontar locais para o aproveitamento hidroenergético remanescente em três bacias hidrográficas a partir de dados altimétricos e regionalização de vazões. Os resultados foram verificados a partir da comparação dos potenciais hidroenergéticos identificados com locais previamente inventariados. Da análise comparativa realizada verificou-se a possibilidade de identificar os potenciais remanescentes de forma eficiente e rápida sendo esta ferramenta um importante instrumento para auxiliar a prospecção de potenciais hidroenergéticos. Palavras Chaves: SIG; Hidroenergia; Recursos hídricos Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 427 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad 1 Introduction Together with other renewable sources of energy, small hydroelectric exploitation represent a good alternative to large hydroelectric, thermoelectric and nuclear power stations due to economic, social and environmental factors, as well as market price guarantees, governmental articulation and well deined public policies (Larentis et al., 2010). A small hydropower plant has some advantages over thermal generation, such as lower installation costs, smaller investments for generation and the life cycle of hydroelectric energy is greater than in thermal power station (Abbasi & Abbasi, 2011). Punys et al. (2011) evaluated tools and methodologies for planning small hydropower plants in Canada, Germany, France, the Netherlands and the in United States. They concluded that the main advances in the last decades occurred due to the integration with geographic information systems, as well as the improvement of topographic and precipitation data. Sachdev et al. (2015) presented a review on the main sources of renewable energy and they commented that the small hydropower plants are costefective to be implemented in remote communities that have waterfalls. The greatest diiculty of a model is to be able to describe the minimum parameters for assessment of technical viability, such as safety in the supply, sizing of pumps and turbines and scope of changes in the hydrological regime. The expansion of the hydroelectric system within the sustainability assumptions should be in line with public policies on water resources and the environment, social development plans, international agreements and conventions (e.g. Climate convention). The largest hydroelectric potential in Brazil is found in the Amazon region, some parts in protected areas, Environmental Conservation Units and indigenous lands, which makes the licensing of those large generating units very diicult (Brazil, 2007). Thus, the prospections of small generating units represent an alternative to guarantee and assure the demands for energy. In the preliminary studies for the prospection of electric energy, the terrain morphology and 428 the low of the watercourse are relevant. They determine whether the power plant will be high or low, as well as the energy potential and the surface area that will be looded and will afect directly on the implementation costs of the enterprise due to environmental and land demands. In a conventional manner, the hydro-energetic potential is identiied from the low duration curve FDC (Gustard et al., 1992). That methodology was used by Salford Civil Engineering (1989) to identify small-scale energy potential in Scotland (<100 kW), to estimate the base low index (BFI) and the annual low from the combination of the use of the soil, average evapotranspiration and standard average annual rainfall. Hydrobot is a geographic information system to identify hydropower potential in Scotland. The algorithm performs the search from a location and extends up to 1.5 km with increments of 20 meters. Criteria for legal, environmental, and inancial constraints were added to the algorithm and the results could be compared with existing schemes (Forrest & Wallace, 2009; Sample et al., 2015). Simulated hydrological data in the WatBal software were applied to the HEC-5 package (Hydrologic Engineer Center of USACE) to assess the impacts of climate change on the power supply of the Batoka George power plant (1.6 GW) in the United States. The study suggests signiicant reductions in river outlows that will lead to a decline in the production and sale of electricity as well as impacts on long-term investment projections (Harrison & Whittington, 2002). Tools for the evaluation of remaining electric power should detect the variability of low and precipitation due to long-term climate change to support investment projection and in the short-term to ensure stability in the supply and management of resources. Simulations conducted in the Colorado Basin in the United States have shown that the decrease in summer low was ofset by increased winter precipitation, so robust tools for uncertainty analysis are needed (Christensen et al., 2004; Christensen & Lettenmaier, 2007) Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad The products delivered by geographic information systems (GIS) allow the analyst to evaluate the locations to advance the electric energy expansion projects. However, the uncertainties are associated with the spatial resolution of the images for the construction of the digital elevation model (DEM) that will inluence the estimation of the amount of energy to be generated and looded areas. The objective of this study was to develop and apply a tool in a GIS environment capable of pointing out sites for the remaining hydro-energy use in three Brazilian river basins. The results were veriied by comparing hydro-energetic potentials identiied with previously inventoried sites. 2 Materials and Methods Three hydrographic basins were selected as a study area for the application of the tool. The Hydrographic Basin of Rio Preto located in Minas Gerais State, where there are three electric power generation units: Hydropower Plant (HPP) Queimado, Small Hydropower Plant (SHP) Mata Velha andSmall Hydropower Plant (SHP) Unaí Baixo. The Hydrographic Basin of Alto Teles Pires River located in the Amazonas State, where there is an inventory point (Salto do Magessi) and the SHP Canoa Quebrada; and the Açungui River Basin, between the states of São Paulo and Paraná, with several inventory points. The methodology for prospecting remaining potentials from digital elevation models by automated GIS procedures initially consisted of obtaining Digital Elevation Models (DEM) from study areas from the Shuttle Radar Topography Mission (SRTM) of February 2000 with a spatial resolution of 1 arc of a second (approximately 30 meters) made available to the region of Brazil from the year 2014. The regions studied include areas where hydro-energetic studies or inventories already existed or where hydraulic works were built for Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 energy utilization after the year 2000, date of the SRTM mission. The DEMs initially underwent the procedure of removal of areas of depression and small imperfections through the Fill Sinks tool, as described by Taborton et al. (1991). That procedure has the purpose of improving the hydrological consistency of the model for the next stage in the deinition of low directions of the DEM. The low directions were determined, pixel by pixel, taking into account the eight neighbors adjacent to the pixel studied in the direction of greater slope. That procedure is known as FD8 and it was presented by Jenson & Domingue (1988). Using the topology deined by the calculated low directions, the cells that contribute upstream to each pixel were counted, generating a raster of accumulated lows. A threshold of 0.5% of the cumulative value in the mouth of the study basin was used for the selection of the synthetic drainage network pixel. The gaps between the upstream and downstream pixel were then analyzed only along this drainage network. By means of a map algebra operation, the dimension value (corrected by the Fill Sink) was assigned along the drainage. In order to calculate the diference, it was necessary to deine the pixel dimension values immediately downstream. When analyzing a single pixel along the drainage network between its eight possible neighbors, necessarily the neighboring pixel of lower dimension value corresponds to the downstream pixel. In order to identify that pixel, it was used a spatial analysis function called Focal Statistics, in which the 3x3 pixel area was deined as the analysis window and the minimum function to identify the downstream dimension value. That methodology was presented by Carvalho Jr. et al. (2008). The calculated slope value (H), for the subsequent evaluation of the hydro-energetic potential, it is the result of a map algebra operation consisting of the subtraction of the dimension raster and the upstream dimension plus a constant value, which is a parameter deined by the analyst and it corresponds to the maximum height allowed for the dam. 429 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad For the calculation of the low, the regionalized low value was used as reference value for the area of the hydrographic basin of interest according to the respective areas of contribution. DEM does not change, and in the places where the dams were selected when the DEM value is lower than the raster value of the dams, the dam is the one that passes to the new modiied DEM. From the spatial resolution of the DEM, the corresponding area of one is calculated. By multiplying the value of the pixel area by the raster of accumulated lows of the drainage network, by means of a map algebra operation, the contribution areas for each pixel of the drainage network are calculated. Applying the regionalization coeicients, the low values are obtained at each pixel of the drainage. From the new DEM and the dam dimension values, one must then identify the cells that constitute Sinks (depressions). For those cells, the upstream areas constituting the respective watersheds (contributed hydrographic basin of each Sink) are determined. Therefore, due to the modiications incorporated to the DEM, a new map of direction low must be constructed. With the rasters referring to the unevenness and the low, the equation 1 can be applied to establish the potential. P = ρ .Q.H .η (1) P is the hydro-energetic potential [MW]; ρ = speciic weight of water [Kg.m-3]; Q = low [m³.s-1]; H = slope [m] and η = yield of the turbines. With the established potentials, it was possible to select those that have the highest values by means of a predetermined parameter that represents a percentage on the highest calculated potential. The selected sites were then converted from raster format to a vector and their attribute table was illed with dimension values, slope, low, low directions and potential. From the coordinates of the selected points, lines representing the dam at each point are constructed, perpendicular to the lines deining the drainage network, with lengths pre-established by the analyst, in number of pixels always odd (1, 3, 5, ...). Thus, each dam has as an attribute of the quota of the dam, the quota value in the drainage network and the value of the height of the dam previously deined. Polygons that surround the dam lines are then built through bufer with one pixel distance and those are converted to raster with calculated dam quota values. With that raster, the hydrologically consisted DEM (DEM Fill) is altered so that it incorporates the dam quota values. Where there are no dams, the 430 By means of raster values of dam dimensions, these values are transferred to the watershed raster and the cross between that raster and the consisted DEM (subtraction between both), it can be determined the looded areas as a function of the dams by selecting the pixels that result in negative values when subtracting, which are the depth values. The absolute value of the depths multiplied by the pixel area results in the looded volume in each pixel, which will later be aggregated for each reservoir resulting in its total volume. From this selection, the depth raster is converted into polygons (vector), which are aggregated as a function of the respective identiier to each dam. Thus, the attribute table may contain the area and volume values of the reservoirs of each dam. In order to automate the procedures, to allow better eiciency and to minimize the processing time, a tool was built in the ArcGis software through a functionality that allows creating models from lows that join a sequence of tools, necessarily present in the ArcToolbox, and the database. Model Builder allows creating worklow routines and new tools, and even integrates scripts into the lowchart. The complete lowchart of the developed tool involves 39 spatial analysis operations between map algebra and attribute selections and GIS format conversions. It also includes a Python language script that was written speciically to generate the dam lines at each selected site, operating directly from the coordinates of the start vertices and the end of the dam, starting from the coordinates of the site and the number of cells established for the dams, as well as their spatial resolution. Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad 3 Results and Discussion The Rio Preto watershed is part of the Area of the Plateaus in Concordant Sedimentary Structures that covers the Goiás Minas Plateau, with altitudes ranging from 400 to 1400 meters (Brazil, 1982). The geomorphological compartmentalization is subdivided into ive classes: Upstream Plateau, Dissected Plateau, Peak of the Unaí, Terraces and Fluvial Plain downstream where it lows into the Paracatu River, Minas Gerais (Borges, 2007). Figure 1 shows the regionalization for the low with permanence of 90% (Q90%) of the data from the monitoring stations of the Rio Preto Basin in the state of Minas Gerais. In addition, Figure 2 displays the map with the places where there is the potential energy. Figure 1 Regionalization curve of low rates of Rio Preto, MG. Figure 2 Map of places where there are potential energy in Rio Preto, MG. Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 431 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad The geomorphology of the upper basin of the Upper Teles Pires River called the Graben of the Caiabis formed by sedimentary rocks of the Caiabis Group, which in the edges form plateaus with average elevations of 500 meters and, in the interior, the plateaus formation predominates (Bias et al., 2006). Figure 3 shows the regionalization of the lows (Q90%) of the Alto Teles Pires River stretch in the Amazonas State according to the data of the luviometric monitoring stations of the National Water Agency (ANA, 2014). Figure 4 displays all the potential energies of the Alto Teles Pires River identiied by the tool according to the parameters provided in Coniguration 1. The data processing took 26 minutes to complete all the calculations. The relief of the Teles Pires River basin is not very rugged, so even low dams generate large looded areas and high potentials due to the high lows present in the basin. The inventory presented by the Energy Research Company 2005 already indicated the construction of UHEs in the basin with run-of-river conigurations. The PCHs Canoa Quebrada (built) and Salto Magessi (inventoried) were identiied by the tool. In addition, many points were identiied at the mouth of this subbasin, which is in the area covered (looded) of the Sinop HPP. Altering the coniguration of height of dams and minimum slope (coniguration 2), a new map of the potential energies of the Alto of Teles Pires River basin was obtained (Figure 5). The Açungui River Basin is located in the geomorphological unit called First Paranaense Plateau, with a predominant declivity class of 6%, varying in altitudes from 560 to 1240 meters (Paraná, 2006a). The permanence curve of the Açungui River is shown in green in Figure 6, according to the low studies of the surface of the Upper Iguaçu basin and the tributaries of the Upper Ribeira River (Paraná, 2006b). The speciic low for the Açungui River is calculated by the equation 2. Qspec% = -12,81 * Ln(per %) + 63,588 (2) Qspec% is the speciic low for the retention time (% PER) in L.s-1 * Km2 and %PER is the retention time for a percentage value between 1 and 100. Figure 7 show the results of the calculation of the potential energy for the Açungui River. After the selection of some points, new calculations were made for the formed reservoirs, as shown in Figure 8. It noteworthy that, for the three study areas, the tool pointed to possible hydroelectric potential points close to places where there are Small Hydroelectric Plants already installed or inventoried. It demonstrates that the tool can help managers in the preliminary deinition of areas that provide greater potential in the generation of energy, thus taking better advantage of the looded area. Obviously, such a tool does not eliminate ieldwork since there is need for hydrosedimentological studies. 4 Conclusion The proposed tool for the location and the calculation of the hydro-energy potentials proved Figure 3 Regionalization curve of low rates of Upper Teles Pires River 432 Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad Figure 4 Map of the hydro-energetic potentials of the Upper Teles Pires River, AM in coniguration 1 Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 433 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad Figure 5 Map of the hydro-energetic potential of the Upper Teles Pires River, AM in coniguration 2 434 Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad Figure 6 Curve of permanence of speciic lows of the Açungui River (in green). Figure 7 Map of all of the hydropower potential of the Açungui River Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 435 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad Figure 8 Map of the hydropower potential after recalculation 436 Anuário do Instituto de Geociências - UFRJ ISSN 0101-9759 e-ISSN 1982-3908 - Vol. 41 - 2 / 2018 p. 427-437 Tool for Prospecting of Remaining Hydro-Energetic Potential Claudio Bielenki Junior; Dalva Maria de Castro Vitti; Bruno Bernardo dos Santos & Frederico Fábio Mauad to be very efective, since it was able to identify, in the studied basins, the points where inventories were already carried out and the power plants were installed in a fast and automated manner. The spatial resolution of the digital terrain models and the consistency of the low data inluence the accuracy of site identiication and estimation of potentials, as well as the conigurations in terms of height and size of the dams. The obtained results showed that this tool assists in the preliminary evaluation of the hydropower potential for any basin of interest. Once the locations of the largest remaining hydroelectric potential have been identiied, eforts can be made to investigate other variables such as environmental impacts, climate change efects on pluviometric indices, low, sediment production, costs and amortization of investments. 5 Acknowledgments The authors would like to thank the Foundation for Industrial Research and Advancement (FIPAI), the Hydrometry Nucleus of the Water Resources Center and the Environmental Studies of the São Carlos School of Engineering of the University of São Paulo (EESC / USP). 6 References Abbasi, T. & Abbasi, S.A. 2011. Small Hydro and the Environmental implications of its extensive utilization. Renewable and Sustainable Energy Reviews, 15: 2134 - 2143. Agência Nacional de Águas - ANA. 2014. Situação dos Recursos Hídricos no Brasil 2014”. Brasília, DF. Bias, E.S.; Torres, M.G.; Baptista, G.M.M.; Ribeiro, R.J.C. & Resende, M.G. 2006. Análise da evolução geomorfológica na Bacia Teles Pires a partir de dados SRTM - Shuttle Radar Topography Mission. In: SIMPÓSIO NACIONAL DE GEOMORFOLOGIA, VI, Goiânia, 2006. Artigos completos, Goiânia, Universidade Federal de Goiás, p. 1 – 14. Borges, M.E.S.; Soares, F.S.; Carvalho Jr.; O.A.; Martins, E.S.; Guimarães, R.F.; Gomes, R.F. & Gomes, R.A.T. 2007. Relação dos compartimentos geomorfológicos com o uso agrícola na bacia do Rio Preto. Rev. Espaço e Geograia, 10(2): 453 - 476. Brasil. Ministério das Minas e Energia 1982. Secretaria Geral. Projeto RADAMBRASIL. Folha SD-23, Brasília; Geomorfologia, pedologia, vegetação e uso potencial da terra. 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https://openalex.org/W3155175008	https://www.frontiersin.org/articles/10.3389/fphys.2021.659777/pdf	English	null	Hepatic Oxidative Stress, Apoptosis, and Inflammation in Broiler Chickens With Wooden Breast Myopathy	Frontiers in physiology	2,021	cc-by	9,402	Hepatic Oxidative Stress, Apoptosis, and Inﬂammation in Broiler Chickens With Wooden Breast Myopathy Tong Xing, Xiaona Pan, Lin Zhang and Feng Gao* Key Laboratory of Animal Origin Food Production and Safety Guarantee of Jiangsu Province, Joint International Research Laboratory of Animal Health and Food Safety, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, National Experimental Teaching Demonstration Center of Animal Science, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China Wooden breast (WB) syndrome has emerged as a global myopathy in modern commercial broiler chickens, mainly affecting the pectoralis major muscle. Recent evidence suggests that WB myopathy is a systemic disease, which might be accompanied by other physiological disparities and metabolic changes. This study was conducted to systemically investigate the potential physiological changes in liver tissues as well as the possible mechanisms involved to enhance the understanding of the etiology. A total of 93 market-age Arbor Acres male broiler chickens were sampled and categorized into control (CON) and WB groups based on the evaluation of myopathic lesions. Liver samples were collected (n = 10 in each group) for histopathological evaluation and biochemical analyses. Results indicated that WB birds exhibited signiﬁcantly higher plasma aspartate amino transferase, alkaline phosphatase, and gamma glutamyl transpeptidase activities. Histopathological changes in hydropic/fatty degeneration, inﬂammatory cell inﬁltration, intrahepatic hemorrhages, elevated myeloperoxidase activity, and overproduction of nitric oxide were observed in WB liver compared with CON, suggesting the occurrence of liver injury in birds affected by WB myopathy. The WB group showed increased levels of reactive oxygen species, oxidative products, as well as enhanced antioxidant capacities in the liver. These changes were associated with impaired mitochondria morphology and mitochondrial dysfunction. WB myopathy also induced mitochondria-mediated hepatic apoptosis by upregulating levels of caspases 3 and 9, altering the expressions of apoptotic B-cell lymphoma-2 family regulators, as well as increasing the release of cytochrome c. The activation of nuclear factor kappa-light-chain-enhancer of activated B cell signaling enhanced the mRNA expression of downstream inﬂammatory mediators, contributing to the production of inﬂammatory cytokines in WB liver. Combined, these ﬁndings suggest that hepatic disorders may be conjoined with WB myopathy in broiler chickens and indicating systemic physiological disparities, and other metabolic changes accompanying this myopathy need further assessment. ORIGINAL RESEARCH published: 14 April 2021 doi: 10.3389/fphys.2021.659777 Keywords: wooden breast, broiler chicken, liver, oxidative stress, apoptosis, inﬂammation Edited by: Massimiliano Petracci, University of Bologna, Italy Reviewed by: Maurizio Mazzoni, University of Bologna, Italy Alessandra Piccirillo, University of Padua, Italy Correspondence: Feng Gao gaofeng0629@sina.com Reviewed by: Maurizio Mazzoni, University of Bologna, Italy Alessandra Piccirillo, University of Padua, Italy Correspondence: Feng Gao gaofeng0629@sina.com Specialty section: This article was submitted to Avian Physiology, a section of the journal Frontiers in Physiology Received: 28 January 2021 Accepted: 10 March 2021 Published: 14 April 2021 MATERIALS AND METHODS p y y Extensive studies have been carried out to investigate the histological lesions, physiological properties, and molecular changes involved in the development of WB myopathy. The ﬁndings imply that the underlying mechanisms of this growth-related muscular abnormality are complicated and multifaceted processes, which might be related with the abnormal accumulation of endomysial and perimysial connective tissue and the consequent ﬁbrosis, hypoxia, oxidative stress, and inﬂammatory response as well as metabolic shift (Sihvo et al., 2014; Mutryn et al., 2015; Soglia et al., 2017; Liu et al., 2020). Recently, accumulating evidence suggests that the etiology of WB myopathy is not limited to the PM muscle, but is also associated with perturbations in blood circulation and other organs. Greene et al. (2019) demonstrated that the circulatory oxygen homeostasis was dysregulated in WB myopathic broiler chickens as indicated by the altered pressure of blood gases and hemoglobin levels. Our recent study revealed that the inﬂammatory cytokines including interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were enhanced in the serum of WB aﬀected birds (Xing et al., 2021). Furthermore, lung histopathology of WB myopathic birds exhibited occasional localized multifocal lymphoplasmacytic phlebitis and more foci of chondro-osseous metaplasia compared with the unaﬀected birds (Lake et al., 2020). WB myopathy increased stress hormone corticosterone levels in plasma and altered expression patterns of stress response- related genes in the liver (Kang et al., 2020). Assessment of potential systemic physiological disparities accompanying WB myopathy might contribute to a profound understanding of its etiology. Experimental Broiler Chickens and Tissue Collection All experimental procedures and bird managements were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University. Broiler chickens used in the current study were all Arbor Acres males raised in three layered cages and received commercially formulated feed and husbandry. Birds were provided ad libitum access to feed and water. Birds were vaccinated against Newcastle disease virus, infectious bronchitis virus, and infectious bursal disease virus through neck injection at 11 days of age, using commercially available vaccines. At 42 days of age, a total of 300 live birds were clinically examined for WB myopathy involving visual observations for posture and wing contact as well as bilateral manual palpation for hardness of the pectoralis major (PM) in a cranio-caudal direction by two trained personnel (Papah et al., 2017). This resulted in 63 suspected WB-aﬀected and 30 WB- unaﬀected broilers. These broilers were electrically stunned (50 V, alternating current, 400 Hz for 5 s each) and exsanguinated via the carotid arteries and jugular veins. Immediately after execution, birds were necropsied, and samples of PM muscle and liver tissues were collected and labeled. Liver tissues from the caudal region of the left lobe were taken and ﬁxed in 4% paraformaldehyde or 2.5% glutaraldehyde for histological evaluation or ultrastructural observation. The remaining liver tissues were frozen in liquid nitrogen and stored at −80◦C for biochemical analysis. The liver is a primary metabolic organ, which has important physiological functions such as biosynthesis, clearance, detoxiﬁcation, and host defense. Liver damage has become a common disease, which can be caused by various risk factors of xenobiotics, malnutrition, and other chronic diseases (Malhi and Citation: Xing T, Pan X, Zhang L and Gao F (2021) Hepatic Oxidative Stress, Apoptosis, and Inﬂammation in Broiler Chickens With Wooden Breast Myopathy. Front. Physiol. 12:659777. doi: 10.3389/fphys.2021.659777 April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 1 Wooden Breast Liver Disorder Xing et al. INTRODUCTION Gores, 2008). In patients with Duchenne muscular dystrophy (DMD), liver atrophy was shown to occur concomitantly with skeletal muscle wasting (Moriuchi et al., 1991). Liver abnormalities in mdx dystrophic mice, including decreased glycogen levels and hyperglycemia, have been observed (David et al., 2014). Furthermore, patients with muscular dystrophies showed an increased susceptibility to acute liver failure upon therapeutic paracetamol administration (Pearce and Grant, 2010). It appears that there is a strong link between hepatic disorders and muscular diseases. Interestingly, PM muscle, and liver transcriptome through the ingenuity pathway analysis identiﬁed critical transcriptional response network associations in WB myopathic birds (Phillips et al., 2020), suggesting the systemic pathology involved in the progression of this myopathy. To date, limited data exist on the hepatic changes associated with WB myopathy. The current study was designed to systemically compare the histological and biochemical characteristics and the underlying mechanism causing these diﬀerences, if any, in the liver between normal and WB myopathic broiler chickens. Over the past few decades, the demand for poultry meat has increased notably. Consequently, genetic selection of modern commercial broiler chickens has been pushed toward fast growth and enhanced breast muscle yield (Petracci et al., 2015). Despite achieving extraordinary improvements, this selection pressure accompanied with the modern intense breeding programs have caused the increasing incidence of spontaneous breast muscle abnormalities. These emerging myopathies have drawn worldwide attention due to their high occurrence and negative impacts on meat quality (Petracci et al., 2019). One abnormality, wooden breast (WB) myopathy, is macroscopically characterized by hardened areas, pale ridge-like bulges, as well as occasional appearance of clear viscous ﬂuid, small hemorrhages, and white striping in the pectoralis major (PM) muscle (Sihvo et al., 2014). Due to the unappealing appearance, WB ﬁllets are usually downgraded and used only for highly processed products. Although still usable, WB myopathy seriously impairs the quality and nutritional value of breast meat (Mudalal et al., 2015; Soglia et al., 2016), thereby causing substantial economic losses to the poultry industry. Frontiers in Physiology \| www.frontiersin.org Determination of Reactive Oxygen Species Serum enzymatic activities of alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (AKP), and gamma glutamyl transferase (γ-GT) were determined by using the corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. Intracellular reactive oxygen species (ROS) in liver was measured using a ﬂuorescent probe, 2,7-dichloroﬂuorescein diacetate (DCFH-DA, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as previously described (Xing et al., 2017b). The ﬂuorescence intensity was detected at an excitation wavelength of 500 nm and emission wavelength of 525 nm, respectively, using a ﬂuorescence Microplate Reader (Spectramax M2; Molecular Devices, Sunnyvale, CA, United States). Wooden Breast Myopathy Scoring and Sample Selection During necropsy, the dissected PM muscle was further evaluated using a more accurate WB myopathy scoring system based April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 2 Xing et al. Wooden Breast Liver Disorder Histopathological Evaluation p g For histological analysis, liver tissues were ﬁxed in 4% paraformaldehyde for more than 24 h at room temperature, dehydrated in a graded series of ethanol, trimmed, and embedded in paraﬃn blocks. Liver sections were cut into 8-µm thickness and mounted on polylysine-coated slides. Subsequently, the slides were rehydrated by a series of incubations in xylene and ethanol solutions and then subjected to hematoxylin and eosin (H&E) and Masson trichrome staining according to the procedures described by Huang et al. (2010) and Xing et al. (2017a). Images were acquired under identical conditions and at the same magniﬁcation using a light microscope (Axio Scope.A1, Carl Zeiss, Oberbochen, Germany). Liver tissue was examined for histopathologic changes including the presence of inﬂammation, intrahepatic hemorrhages, or ﬁbrosis. Assessment of all slides was performed as a blind study to prevent bias in the examination of tissues. Ultrastructural Observation Liver tissue specimens were ﬁxed in 2.5% glutaraldehyde solution and washed with 0.1 M PBS, followed by postﬁxing with 1% osmium tetroxide. After washing with PBS, the tissues were hierarchically dehydrated with gradually increasing concentrations of ethanol (30–70%) and then embedded in Spurr’s resin. Samples were embedded in Epon812 and sectioned using an ultra-microtome (RMC Power Tome XL, Leica, Wet- zlar, Germany). Ultrathin sections (30 nm) were collected and stained with 3% uranyl acetate and lead citrate. Ultrastructural changes were examined using a transmission electron microscope (TEM, Hi-tachi H-7650, Tokyo, Japan). Mitochondria Isolation and Myeloperoxidase (MPO) activity and nitric oxide (NO) level were assessed spectrophotometrically with commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), per the manufacturer’s instructions. The protein concentration of the liver was determined using a BCA protein assay kit (Pierce Chemical Co., Rockford, IL, United States). The activity of MPO was expressed as units per gram of wet tissue, and the level of NO was expressed as micromoles per gram of protein. Analysis of Hepatic Oxidative Products and Antioxidant Ability on gross lesions and palpable ﬁrmness as described by Livingston et al. (2019). Brieﬂy, the ordinal scale ranged from 0 to 3 points, where a score of (0) was used when there was no presence of macroscopic myopathic lesion (normal), (1) was used when the ﬁllets were hard primarily in the cranial region but otherwise pliable (mild), (2) was used when the ﬁllets were hard throughout but ﬂexible in the mid to caudal region (moderate), and (3) was used when the ﬁllets were extremely hard and rigid throughout from cranial region to caudal tip (severe). We found 30 normal, 31 mild, 20 moderate, and 12 severe WB myopathy aﬀected PM muscles among the 93 selected birds. After scoring, 10 liver tissues from birds with normal PM muscle (CON) and 10 samples from birds aﬀected by moderate-to-severe WB myopathy (WB) were randomly selected to evaluate the subsequent biochemical parameters. Liver tissues were homogenized in 0.9% NaCl buﬀer and centrifuged at 2,000 × g for 10 min at 4◦C. The supernatants were collected for the determination of oxidative products and antioxidant ability. Protein concentration was determined using a BCA protein assay kit. The measurements of malondialdehyde (MDA), lipid peroxidation (LPO), and protein carbonyl were performed using corresponding commercial kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The content of the 8-hydroxydeoxyguanosine (8- OHdG) was determined using an ELISA kit obtained from Aogene Bioengineering Institute (Nanjing, China). Results of MDA and protein carbonyl were expressed as micromoles per milligram protein. The content of LPO and 8-OHdG were expressed as moles per milligram protein and nanogram per milligram protein, respectively. The activities of total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GSH-ST) were measured using the corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The results were all expressed as units per milligram protein. Mitochondrial Function Assay The liver mitochondria were extracted as described by Frolova et al. (2019) with some modiﬁcations. Brieﬂy, fresh liver tissues were rinsed using phosphate buﬀer solution (PBS) and minced into mash. Minced tissues were homogenized in chilled isolation buﬀer (20 mM Tris-HCl, 250 mM sucrose, and 1 mM EDTA, pH 7.4) and then centrifuged at 1,000 × g for 15 min at 4◦C. The supernatants were collected and April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 3 Wooden Breast Liver Disorder Xing et al. at 260 nm with a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Rockland, DE, United State), and the purity was assessed by determining the ratios of optical density (OD) value at 260 and 280 nm. cDNA was reverse transcribed using a commercial cDNA Synthesis Kit (PrimeScriptTM RT Master Mix, Takara) and diluted 20 times with DEPC water before use. Quantitative real-time PCR was performed on an Applied Biosystems 7500 instrument (Foster City, CA, United State) using SYBR Premix EX Taq (Takara, United State). The PCR reaction conditions consisted of denaturation at 95◦C for 10 min, followed by 40 cycles of 95◦C for 15 s, annealing at 60◦C for 1 min, and extension at 60◦C for 20 s. Primer sets used for quantitative RT-PCR analysis are listed in Supplementary Table 1. All gene expressions are calculated as the relative fold changes compared with CON, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference to normalize the expression of target genes. Relative mRNA expression was calculated according to the 2−11CT method. centrifuged at 12,000 × g for 15 min at 4◦C, where mature heavy mitochondria were precipitated. Subsequently, the mitochondria pellets were washed thrice using the isolation buﬀer. Finally, all mitochondrial fractions were suspended in ice-cold isolation buﬀer and diluted to a protein concentration of 1 mg/ml. Protein concentration was determined using the BCA protein assay kit. Aliquots of the mitochondrial suspension were stored at −80◦C for further use. Mitochondria membrane potential (1ψm) assay was performed using a JC-1 kit (Solarbio Science & Technology, Co., Ltd, Beijing, China), per the manufacturer’s instructions. Brieﬂy, mitochondrial suspension (20 µl) with a total protein content of 20 µg was mixed with 180 µl of JC-1 dyeing working solution for 10 min, and the ﬂuorescence intensity was measured using a microplate reader. Mitochondrial Function Assay The wavelengths for the detection of the monomeric and aggregated forms of JC-1 were 514/529 and 585/590 nm (excitation/emission). The mitochondrial swelling was assessed according to Zhang et al. (2015). The obtained liver mitochondria suspension (20 µl) was incubated with 170 µl of swelling assay buﬀer containing 150 mM KCl, mM HEPES, 2 mM K2HPO4, 5 mM glutamate, and 5 mM malate to get a 20-µg total protein content. The mitochondrial swelling was triggered by the addition of 10 µl of calcium solution (1 mM). The absorbance was continuously determined at 540 nm for 18 min with an interval of 45 s using a microplate reader. Low mitochondria swelling exhibits high absorbance, and mitochondria with high swelling has low absorbance. Total Protein Extraction and Western Blot Analysis y Frozen liver tissues were homogenized in RIPA lysis buﬀer (Beyotime Biotechnology, Jiangsu, China) containing 1 mM PMSF. The homogenate was centrifuged at 12,000 × g for 20 min at 4◦C, and the supernatant was collected. The BCA assay was used to determine protein concentration. Equal amounts of total protein (40 µg) were resolved on 10% SDS- PAGE using a BioRad Electrophoresis System (Richmond, CA, United State) and transferred to a nitrocellulose membrane (Millipore, Merck, Germany). The membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated in primary antibodies against kappa-light-chain- enhancer of activated B cells (NF-κB), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), cytochrome c (Cytc), GAPDH (Servicebio Biological Technology, Wuhan, China), B-cell lymphoma (Bcl)-2 (Boster Biological Technology, Wuhan, China), and caspase3 (Absin Bioscience Inc., Shanghai, China) overnight at 4◦C followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (Bioworld, Nanjing, China) for 1 h. Finally, the membranes were visualized using ECL reagents (Pierce, IL, United States) and scanned using ImageQuant LAS4000 (GE, CT, United State). The density of each band was quantiﬁed by using Quantity One software (Bio-rad) and normalized to its respective housekeeping protein (GAPDH). All protein contents are calculated as the relative fold changes compared with CON. Apoptotic Nuclei Analysis The detection of nuclei exhibiting apoptosis was performed using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) kit according to the manufacturer’s instructions (Vazyme Biotech Co., Ltd., Nanjing, China) with minor modiﬁcations. Brieﬂy, the paraﬃn-embedded liver tissues were sectioned at 5 µm, rehydrated by a series of incubations in xylene and ethanol solutions, and permeabilized using proteinase K (20 µg/ml) at 37◦C for 25 min. After rinsing in PBS, the sections were incubated with mixed reagents consisting of TdT and dUTP at 37◦C for 1 h. The sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China) to label the nuclei. Finally, the TUNEL-positive cells were visualized using a ﬂuorescence microscope (Axio Scope.A1, Carl Zeiss, Oberbochen, Germany). For apoptotic nuclei evaluation, four ﬁelds of 137,600 square micrometers per section were randomly selected and analyzed using the Image- Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, United State). The hepatic apoptotic index was calculated as percentage of the total number of nuclei. Histopathological Observation and Biochemical Parameters in the Liver of Wooden Breast Myopathic Birds We stained the liver tissues using H&E and Masson staining to reveal the damage caused by WB myopathy (Figure 1A). Histology of the liver tissues from the CON group showed normal structures with regular morphology. On the contrary, the liver tissue of WB broiler chickens showed widespread lesions with hydropic/fatty degeneration (indicated by black arrows), inﬁltration of inﬂammatory cells (indicated by ∗), and severe intrahepatic hemorrhages (indicated by white arrows). In addition, occasional collagen deposition or ﬁbrosis (indicated by black triangle) were simultaneously observed in the liver of WB birds. Both MPO activity and NO production were signiﬁcantly elevated in WB compared with CON (P < 0.05; Figures 1B,C). FIGURE 1 \| Histopathological observation and biochemical parameters in liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative images of hematoxylin and eosin (H&E) and Masson staining of liver, indicating the widespread lesions with hydropic/fatty degeneration (black arrowhead), inﬂammatory cell inﬁltration (), and intrahepatic hemorrhages (white arrowhead) as well as occasional ﬁbrosis (black triangle) in WB. (B) Myeloperoxidase (MPO) activity and (C) nitric oxide (NO) level. Data are expressed as the mean ± SE (n = 10). P < 0.05. Enzymatic Activities in the Serum of Wooden Breast Myopathic Birds Measuring levels of serum ALT, AST, AKP, and other enzymes provides a clinical sign of liver injury and ascertains the severity of liver disease. As exhibited in Table 1, the activities of AST, AKP, and γ-GT were elevated by 73.3 ± 15.3% (P < 0.01), 63.7 ± 10.6% (P < 0.01), and 46.1 ± 3.0% (P < 0.01), respectively, in the serum of WB aﬀected broiler chickens compared with CON. No signiﬁcant diﬀerence in ALT activity was observed between the two groups (P > 0.05). RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR Data were analyzed by one-way analysis of variance (ANOVA) using SAS 9.12 (2003; SAS Inst. Inc., Cary, NC), and the diﬀerences between individuals were compared using Student’s t-tests. Data were reported as means ± SE. Signiﬁcance was considered when P ≤0.05, and a trend was indicated when 0.05 < P < 0.1. Total RNA was isolated from the liver tissues of broiler chickens using RNAiso Plus reagent (Takara Biotechnology Co., Ltd, Dalian, China) following the manufacturer’s instructions. Total RNA concentration was quantiﬁed by measuring the absorbance April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 4 Wooden Breast Liver Disorder Xing et al. FIGURE 1 \| Histopathological observation and biochemical parameters in liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative images of hematoxylin and eosin (H&E) and Masson staining of liver, indicating the widespread lesions with hydropic/fatty degeneration (black arrowhead), inﬂammatory cell inﬁltration (), and intrahepatic hemorrhages (white arrowhead) as well as occasional ﬁbrosis (black triangle) in WB. (B) Myeloperoxidase (MPO) activity and (C) nitric oxide (NO) level. Data are expressed as the mean ± SE (n = 10). P < 0.05. RESULTS Enzymatic Activities in the Serum of Wooden Breast Myopathic Birds Measuring levels of serum ALT, AST, AKP, and other enzymes provides a clinical sign of liver injury and ascertains the severity of liver disease. As exhibited in Table 1, the activities of AST, AKP, and γ-GT were elevated by 73.3 ± 15.3% (P < 0.01), 63.7 ± 10.6% (P < 0.01), and 46.1 ± 3.0% (P < 0.01), respectively, in the serum of WB aﬀected broiler chickens compared with CON. No signiﬁcant diﬀerence in ALT activity was observed between the two groups (P > 0.05). Enzymatic Activities in the Serum of Wooden Breast Myopathic Birds Hepatic Oxidative Products and Antioxidant Ability of Wooden Breast Myopathic Birds April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 5 Wooden Breast Liver Disorder Xing et al. CAT, SOD, GSH-Px, and GSH-ST than those from the CON birds. These results implied a disturbed redox status in the liver tissue of WB myopathic birds. CAT, SOD, GSH-Px, and GSH-ST than those from the CON birds. These results implied a disturbed redox status in the liver tissue of WB myopathic birds. apoptotic index of hepatocyte when compared with the CON group (P < 0.01; Figure 3B). The mRNA expressions of pro- apoptotic factors including Bcl-2-associated X protein (Bax, P < 0.05), Bcl-2 antagonist or killer 1 (Bak1, P < 0.01), and Cyt c (P < 0.01) were upregulated, whereas the antiapoptotic regulators of B cell lymphoma (Bcl)-2 (P < 0.05) and Bcl-xl (P < 0.1) were downregulated in the WB group compared with the CON group (Figure 3C). In addition, the transcription of caspase 9 and caspase 3 were signiﬁcantly enhanced in WB compared with CON (P < 0.01). Consistently, WB myopathy increased the protein contents of Cytc and caspase 3, but decreased the Bcl-2 protein content in comparison with the CON group (P < 0.05; Figures 3D,E). Wooden Breast Myopathy Induced Liver Mitochondria Morphology Changes and Mitochondrial Dysfunction y The ultrastructure of the mitochondria of liver tissues from the CON group was well developed with intact membrane integrity and rich cristae density, whereas the collapse of cristae and membrane swelling was observed in the liver tissues of the WB group (Figure 2A). The production of ROS was signiﬁcantly increased in WB when compared with CON (P < 0.01; Figure 2B). We further detected 1ψm and mitochondrial swelling to evaluate mitochondrial function changes. Results indicated that 1ψm presented a signiﬁcant decrease in the WB group compared with the CON group (P < 0.05). Consistently, the mitochondria in the WB birds had signiﬁcantly decreased OD values at 540 nm triggered by calcium compared with those of the CON birds (P < 0.01), showing that the mitochondria in the liver tissue of birds aﬀected by WB myopathy were prone to swelling. Wooden Breast Myopathy Induced Inﬂammatory Responses in Chicken Liver The protein contents of NF-κB, iNOS, and COX-2 were signiﬁcantly increased in the liver of WB birds compared with the CON birds (P < 0.05, Figures 4A,B). Similarly, WB myopathy enhanced the transcription of NF-κB, iNOS, COX-2, and prostaglandin E synthetases (PTGEs) in chicken liver in comparison with the CON group (P < 0.05, Figure 4C). Furthermore, WB broiler chickens exhibited signiﬁcantly increased mRNA expressions of pro-inﬂammatory cytokines including IL-1β, IL-6, IL-8, and TNF-α in the liver compared Hepatic Oxidative Products and Antioxidant Ability of Wooden Breast Myopathic Birds As exhibited in Table 2, WB myopathy induced oxidative stress and led to damage in the cellular biomacromolecules in the liver tissues as indicated by the considerable elevated (P < 0.05) contents of MDA, LPO, protein carbonyl, and 8- OHdG. Additionally, the liver samples from WB broiler chickens exhibited signiﬁcantly higher (P < 0.05) activities of T-AOC, TABLE 2 \| Oxidative products and antioxidant ability in liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle (n = 10). TABLE 2 \| Oxidative products and antioxidant ability in liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle (n = 10). chickens with normal (CON) and wooden breast (WB) pectoralis major muscle (n = 10). Items1 Category SEM P value CON WB MDA (nmol/mg protein) 0.99b 1.16a 0.06 * LPO (mol/mg protein) 0.33b 0.42a 0.03 * Protein carbonyl (nmol/mg protein) 1.44b 1.70a 0.08 * 8-OHdG (ng/g protein) 0.41b 0.51a 0.06 * T-AOC (U/mg protein) 3.67b 4.45a 0.15 CAT (U/mg protein) 32.60b 47.01a 2.56 SOD (U/mg protein) 186.13b 211.32a 6.98 * GSH-Px (U/mg protein) 25.81b 31.21a 1.55 * GSH-ST (U/mg protein) 14.86b 23.53a 1.40 *** a,bMean values within the same row followed by different superscript letters indicating signiﬁcance (P < 0.05). P < 0.05. Signiﬁcant difference at P < 0.01. signiﬁcant difference at P < 0.001. 1SEM, standard error of mean; MDA, malondialdehyde; LPO, lipid peroxidation; 8-OHdG, 8-hydroxydeoxyguanosine; T-AOC, total antioxidant capacity; CAT, catalase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; GSH-ST, glutathione S-transferase. TABLE 1 \| Enzymatic activities in serum of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle (n = 10). Items1 Category SEM P value CON WB ALT activity (U/L) 1.74 1.76 0.12 ns AST activity (U/L) 21.07b 36.52a 2.44 * AKP activity (U/L) 455.45b 745.77a 39.25 * γ-GT activity (U/L) 24.50b 35.81a 0.62 * a,bMean values within the same row followed by different superscript letters indicating signiﬁcance (P < 0.05). *Signiﬁcant difference at P < 0.001. ns, indicates no signiﬁcant difference. 1SEM, standard error of mean; ALT, alanine amino transferase; AST, aspartate amino transferase; AKP, alkaline phosphatase; γ-GT, gamma glutamyl transpeptidase. TABLE 1 \| Enzymatic activities in serum of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle (n = 10). Wooden Breast Myopathy Induced Apoptosis in Chicken Liver Apoptosis in Chicken Liver Apoptotic hepatocytes were detected using TUNEL staining as exhibited in Figure 3A. WB myopathy signiﬁcantly increased FIGURE 2 \| Mitochondria morphology and mitochondrial function changes in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative transmission electron microscope images of liver. (B) Relative reactive oxygen species (ROS) level. (C) Relative mitochondrial membrane potential. (D) Relative mitochondrial swelling. Data are expressed as the mean ± SE (n = 10). P < 0.01 and P < 0.05. FIGURE 2 \| Mitochondria morphology and mitochondrial function changes in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative transmission electron microscope images of liver. (B) Relative reactive oxygen species (ROS) level. (C) Relative mitochondrial membrane potential. (D) Relative mitochondrial swelling. Data are expressed as the mean ± SE (n = 10). P < 0.01 and P < 0.05. April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 6 Wooden Breast Liver Disorder Xing et al. Xing et al. RE 3 \| Apoptotic status and apoptosis-related mediators in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major le. (A) Representative images of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of liver. (B) Relative percentage of ocyte apoptosis. (C) Relative mRNA expression of Bcl-2-associated X protein (Bax), Bcl-2 antagonist or killer 1 (Bak1), B cell lymphoma (Bcl)-2, Bcl-xl, hrome c (Cytc), caspase 9, and caspase3. (D) Representative Western blotting images of Cytc, Bcl-2, and caspase 3. (E) Relative protein content of Cytc, and caspase 3. Data are expressed as the mean ± SE (n = 10). *P < 0.01, P < 0.05, and #0.05 < P < 0.1. RE 4 \| Inﬂammatory mediators and proinﬂammatory cytokines in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis muscle. (A) Representative Western blotting images of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducible nitric oxide synthase ), and cyclooxygenase-2 (COX-2). (B) Relative protein content of NF-κB, iNOS, and COX-2. (C) Relative mRNA expression of NF-κB, iNOS, COX-2, and aglandin E synthetases (PTGEs). (D) Relative mRNA expression of pro-inﬂammatory cytokines including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor -α. Data are expressed as the mean ± SE (n = 10). *P < 0.01 and P < 0.05. Wooden Breast Myopathy Induced Apoptosis in Chicken Liver FIGURE 3 \| Apoptotic status and apoptosis-related mediators in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative images of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of liver. (B) Relative percentage of hepatocyte apoptosis. (C) Relative mRNA expression of Bcl-2-associated X protein (Bax), Bcl-2 antagonist or killer 1 (Bak1), B cell lymphoma (Bcl)-2, Bcl-xl, cytochrome c (Cytc), caspase 9, and caspase3. (D) Representative Western blotting images of Cytc, Bcl-2, and caspase 3. (E) Relative protein content of Cytc, Bcl-2, and caspase 3. Data are expressed as the mean ± SE (n = 10). *P < 0.01, P < 0.05, and #0.05 < P < 0.1. FIGURE 3 \| Apoptotic status and apoptosis-related mediators in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative images of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of liver. (B) Relative percentage of hepatocyte apoptosis. (C) Relative mRNA expression of Bcl-2-associated X protein (Bax), Bcl-2 antagonist or killer 1 (Bak1), B cell lymphoma (Bcl)-2, Bcl-xl, cytochrome c (Cytc), caspase 9, and caspase3. (D) Representative Western blotting images of Cytc, Bcl-2, and caspase 3. (E) Relative protein content of Cytc, Bcl-2, and caspase 3. Data are expressed as the mean ± SE (n = 10). *P < 0.01, P < 0.05, and #0.05 < P < 0.1. FIGURE 4 \| Inﬂammatory mediators and proinﬂammatory cytokines in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative Western blotting images of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). (B) Relative protein content of NF-κB, iNOS, and COX-2. (C) Relative mRNA expression of NF-κB, iNOS, COX-2, and prostaglandin E synthetases (PTGEs). (D) Relative mRNA expression of pro-inﬂammatory cytokines including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Data are expressed as the mean ± SE (n = 10). *P < 0.01 and P < 0.05. FIGURE 4 \| Inﬂammatory mediators and proinﬂammatory cytokines in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative Western blotting images of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). (B) Relative protein content of NF-κB, iNOS, and COX-2. DISCUSSION WB myopathy has been identiﬁed as an emerging muscular disease in modern broiler chickens over the past decade. Chronic WB myopathy-aﬀected muscles exhibit histological lesions of polyphasic myodegeneration, myoﬁber necrosis, small regenerating myoﬁbers, inﬂammatory cell inﬁltration, as well as deposition of lipid and connective tissue (Sihvo et al., 2014). Growing evidence suggests that the excessive development of breast muscle leads to the hypertrophy of myoﬁber, thereby reducing perimysial and endomysial space available for capillaries and compromising blood supply (Velleman, 2019), which possibly trigger an intricate pathogenesis including oxidative stress, impaired calcium homeostasis, inﬂammatory responses, and metabolic shifts (Petracci et al., 2019). Meanwhile, the aberrant accumulation of interstitial ﬁbrotic tissues and a consequent increase in the spaces between muscle ﬁbers might be associated with a lower capillary density and a greater intercapillary distance, which could further deteriorate the microvascular architecture and aggravate this situation (Soglia et al., 2017). Besides breast muscle abnormalities, broilers aﬄicted with WB myopathy also exhibit secondary pathophysiological perturbations in blood circulation and in other organ systems (Lake et al., 2020; Phillips et al., 2020). Oxidative stress occurs when the balance of pro-oxidants and endogenous antioxidants in a living system is disturbed, which can lead to the overproduction of free radicals (Sies et al., 2017). Previous studies suggested the presence of altered redox homeostasis and oxidative stress as possible biological processes linked with the pathogenesis of WB disease (Abasht et al., 2016; Papah et al., 2018). In addition, the excessive formation of ROS was directly observed in WS and WB-aﬀected PM muscle (Salles et al., 2019; Pan et al., 2020). Intracellular macromolecules are vulnerable to free radicals, and the resultant lipid peroxidation, modiﬁcation of proteins, and nucleic acid breaks may further contribute to structural collapse and dysfunction. Herein, we observed an aberrant ROS accumulation and augmented levels of MDA, LPO, protein carbonyl, and 8-OHdG, indicating that oxidative stress occurs in the liver of WB-aﬀected birds. Oxidative stress or disturbed redox state has been implicated as a crucial mediator contributing to hepatic damage and the progression of pathological liver disorders (Zhu et al., 2012). Aﬂatoxin B1 administration induced oxidative stress in the liver of broilers, which contributed to hepatic dysfunction characterized by pallor discoloration, enlargement, and necrosis (Li et al., 2019). Wooden Breast Myopathy Induced Apoptosis in Chicken Liver (C) Relative mRNA expression of NF-κB, iNOS, COX-2, and prostaglandin E synthetases (PTGEs). (D) Relative mRNA expression of pro-inﬂammatory cytokines including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Data are expressed as the mean ± SE (n = 10). *P < 0.01 and P < 0.05. FIGURE 4 \| Inﬂammatory mediators and proinﬂammatory cytokines in the liver tissues of broiler chickens with normal (CON) and wooden breast (WB) pectoralis major muscle. (A) Representative Western blotting images of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). (B) Relative protein content of NF-κB, iNOS, and COX-2. (C) Relative mRNA expression of NF-κB, iNOS, COX-2, and prostaglandin E synthetases (PTGEs). (D) Relative mRNA expression of pro-inﬂammatory cytokines including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Data are expressed as the mean ± SE (n = 10). *P < 0.01 and P < 0.05. April 2021 \| Volume 12 \| Article 659777 7 Frontiers in Physiology \| www.frontiersin.org Wooden Breast Liver Disorder Xing et al. myopathies such as white striping (WS) and dorsal cranial myopathy exhibited increased levels of ALT and AST in the serum (Kuttappan et al., 2013; Sesterhenn et al., 2017). However, Kuttappan et al. (2013) observed the unchanged γ-GT and decreased AKP activity in the serum of WS-aﬀected birds compared with the normal. We ascribed this inconsistency to the diﬀerent stages of myopathy progression. Since WS and WB are both growth-associated myopathies sharing similar histological lesions (Sihvo et al., 2014), the myopathic aberrations of PM muscle might start with the onset of WS and progress into WB during the whole growth period (Griﬃn et al., 2018). Consistent with the elevations of indicators of hepatic damage in the plasma, we observed widespread lesions with hydropic/fatty degeneration, inﬁltration of inﬂammatory cells, occasional ﬁbrosis, and severe intrahepatic hemorrhages in liver sections of WB myopathic birds. The increase in MPO activity and the overproduction of NO further implied the inﬁltration of neutrophil and mononuclear cells, and conﬁrmed the induction of the inﬂammatory process in the liver tissue (Aktan, 2004; Loria et al., 2008). with the CON birds (P < 0.05, Figure 4D). These results suggested that WB myopathy induced inﬂammatory responses in the liver by activating inﬂammatory mediators and enhancing production of proinﬂammatory cytokines. Frontiers in Physiology \| www.frontiersin.org DISCUSSION Similarly, broilers aﬀected by other April 2021 \| Volume 12 \| Article 659777 8 Wooden Breast Liver Disorder Xing et al. broilers (Pan et al., 2020). The inhibited activities of GSH- Px and GSH-ST were also observed in the PM muscle of severe WS-aﬀected birds (Salles et al., 2019), suggesting that the activation of antioxidant armamentarium depends on the stage and severity of injury or disease. The mitochondria not only constitute primary sources of ROS but also are vulnerable to oxidative attack due to the high content of phospholipid and protein in their membranes (Cadenas and Davies, 2000). In the current study, the ultrastructural examination indicated the damaged mitochondrial structure in WB liver. In addition, the WB liver mitochondria exhibited loss of 1ψm and were prone to go through swelling, indicating an impaired function. In the meantime, mitochondria dysfunction may interact with cellular redox environment, contributing to ROS overproduction and the impaired antioxidant defense system (Balaban et al., 2005). Collectively, these results demonstrated the occurrence of oxidative stress in the liver of WB myopathic birds as evidenced by the disturbed redox homeostasis and mitochondria damage, which possibly contribute to hepatic pathological changes. important roles in the pathogenesis of liver injury (Schwabe and Brenner, 2006). IL-1β might contribute to the pathogenesis of liver damage as IL-1β knockout mice showed attenuated hepatocellular damage, steatosis, and ﬁbrosis in atherogenic diet-induced steatohepatitis (Kamari et al., 2011). Therefore, these dysregulated cytokines could further lead to immune disorder and contribute to the aggravation of liver damage. This result also supports our recent ﬁnding of the systemic inﬂammatory response in WB myopathic broilers as implied by the elevation of circulating cytokines (Xing et al., 2021). Besides the secretion of cytokines, inﬂammatory cells can also produce excessive oxygen free radicals to attack host cells, leading to hepatocyte damage (Zhu et al., 2012). NF-κB is a central regulator in mediating liver inﬂammatory responses by controlling the expression of cytokines; the activation of NF-κB signaling has been implicated in various liver diseases (Luedde and Schwabe, 2011). In accordance with these studies, the expression of NF-κB was enhanced, and the expressions of its downstream targets including iNOS, COX-2, and PTGEs were upregulated in WB compared with those in CON. Meanwhile, these increased inﬂammatory mediators could further contribute to the exacerbation of inﬂammatory progression and cytokine production (Aktan, 2004; Subbaramaiah et al., 2012). 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. Hepatocytes undergoing injury can promote the release of cytokines and recruit inﬂammatory cells, such as neutrophils and macrophages to clean up debris and stimulate regeneration (Malhi and Gores, 2008). In this study, we observed the upregulated mRNA expression of proinﬂammatory cytokines including IL-1β, IL-6, TNF-α, and IL-8 in the liver of WB compared with CON. TNF-α acts as a potent activator of both proinﬂammatory and proapoptotic pathways, exerting CONCLUSION In summary, this study provides evidence of liver damage in birds aﬀected by WB myopathy primarily by impaired liver morphology as well as elevated serum AST, AKP, and γ-GT activities. Oxidative stress in WB liver triggered by the excessive ROS accumulation might be associated with disturbance of redox status and mitochondrial dysfunction. Additionally, the present study conﬁrms the mitochondria-mediated hepatocyte apoptosis and NF-κB signaling-regulated inﬂammatory response, which possibly contribute to the aggravation of liver injury of WB myopathic birds. In general, our results strongly suggest that hepatic disorders might be strongly correlated with WB myopathy and provide evidence to explain the possible mechanisms involved in these perturbations. Further studies are needed to assess systemic physiological disparities and other metabolic changes accompanying this myopathy for further recognition of its etiology. DISCUSSION p y p p g g Oxidative stress triggered by various insults or pathological states is closely associated with the induction of programmed cell apoptosis (Ryter et al., 2007). The present study revealed a signiﬁcantly increased number of hepatocytes undergoing apoptosis in WB demonstrated by the TUNEL assay. The apoptosis process is executed through the caspase family, among which, caspase3 plays a vital role in mediating both intrinsic and extrinsic signaling pathways. The present results indicated the mRNA expression of caspase 3 and 9 as well as the protein content of caspase 3 that was upregulated in the WB liver compared with the CON. Similarly, the occurrence of apoptosis is involved in liver injury, alcoholic and non-alcoholic steatohepatitis, and chronic liver disease (Aizawa et al., 2020). The mitochondrial and endoplasmic reticulum-mediated intrinsic apoptosis is usually triggered by a variety of stimuli such as calcium overload, ROS overproduction, and unfolded protein response. In addition, loss of 1ψm is an important hallmark in apoptosis and occurs in the early phase of mitochondria-mediated apoptosis (Kinnally et al., 2011). Based on the elevated ROS accumulation and mitochondrial dysfunction in WB liver, we speculated that the activation of apoptosis was mitochondria mediated caspase dependent. The mitochondrial pathway is regulated by pro- and antiapoptotic Bcl-2 family members. The imbalance of these regulators causes the permeation of the mitochondrial outer membrane and promotes the release of pro-apoptotic protein, thereby activating the caspase cascade (Xing et al., 2019). Therefore, the increased levels of Bax and Bak1, decreased levels of Bcl-2 and Bcl-xl, as well as the release of Cytc conﬁrmed the activated mitochondrial pathway were involved in liver apoptosis of WB myopathic birds. DISCUSSION Intraperitoneal injection of hydrogen peroxide triggered hepatic oxidative stress by increasing ROS level and contents of oxidative products, thereby exerting a negative impact on the histomorphology and redox status in the liver, as well as the resultant decline in growth performance of broilers (Chen et al., 2018). Furthermore, oxidative markers could serve as prognostic indicators of liver damage and chronic hepatic diseases such as non-alcoholic steatohepatitis and liver ﬁbro-proliferative disease (Cicho˙z-Lach and Michalak, 2014). To counteract oxidative stress, organisms generally stimulate multiple layers of antioxidant defense system to reduce the formation of excessive free radicals. As expected, we observed signiﬁcantly enhanced activities of T-AOC, CAT, SOD, GSH- Px, and GSH-ST in the liver of the WB group compared with the CON group. Accordingly, the antioxidant enzyme defensive system was activated in the PM muscle of WB myopathic The liver is deﬁned as the primary internal organ for poultry exerting a variety of metabolic and homeostatic functions including digestion, metabolism, biosynthesis, excretion, and detoxiﬁcation. The disruption of hepatic function has been implicated to reduce growth performance and threaten the health of birds, causing economic losses to the poultry industry (Zaefarian et al., 2019). In addition, mammalian studies indicate that liver disease might be implicated as a conjoint pathological mechanism underlying several metabolic disorders including neuromuscular disease (Pearce and Grant, 2010; David et al., 2014). Recent studies have likewise assessed the alterations of transcriptome and stress response genes in both liver and muscle of WB myopathic birds and revealed that the etiology of this myopathy is not limited to muscle, but is a systemic pathology (Kang et al., 2020; Phillips et al., 2020). Therefore, this study was conducted to investigate the histological and biochemical status and to depict the possible mechanistic changes involved in the liver of myopathic birds for better understanding of the molecular basis of the underlying pathological process and thereby promoting the healthy production of broiler chickens. Injury to the liver tissue can lead to the release of various hepatic enzymes into the bloodstream. Increases in plasma levels of aminotransferases, AKP, and γ-GT are widely used as diagnostic markers of hepatic damage (Li et al., 2019). 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Gastroenterol. 20, 8082–8091. doi: 10.3748/wjg.v20.i25. 8082 David, I., Lau, X., Flores, M., Trieu, J., Gehrig, S. M., Chee, A., et al. (2014). ETHICS STATEMENT The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Nanjing Agricultural University. April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 9 Xing et al. Wooden Breast Liver Disorder FUNDING This study was supported by the China Postdoctoral Science Foundation Grant (2018M640492 and 2019T120432), the Jiangsu Postdoctoral Science Foundation (2019K013), the Natural Science Foundation of Jiangsu Province in China (BK20190516), the National Natural Science Foundation of China (31872374 and 32072780), the National Key Research and Development Program of China (2018YFD0500405), and the Earmarked This study was supported by the China Postdoctoral Science Foundation Grant (2018M640492 and 2019T120432), the Jiangsu Postdoctoral Science Foundation (2019K013), the Natural Science Foundation of Jiangsu Province in China (BK20190516), the National Natural Science Foundation of China (31872374 and 32072780), the National Key Research and Development Program of China (2018YFD0500405), and the Earmarked ACKNOWLEDGMENTS We thank Mrs. Jeanene M. de Avila (Department of Animal Sciences, and School of Molecular Bioscience, Washington State University, United State) for revising and editing this manuscript to provide grammatical coherence. AUTHOR CONTRIBUTIONS Fund for Jiangsu Agricultural Industry Technology System (JATS[2020]407). Fund for Jiangsu Agricultural Industry Technology System (JATS[2020]407). Fund for Jiangsu Agricultural Industry Technology System (JATS[2020]407). TX and FG conceived and designed the study. TX, XP, and LZ performed the experiments and conducted the data analysis. TX drafted the manuscript. All authors contributed to the article and approved the submitted version. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2021.659777/full#supplementary-material REFERENCES M., Fu, W., Lee, W. R., and Abasht, B. (2015). Characterization of a novel chicken muscle disorder through diﬀerential gene expression and pathway analysis using RNA-sequencing. BMC Genomics 16:399. doi: 10.1186/s12864-015-1623-0 April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 10 Xing et al. 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Physiol. Gastr. Liver Physiol. 290, G583–G589. doi: 10.1152/ajpgi.00422. 2005 Zhu, R., Wang, Y., Zhang, L., and Guo, Q. (2012). Oxidative stress and liver disease. Hepatol. Res. 42, 741–749. doi: 10.1111/j.1872-034X.2012.00996.x Sesterhenn, R., Siqueira, F., Hamerski, A., Driemeier, D., Valle, S., Vieira, S., et al. (2017). REFERENCES Histomorphometric study of the anterior latissimus dorsi muscle and evaluation of enzymatic markers of broilers aﬀected with dorsal cranial myopathy. Poult. Sci. 96, 4217–4223. doi: 10.3382/ps/pex252 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. Sies, H., Berndt, C., and Jones, D. P. (2017). Oxidative stress. Annu. Rev. Biochem. 86, 715–748. doi: 10.1146/annurev-biochem-061516-045037 Copyright © 2021 Xing, Pan, Zhang and Gao. 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. Sihvo, H. K., Immonen, K., and Puolanne, E. (2014). Myodegeneration with ﬁbrosis and regeneration in the pectoralis major muscle of broilers. Vet. Pathol. 51, 619–623. doi: 10.1177/0300985813497488 Soglia, F., Gao, J., Mazzoni, M., Puolanne, E., Cavani, C., Petracci, M., et al. (2017). Superﬁcial and deep changes of histology, texture and particle size distribution April 2021 \| Volume 12 \| Article 659777 Frontiers in Physiology \| www.frontiersin.org 11
https://openalex.org/W2002707427	http://www.scielo.br/pdf/rsp/v40n1/en_27123.pdf	English	null	Modificações nos padrões de consumo de psicofármacos em localidade do Sul do Brasil	Revista de saúde pública/Revista de Saúde Pública	2,006	cc-by	4,908	Conclusions A decade later, prevalence remained high, yet psychotropic drug use did not increase. The association between health service utilization and consumption shows the importance of the appropriate prescription of psychotropic drugs and regular follow- up of those prescribed them by physicians. Lima M. Morbidade Psiquiátrica Menor e Consumo de Psicofármacos em Pelotas, RS [Master’s dissertation]. Pelotas: Universidade Federal de Pelotas; 1995. Based on Master’s dissertation presented at the Faculdade de Medicina of Universidade Federal de Pelotas, in 2004. Received on 10/1/2005. Reviewed on 2/8/2005. Approved on 8/8/2005. Methods This is a population-based cross-sectional study carried out in 2003 with 3,542 participants aged 15 or older who lived in an urban area in Southern Brazil. Two-week recall data were collected in household interviews through the same questionnaire used in the 1994 study. The variables studied were age, gender, race, education, family income, marital status, smoking, medical diagnosis of hypertension, and physician visit at last three months. Pearson’s Chi-square and linear tendency were used in the bivariate analysis. Four levels of multivariate analysis was performed. Results The overall prevalence of psychotropic use was 9.9% (CI 95%: 8.9-10.9). There was no significant difference among standardized age groups when compared to the prevalence rates observed in 1994. Higher psychotropic use was associated with being female, older age, medical diagnosis of hypertension, and health service utilization. Of those interviewed, 74% of those drug users were using psychotropic drugs for over three months. Keywords Psychotropic drugs, administration & dosage. Drug utilization, trends. Pharmacoepidemiology. Cross- sectional studies. Psychopharmacology. Abstract Keywords Psychotropic drugs, administration & dosage. Drug utilization, trends. Pharmacoepidemiology. Cross- sectional studies. Psychopharmacology. Maria Aparecida P Rodrigues, Luiz Augusto Facchini and Maurício Silva de Lima Programa de Pós-Graduação em Epidemiologia. Faculdade de Medicina. Universidade Federal de Pelotas. Pelotas, RS, Brasil Based on Master’s dissertation presented at the Faculdade de Medicina of Universidade Federal de Pelotas, in 2004. Received on 10/1/2005. Reviewed on 2/8/2005. Approved on 8/8/2005. Objective j To assess the prevalence and patterns of psychotropic use by population and to compare the results with a study carried out in 1994. METHODS The study was conducted in Pelotas, a mid-sized port city located in the extreme south of Brazil with a population of approximately 320,000, of which 93.2% reside in urban areas (Demographic Census 2000 IBGE). Fieldwork was conducted during the months of October through December of 2003 and data was collected directly with eligible individuals through interviews in their homes. In cases of absence or refusal, the interviewers returned at least two more times to the household. In an attempt to revert re- fusals, the district supervisor made an attempt to encourage participation. This cross-sectional study was conducted in 2003 with individuals 15 years and older who were resi- dents in the urban areas of the city. The process of defining the sample was conducted in various stages. The groups were selected based on the municipal districts defined in the 2000 IBGE Demographic Census. After excluding the collective districts, 144 of the 404 municipal districts were sys- tematically sorted and stratified according to the av- erage income of the head of household. Each district was then visited to identify the households. The households were chosen proportionately based on the size of each district, after the exclusion of those which were abandoned or commercial spaces, lead- ing to the definition of a sample of 1,530 eligible households. All of those living in the households who were 15 or older were included in the sample. Data entry was conducted using EpiInfo 6.04d with double data entry and automatic checks for consist- ency and range. The dependent variable was defined by the follow- ing question: “Since <day of the week> the week before last, have you taken any medication for nerv- ousness or to sleep or any other medication that is only sold with a prescription?”. Respondents were asked to show their prescription, packaging, or in- structions so that interviewers could note and code the medication in the future based on the Ministry of Health’s National Agency of Sanitary Vigilance (ANVISA) list of controlled medicines (A3, B1, B2, and C1). If the respondent took more than one medi- cation, the one that was most recently used was in- cluded. The length of time using the medication, place of acquisition, person responsible for recommending the medication, and if the person used any other psy- chotropic drugs other than that already indicated were also included in the questionnaire. INTRODUCTION 1988 study1 in Ilha do Governador (state of Rio de Janeiro), was 5.2% and 10% in a 1993 study in the city of São Paulo.7 Psychotropic drug use has increased in the last few decades in various Occidental countries4,13 and even in some countries in the Orient.14,15 This increase has been attributed to an increase in the frequency of the diagnosis of psychiatric disturbances in the popula- tion, the introduction of new psychotropic drugs in the pharmaceutical market, and new therapeutic rec- ommendations of already existent psychotropic drugs. In Pelotas, a cross-sectional study* conducted in 1994 identified the prevalence rate of psychotropic drug use to be 11.9%. In this same year, the country established the Law of Generic Medicines, thereby reducing the cost of medicines. Also during this pe- riod, the appearance of new psychotropic drugs in the pharmaceutical market diversified the psycho- tropic drugs available. The prevalence of psychotropic use found in a Correspondence: Maria Aparecida P Rodrigues Avenida Duque de Caxias, 250 3o piso 96030-002 Pelotas, RS, Brasil E-mail: rodriguesmapa@aol.com Correspondence: Maria Aparecida P Rodrigues Avenida Duque de Caxias, 250 3o piso 96030-002 Pelotas, RS, Brasil E-mail: rodriguesmapa@aol.com Modifications in psychotropic drug use Rodrigues MAP et al dence interval. In addition, it permitted the detection of relative risks equal to or greater than 1.6 with 80% power for predictors with frequencies between 20% and 80% and psychotropic use prevalence rate of 7% for those unexposed, with a significance level of 5%. The calculation was estimated using EpiInfo 6.04d. During this same period, the Pelotas mental health services were expanded through the creation of vari- ous Psychosocial Care Centers (CAPS). This was ac- companied by the creation of the Municipal Phar- macy which caused an increase in free access to psy- chotropic drugs among those who use the One Health System (SUS). The data collection instrument used was the same instrument used in the 1994* study. It consisted of a structured and pre-coded questionnaire and included specific aspects of psychotropic drug use including demographic, socioeconomic, and health variables. Thirty-two interviewers were selected who were all 18 or older and had completed high school. They completed a 40 hour training session and a theory and practice exam. A pilot study to test the question- naire, instructions manual, and interview training was conducted in a census district, which was not included in the sample. Instituto Brasileiro de Geografia e Estatística. Censo demográfico de 2000. Available from: http://www.ibge.gov.br/cidadesat [2005 nov 18] Lima M. Morbidade Psiquiátrica Menor e Consumo de Psicofármacos em Pelotas, RS [Master’s dissertation]. Pelotas: Universidade Federal de Pelotas; 1995. INTRODUCTION Even though the facts cited indicate a likely in- crease in the use of psychotropic, there is no study in the country that has evaluated the use of psycho- tropic drugs among the same population group dur- ing a set amount of time. The objective of the current study was to verify the current prevalence and pat- tern of psychotropic drug use in comparison to the 1994 findings. METHODS Chronic use was considered those who used the drug intermittently for at least three months. The sample size calculation was established based on the largest value found between two calculations: estimate of the prevalence of psychotropic drug use and an evaluation of the associations between the dependent variable and the studied independent vari- ables. The calculation included an increase of 10% for losses, 15% for confounders and 1.2% for the study delineation effect. The sample obtained permitted an estimate of the prevalence of psychotropic consump- tion of 9% with a 1% margin of error and 95% confi- The following independent variables were studied: Modifications in psychotropic drug use Rodrigues MAP et al 57.9% 11.8% 9.0% 8.4% 7.9% 5.0% Benzodiazepínes Antipsychotic Amphetamine derivative Antidepressant Anticonvulsant Others 52.1% 31.6% 6.7% 3.7% 4.0% 1.9% 1994 2003 178 426 Figure - Distribution of psychotropic drug groups consumed within the 14 days prior to the interview. Pelotas, Brazil, 1994 and 2003. 57.9% 11.8% 9.0% 8.4% 7.9% 5.0% 1994 178 sex; age (years completed); skin-color (white or non-white); marital status (with, without companion, separated, or widowed; educa- tion (years of school completed); family in- come (monthly minimum wage according to the value of the national minimum wage during the period of data collection, in quar- tiles); smoking (non-smoker, smoker, or former smoker); medical diagnosis of hyper- tension, and medical consultation in the last three months. 178 426 Figure - Distribution of psychotropic drug groups consumed within the 14 days prior to the interview. Pelotas, Brazil, 1994 and 2003. and intraclass correlation of 0.005. In 1994, the preva- lence rate was 11.9% (95% CI: 10.1-13.7). After the standardization of the data by age, the prevalence went from 11.5% in 1994 to 10.0% in 2003 and there was no significant difference between the two (p=0.13). Data analysis was conducted using Stata 8.0. First, a description of the sample was conducted. Pearson’s Chi-square and linear tendency were used in the bivariate analysis. The adjusted analysis was conducted using linear regression based on a conceptual linear analysis model composed of four levels. In the first level were the demographic variables (sex, age, skin color), the second level were the socioeconomic variables (mari- tal status, education, family income), the third level included smoking status and medical diagnosis of systemic arterial hypertension, and in the fourth level, medical consultation in the last three months. METHODS The variables associated with exposure and the depend- ent variable were kept in the analysis with p≤0.20 for control of the confounders. In 1994, 92% of psychotropic drug users received a prescription for the medication from their physi- cian. The remaining 8% decided to take the medica- tion on their own. The percentage of psychotropic use with a medical prescription was 92%, of which 41% was through a general clinic, 20% through psy- chiatrists, and 31% through other specialists. The figure shows the comparison by group of psy- chotropic drugs consumed in 1994 and 2003. In both studies, the benzodiazepines represent more than half of the psychotropic drugs consumed. In 2003, there was a reduction in the proportion of antipsychotic use (p=0.09) and anorexigens (p=0.0006), in com- parison with the 1994 study. On the other hand, there was a significant increase in the antidepressant use in 2003 in comparison with 1994 (p=0.0003). The comparison of total prevalence of psychotropic use and the proportion of usage in the variables of exposure of the 1994 and 2003 studies used the pro- portional comparison test. The study was submitted to and approved by the Universidade Federal de Pelotas Commission of E- thics and Research. Before of the questionnaire was administered, verbal consent was obtained from the respondents and the confidentiality of the data col- lected was guaranteed. In 1994, 7.2% of the individuals utilized another psychotropic drug. The percentage increased signifi- cantly (p<0.001) to 22.2% in 2003. Chronic use did not vary significantly between the two studies even though it increased from 70.4% in 1994 to 73.9% in 2003 (p=0.35). Nonetheless, access to psychotropic drugs in the SUS pharmacy presented a highly sig- nificant increase from 3.5% in 1994 to 11.1% in 2003 (p<0.001). RESULTS A total of 3,542 individuals were included in the study, excluding those missing and refusals (3.8%). The medical diagnosis of hypertension variable had 446 missing values, of which 439 related to the group under 20 years old. Data for these individuals was only collected among those who used psychotropic drugs, which only was the case for three individuals of whom none of them had a medical diagnosis of hypertension. In 1994, the sample was 1,277 people. Table 1 describes the study samples in 1994 and 2003 according to the demographic, socioeconomic, and health variables. The distribution of the character- istics between the variable categories was similar in both studies. More than half of the individuals were women in 1994 and in 2003. In 1994, 22% of the indi- viduals were between 15 and 24, and in 2003, the per- centage was 25%. In 1994, 59% had a partner and in 2003, the percentage was 56%. With regard to educa- tion, the percentage of individuals who reported be- The prevalence of psychotropic use was 9.9% (95% CI: 8.9-10.9), with a study delineation effect of 1.03 Modifications in psychotropic drug use Rodrigues MAP et al tween 9 and 11 years of education increased from 17% to 28% in 2003. Note that hypertension went from 20% in 1994 to 23% in 2003. With regard to medical visits in the last three months, note that the percentage went from 44% to 54% in 2003. In 2003, 55% of re- spondents did not smoke and 80% were white. when compared to 1994, which was significant in the age brackets between 15 and 24 and 35 to 44. As the age group 15 to 24 was the reference category, the odds ratio increased in 2003 as compared to 1994. However, in both studies the levels of usage were simi- lar among the age bracket 45 and older. Table 2 presents the patterns of psychotropic drug use in 1994 and 2003, stratified by demographic, so- cioeconomic, and health variables. It examines their differences using the proportions comparison test. In 1994, the individuals with partners, separated, and widowed used significantly more psychotropic drugs than those without a partner. Marital status did not present a significant association with psycho- tropic use in 2003 due to the increase in usage within the reference category of “without partner”. RESULTS There was a statistically significant decrease in the widowed category in comparison with the 1994 study. The consumption of psychotropic drugs was sig- nificantly higher among females in both years ana- lyzed and reached more than double that of the males in 2003. There was a statistically significant reduc- tion in psychotropic use among men in 2003 when compared with the 1994 study. Education was not significantly associated with psy- chotropic use in 1994. In 2003, a reduction in psycho- tropic use was observed among individuals with high levels of education when compared to the group with- out education (p<0.001). This change occurred because there was a significant reduction in psychotropic use Psychotropic use increased with age and presented a strong linear tendency in both years. In 2003, there was a reduction in use among the younger groups of sample according to their demographic, socioeconomic, behavioral, and health characteristics. and 2003 Table 1 - Description of sample according to their demographic, socioeconomic, behavioral, and health characteristics. Pelotas, Brazil, 1994 and 2003. 1994 2003 Variable N % N % Gender Male 568 44.5 1,551 43.8 Female 709 55.5 1,991 56.2 Age (years completed) 15-24 278 21.7 875 24.7 25-34 246 19.2 632 17.8 35-44 254 19.8 686 19.4 45-54 181 14.1 610 17.2 55-64 151 11.8 354 10.0 65 or more 167 13.1 385 10.9 Skin color Non white 701 19.8 White 2,841 80.2 Marital status Without partner 340 26.6 1,065 30.1 With partner 759 59.4 1,989 56.2 Separated 104 8.2 228 6.4 Widowed 74 5.8 260 7.3 Education (years of school completed) 0 118 9.5 227 6.4 1- 4 307 24.5 621 17.5 5- 8 439 35.1 1,222 34.5 9-11 217 17.3 1,005 28.4 ≥12 170 13.6 465 13.1 Household income (minimum wages in quartiles) 1 303 24.9 864 24.5 2 304 25.1 904 25.6 3 304 25.1 884 25.1 4 303 24.9 875 24.8 Smoking Non smoker 1,942 54.8 Smoker 888 25.1 Former smoker 712 20.1 Hypertension No 1,028 80.5 2,372 76.6 Yes 249 19.5 724 23.4 Medical consultation in last three months No 717 56.1 1,673 47.2 Yes 560 43.9 1,869 53.8 Total 1,277 100 3,542 100 Note: In 2003, the maximum number of missing values was 446 in the hypertension variable. In 1994, the maximum number of missing values was 63 in the household income variable. In 2003, the maximum number of missing values was 446 in the hypertension variable. In 1994, the maximum number ssing values was 63 in the household income variable. RESULTS 1 - Description of sample according to their demographic, socioeconomic, behavioral, and health characteristics. s, Brazil, 1994 and 2003. Modifications in psychotropic drug use Rodrigues MAP et al in the categories of 1 to 4, 5 to 8, and 9 to 11 years of school in comparison to the 1994 study. in the categories of 1 to 4, 5 to 8, and 9 to 11 years of school in comparison to the 1994 study. In 2003, white individuals reported higher psycho- tropic use than non-whites (p=0.003) and with re- gards to smoking, no association with psychotropic use was found. The smoking and skin color data from the 1994 study were not released. Psychotropic use was not significantly associated with family income in 1994 and 2003. However, a sta- tistically significant reduction was observed in psy- chotropic use in the first and third quartiles of family income in 2003 when compared to the 1994 study. After controlling for confounders, being female, older age, hypertension, and a medical consultation in the last three months continued to be significantly associated with psychotropic use in the 1994 and 2003 studies. Marital status was no longer signifi- cantly associated with psychotropic use in 1994. The adjustment made in 2003 maintained a significant association between white skin color with psycho- tropic use and found a significant association be- tween smoking and psychotropic use. Note that individuals with hypertension used more psychotropic drugs than those who did not have hyper- tension in both the 1994 and 2003 studies (p<0.001). Individuals who had a medical consultation in the last three months reported significantly higher psy- chotropic use than those who had not in both studies. Nonetheless, in the 2003 study, a significant reduc- tion in psychotropic use among those who had a medical consultation in the last three months was observed when compared to the 1994 study. DISCUSSION Possible memory bias is inherent in a study of us- Possible memory bias is inherent in a study of us- Table 2 - Prevalence, unadjusted odds ratio for psychotropic use and comparison of prevalence rates between the two years studied. Pelotas, Brazil, 1994 and 2003. 1994 2003 Comparison Variable Prevalence OR (95% CI) p-value Prevalence OR (95% CI) p-value p-value (%) (%) Gender <0.001 <0.001* Male 7.9 1.00 5.5 1.00 <0.001 Female 15.1 1.90(1.37-2.65) 13.3 2.40(1.90-3.00) 0.23 Age <0.001 <0.001 15-24 3.2 1.00 1.8 1.00 <0.001 25-34 4.5 1.38(0.58-3.28) 5.2 2.86(1.55-5.27) 0.06 35-44 11.5 3.53(1.70-7.31) 9.0 4.94(2.71-9.01) <0.001 45-54 15.8 4.85(2.35-10.01) 14.3 7.80(4.48-13.58) 0.62 55-64 23.8 7.34(3.63-14.82) 18.1 9.89(5.77-16.94) 0.14 ≥65 22.8 7.00(3.48-14.11) 23.1 12.64(7.39-21.64) 0.94 Skin color 0.003* Non white 6.9 1.00 White 10.7 1.56(1.16-2.09) Marital status 0.001* 0.87* Without partner 7.6 1.00 10.1 1 <0.001 With partner 12.0 1.57(1.03-2.38) 9.7 0.96(0.76-1.22) 0.15 Separated 18.9 2.47(1.36-4.50) 11.4 1.14(0.74-1.74) 0.07 Widowed 20.0 2.62(1.54-4.45) 10.0 1.00(0.65-1.52) 0.02 Education (years of education) NS <0.001 0 11.8 1.00 18.9 1.00 0.09 1-4 15.6 1.33(0.76-2.32) 10.6 0.56(0.38-0.83) 0.03 5-8 10.2 0.87(0.49-1.53) 9.4 0.50(0.36-0.69) <0.001 9-11 11.1 0.94(0.51-1.76) 7.6 0.40(0.28-0.57) <0.001 12 or more 10.0 0.85(0.44-1.66) 11.0 0.58(0.40-0.84) 0.72 Household income (minimum wage in quartiles) NS 0.68 1 10.9 1.00 9.4 1.00 <0.001 2 12.1 1.11(0.71-1.73) 10.4 1.11(0.82-1.49) 0.41 3 9.6 0.88(0.55-1.41) 9.0 0.97(0.71-1.31) 0.001 4 13.9 1.28(0.83-1.96) 10.5 1.12(0.84-1.49) 0.11 Smoking 0.16 Non smoker 9.2 1.00 Smoker 11.2 1.22(0.99-1.50) Former smoker 10.4 1.13(0.89-1.45) Hypertension <0.001 <0.001 No 9.5 1.00 9.0 1.00 <0.001 Yes 21.7 2.63(1.82-3.79) 18.9 2.10(1.74-2.53) 0.34 Medical consultation (last three months) <0.001* <0.001* No 5.4 1.00 4.4 1.00 <0.001 Yes 20.2 4.40(3.00-6.45) 14.9 3.41(2.66-4.36) 0.003 Note: In 2003, the sample size was 3,542 and the maximum number of missing values was 446 in the hypertension variable. In 1994, the sample size was 1,277 and the maximum number of missing values was 63 in the household income variable. OR: Odds ratio NS: Not significant Modifications in psychotropic drug use Rodrigues MAP et al age relying on recall. Nonetheless, the time period of 14 days is short and various studies in this field have used this time period. The 1994 study used this same time period which allows for comparison. DISCUSSION ported by other literature1,6,7 which relates this find- ing in part to the greater use of health services by women than men.8 On the other hand, gender is also associated with a distinct medical perspective of the need to use psychotropics. In their evaluation of pri- mary care medical visits, Moreno Luna et al9 con- cluded that physicians address symptoms of anxiety and depression differently depending on the sex of their patients by prescribing more ansiolitic drugs and diagnosing more functional causes for women. The absence of a difference between the prevalence patterns of psychotropic use in 1994 and 2003 con- trasts with the rest of global literature, which sug- gests an increase in psychotropic use in recent dec- ades.3,13 One possible explanation is that psychotropic use was already high in the city (11.9% in 1994) when compared to findings in national (5.4% in a 1993 study1 in Rio de Janeiro) and international (6.4% in a 2002 multi-center study11 in various European countries) literature. Psychotropic use increased with older age among the age groups analyzed. This is supported by the literature.1,6 In contrast, the reduction observed in use among the age group of 15-24 is not supported by the literature. This finding may support a tendency found in industrialized countries of an increase in psychotropic use among younger people16 that is still not observed in Brazil. On the other hand, the diffi- culties young people face in accessing specialized neurological public medical and mental health serv- ices may partly explain this finding. With regard to the elderly, the large usage psychotropic drugs is cause for alarm due to the increased risk of cognitive degeneration, syncope, breaks and fractures associ- ated with these medicines, which are already more frequent within this group. The percent of self-medication in 2003 was the same as in 1994, which shows that part of those who use psychotropic drugs (8%) continue to use them with- out medical orientation. This finding points to a need for educational campaigns regarding the risks of self- medication. The increase in antidepressant usage in the last decade demonstrates a tendency supported by other research3,12 which has shown an increase in the diag- nosis of depressive diseases accompanied by an in- crease in new medications and an expansion of thera- peutic uses for these medications. Lima M. Morbidade psiquiátrica menor e consumo de psicofármacos em Pelotas, RS [dissertação de mestrado]. Pelotas: Universidade Federal de Pelotas; 1995. 9. Moreno Luna ME, Clemente Lirola E, Pinero Acín MJ, Martinez Matías MR, Alonso Gómez F, Rodríguez Alcalá FJ. Influencia del género del paciente en el manejo de cuadros ansioso/depresivos. Aten Primaria 2000;26(8):554-8. DISCUSSION The greater use of psychotropic drugs observed among whites in 2003 may be related to cultural fac- tors and inequality in the health care system. Olfson12 had already detected lower indices of treatment for depression among blacks and Hispanics as compared to whites during a ten year period in the United States. The decrease in anorexigen use should be observed with caution. In the 1994 study it was investigated in more detail than in 2003, which increased the possi- bility of the respondents reporting its use. Even though psychotropic use was not associated with education in 1994 and 2003 in the adjusted analysis, use was greater among individuals without education in 2003. This finding may be related to the greater frequency of mental disturbances within this group, as observed by Ludermir.5 The increase in the proportion of individuals using another kind of psychotropic medicine may indicate an increase in outpatient treatment for more serious mental disturbances, which takes advantage of the location of these individuals in their households. The important increase in the percentage of indi- viduals who received psychotropic medicines in the SUS pharmacy indicates that there was greater access to psychotropic drugs among individuals with lower wages. This finding suggests an increase in equal access to this medication and also assists in under- standing why there was no association between psy- chotropic use and family income. With regard to the increased use of psychotropic drugs among smokers in the adjusted analysis of the 2003 study, it is noteworthy that an association be- tween mental illness and nicotine dependence has been documented4,11 and considered an outcome of addictive behavior. As such, it is important to con- sider that psychotropic drugs are frequently prescribed in combination with treatment for tobacco depend- ence. The association between the medical diagnosis for hypertension and psychotropic use may indicate, similar to what was observed in a multi-center study,11 that individuals who have a chronic organic disease may have an increased probability of receiving a psy- The risk of use increased among women even though there was no difference in use among females between 1994 and 2003 and a reduction in use among males. Psychotropic use was greatest among women, being twice that of men in 2003. DISCUSSION This finding is sup- Modifications in psychotropic drug use Rodrigues MAP et al findings will contribute to the advancement of mental health policies and pharmaceutical care including efforts related to services offered, per- manent education for doctors and health profes- sionals, and health education for psychotropic users and the general population. chotropic prescription due to the fact that they go to the doctor more frequently. Further applying this perspective, it is possible to understand the strong connection between medi- cal consultations in the last three months and psy- chotropic use in both the 1994 and 2003 studies. This shows the importance of the medical consul- tations, which should be valued by physicians and patients alike to ensure that the prescription of psy- chotropic drugs is the result of an adequate diag- nosis. In conclusion, this study showed a panorama of current psychotropic use in this location, in ad- dition to comparing findings with a previous study conducted in the same city. It is hoped that the The present study only researched the characteris- tics of psychotropic drug users. In the future, studies regarding other factors which influence the use of psychotropic drugs, such as the characteristics of the physicians and their relationship to the health care system, will be necessary in addition to the use of qualitative techniques to more extensively examine psychotropic drug use. 8. Mendoza-Sassi R, Béria JU. Utilización de los servicios de salud: una revisión sistemática sobre los factores relacionados. Cad Saúde Pública 2001;17(4):819-32. REFERENCES 1. Almeida L, Coutinho E, Pepe V. Consumo de psicofár- macos em uma região administrativa do Rio de Janeiro: a Ilha do Governador. Cad Saúde Pública 1994;10(1):5-16. 2. Cherin P, Colvez A, Deville de Periere G, Sereni D. Risk of syncope in the elderly and consumption of drugs: a case-control study. J Clin Epidemiol 1997;50(3):313-20. 10. Neutel CI, Perry S, Maxwell C. Medication use and risk falls. Pharmacoepidemiol Drug Saf 2002;11(2):97-104. 3. Hemels ME, Koren G, Einarson TR. Increased use of antidepressants in Canada, 1981-2000. Ann Pharmacother 2002;36(9):1375-9. 11. Ohayon MM, Lader MH. Use of psychotropic medication in the general population of France, Germany, Italy and the United Kingdom. J Clin Psychiatry 2002;63(9):817-25. 4. Leon J, Becona E, Gurpegui M, Gonzalez-Pinto A, Diaz FJ. The association between high nicotine dependence and severe mental illness may be consistent across countries. J Clin Psychiatry 2002;63(9):812-6. 12. Olfson M, Marcus SC, Druss B, Elinson L, Tanielian T, Pincus HA. National trends in the outpatient treatment of depression. JAMA 2002;287(2):203-9. 13. Pincus HA, Tanielian T, Marcus SC, Olfson M, Zarin DA, Thompson J et al. Prescribing trends in psychotropic medications: primary care, psychiatry, and other medical specialties. JAMA 1998;279(7):526-31. 5. Ludermir AB, Melo Filho DA. Condições de vida e estrutura ocupacional associadas a transtornos mentais comuns. Rev Saúde Pública 2002;36(2):213-21. 6. Magrini N, Vaccheri A, Parma E, D’Alessandro R, Bottoni A, Occhionero M et al. Use of benzodiazepines in the italian general population: prevalence, pattern of use and risk factors for use. Eur J Clin Pharmacol 1996;50(1-2):19-25. 14. Tajima O. Mental health care in Japan: recognition an treatment of depression and anxiety disorders. J Clin Psychiatry 2001;62 Suppl 13:39-44. 15. Ungvari GS, Chung YG, Chee YK, Fung-Shing N, Kwong TW, Chiu HF. The pharmacological treatment of schizophrenia in chinese patients: a comparison of prescription patterns between 1996 and 1999. Br J Clin Pharmacol 2002;54(4):437-44. 7. Mari JJ, Almeida Filho N, Coutinho E, Andreoli SB, Miranda CT, Streiner D. The epidemiology of psychotropic use in the city of São Paulo. Psychol Med 1993;23(2):467-74. 8. Mendoza-Sassi R, Béria JU. Utilización de los servicios de salud: una revisión sistemática sobre los factores relacionados. Cad Saúde Pública 2001;17(4):819-32. 16. Zito JM, Safer DJ, Reis S, Gardner JF, Magder L, Soeken K et al. Psychotropic practice patterns for youth: a 10-year perspective. Arch Pediatr Adolesc Med 2003;157(1):17-25.
https://openalex.org/W217281711	https://www.biodiversitylibrary.org/itempdf/102691	English	null	THE MOTIONS OF A SPAR BUOY IN REGULAR WAVES	null	1,963	public-domain	9,745	Tap @C. / Tapp @C. / pe Ne./4¢ 99 J. N. Newman J. N. Newman Wom mn i AA May 1963 May 1963 Report 1499 Report 1499 TABLE OF CONTENTS co} Mon oie -WioMo MoM clio olkoiloMio Mie Mioliort ofl eli efeiiel le) lolol tele) VonoleMoll cielo olieMie Mice MioMnoMiol las THE FIRST-ORDER FORCES AND EQUATIONS OF MOTION ..... TEE) ASV ESTIN Grape Ri GS Sai sty esearch wey ve ues coh ou tov dain ts me Meetuetey re ieas Vayce) Rem s\| (cs Ce C/A EVN IEMOINS) JMO WIELD (GIRO WAN (GvILMNPDIT IR 5 ooonccboeo0a IDUSGUSSUOIN ZNINID) (GOINGIGUISMOINIS 6 obecseccvconddoco nm bnnobcon%S Le GLSINON EDLC IMD IN\| IE yee cob eee ananasec oe te Bina) bie alono Giorno eo puoletanee ee 6. o%s EMPIPITIN DID 9/6 6 o} fol lel ef Volloilolilciiolelaeienveliie! fellelitel toile! ole ieihiehiel ollie etlolioliewvoMonieli-Melielpellshiem olveloilolieMeine) OS) All ii List OF FIGURES Figure Figure Figure Figure Figure Figure 1 2 3 4 5 6 - The Coordinate Systems eo 8 © © © © © © © 8 8 we ee ee ee ew -— Plot of the Surge Amplitude-Wave Amplitude Ratio for the Circular Cylinder Ce - Plot of the Pitch Amplitude-Wave Slope Ratio for the Circular Cylinder e+ © © © © © © © © 8 8 ee ee te el -— Plot of the Heave Amplitude-Wave Amplitude Ratio forthe, Undamiped) Ginculaaz Gylind en. ss eae aisaeeeee set - Plot of the Heave Amplitude-Wave Amplitude Ratio for the Damped Circular Cylinder with R/H=0.1..... - Plot of the Heave Phase Lag for the Damped Gineuilarn Gyyilktaclor waldo IR//et SO, MGs eb. diced sce0c 0b. 18 18 20 20 21 Figure Figure Figure Figure Figure Figure 1 2 3 4 5 6 - The Coordinate Systems eo 8 © © © © © © © 8 8 we ee ee ee ew -— Plot of the Surge Amplitude-Wave Amplitude Ratio for the Circular Cylinder Ce - Plot of the Pitch Amplitude-Wave Slope Ratio for the Circular Cylinder e+ © © © © © © © © 8 8 ee ee te el -— Plot of the Heave Amplitude-Wave Amplitude Ratio forthe, Undamiped) Ginculaaz Gylind en. ss eae aisaeeeee set - Plot of the Heave Amplitude-Wave Amplitude Ratio for the Damped Circular Cylinder with R/H=0.1..... - Plot of the Heave Phase Lag for the Damped Gineuilarn Gyyilktaclor waldo IR//et SO, MGs eb. diced sce0c 0b. TABLE OF CONTENTS NOTATION Incident wave amplitude Gravitational acceleration Body draft 18 18 20 20 21 Body moment of inertia in pitch about the center of gravity = Nil Bessel function of the first kind of order zero Wave number, w"/ 2g Radius of gyration, «2 = I/m y Body mass Unit normal vector into the body 0 -H (z = ac S(z) dz ABSTRACT A linearized theory is developed for the motions of a slender body of revolution, with vertical axis, which is float- ing in the presence of regular waves. Equations of motion are derived which are undamped to first order in the body diameter, but second-order damping forces are derived to provide solutions valid at all frequencies including resonance. Calculations made for a particular circular cylinder show extremely stable motions except for the low frequency range where very sharp maxima occur at resonance. “References are listed on page 27. NOTATION iii Pp Pressure Q,(k) = fe (z - 2G)" S(z) eK dz R(z) Sectional radius of the body S(z) Sectional area of the body r Polar radius, re = x + y“ t Time (35 Wp £4) Cartesian coordinate system ZG Coordinate of the center of gravity iC Heave displacement ¢* Free surface elevation 8 Polar coordinate E Surge displacement fe) Fluid density o) Velocity potential xX Vertical prismatic coefficient co) Pitch angle w Frequency of oscillations Pressure iv iv INTRODUCTION The motions of a vertical body of revolution, which is floating in the presence of waves, present a problem of interest in several connections. The motions of a spar buoy, of a wave-height pole, and of floating rocket vehicles are important examples of such a problem. The same methods developed for these motions may be applied to find the forces acting on offshore radar and oil-drilling structures. A theoretical discussion of this problem, which also treats the sta- tistical problem of motions in irregular waves, has been presented by Barakat.’ However, this analysis is restricted to the case of a circular cylinder and is based upon several semi-empirical concepts of applied ship-motion theory. An alternative procedure is toformulate the (inviscid) hydrodynamic problem as a boundary-value problem for the velocity po- tential and to employ slender-body techniques to solve this problem. The latter approach is followed in the present work, leading to linearized equa- tions of motion which may be solved for an arbitrary slender body with a vertical axis of rotational symmetry. The particular case ofa circular cylinder, whose centers of buoyancy and gravity coincide, is treated in detail and curves are presented for the amplitudes of surge, heave, and pitch oscillations. pitch oscillations. In deriving the hydrodynamic forces and moments acting on the body, we shall assume that the incident waves and the oscillations of the body are small, and thus we shall retain only terms of first order in these ampli- tudes. We shall also assume that the body is slender. The analysis with only first-order terms in the body's diameter leads to undamped resonance oscillations of infinite amplitude. To analyze the motions near resonance, it is necessary to introduce damping forces which are of second order with respect to the diameter-length ratio. THE FIRST-ORDER VELOCITY POTENTIAL We shall consider the hydrodynamic problem of a floating slender body of revolution with a vertical axis in the presence of small incident surface waves. Let (x,y,z) be a fixed Cartesian coordinate system with the z-axis positive upwards and the plane z=0 situated at the undisturbed level of the free surface. The x-axis is taken to be the direction of propa- gation of the incident wave system, and the motion of the body is assumed to be confined to the plane y =0. We shali also employ a coordinate sys- tem (x',y',z') fixed in the body, with z' the axis of the body, so that with the body at rest, (x,y,z) = (x', y',z'); and a circular cylindrical system (r,9,z), where x =rcos 0 and y=r sin®@. If €, €, and w are the instan- taneous amplitudes of surge, heave, and pitch, respectively, relative to the body's center of gravity, it follows that se (2 op oe) COS Wh ar (zB! — ZG) sin \| y=y' [1] N iT Ce ater sin J + (z' — z7,) cos Wt 2, [1] N iT Ce ater sin J + (z' — z7,) cos Wt 2, where ZG is the vertical coordinate of the center of gravity in the body- fixed system; see Figure 1. The displacements €, €, and J are assumed to be small oscillatory functions of time; we shall consistently linearize by neglecting terms of second order in these functions or their products with the incident wave amplitude A. Thus Equation [1] may be replaced by Figure 1 - The Coordinate Systems Figure 1 - The Coordinate Systems x= SB was) ae (A = ae) y=y' [2] m= (6 Sad cp aY x= SB was) ae (A = ae) [2] m= (6 Sad cp aY If an ideal incompressible fluid is assumed, there exists a velocity potential, ®(x,y,z,t), satisfying Laplace's equation, such that its gradi- ent is equal to the velocity of the fluid. This function must satisfy the following boundary conditions: (1) On the body, the normal velocity component of the body must equal the normal derivative of ®. For a body of revolution defined by the equa- tien r'=—=R(z'),, where r'= Nx'2 4 y'2, this boundary condition may be expressed by the equation’ [rot S IRB") = e + VO. Vv) [r' — R(z')] = 0 Gin ? S IR( yg") [3] Gin ? THE FIRST-ORDER VELOCITY POTENTIAL S IR( yg") (2) On the free surface, the normal velocity component of the free surface must equal the normal velocity component of the fluid particles in this surface, and the pressure must equal atmospheric pressure. In the linearized theory, these conditions reduc e” to 2 ® co) ) i 0 — — = = O, 4 aD 8 Se 0 on z [4] 4 [4] or in the case of a sinusoidal disturbance with frequency w, or in the case of a sinusoidal disturbance with frequency w, Ran O83 @ on z= 0, [5] Oz Ran O83 @ on z= 0, Oz Ran O83 @ on z= 0, [5] Oz [5] where K = w/e. where K = w/e. (3) At infinite distance from the body, the waves generated by the body are outgoing (the radiation condition). The free surface condition, Equation [5], and the radiation condition are satisfied by the potential of oscillating singularities beneath the free surface; the boundary condition on the body may be satisfied by a proper distribution of these singularities. This distribution may be found from slender-body theory but some care is required in linearizing the present problem. If r'= R(z') is the equation of the body surface over its sub- merged length (-H <,z'< 0), we shall assume that R and its first deriva- tive are continuous, that R(-H) = 0, and that the magnitude of the slope lar /az'\|<< 1. The depth H i Ae sumed finite, and it follows that R is small of the same order, as dR/dz'. In the analysis to follow we shall also require that R be small compared to the wavelength of the incident wave system, or that KR << l. We wish to obtain the velocity potential of leading order in the small parameters of slenderness and oscillation amplitudes in order to obtain a consistent set of linearized equations of motion for the body. However, it will turn out that the potentials of different phases of the motion are of different orders of magnitude with respect to the slenderness parameter. For example, the potential due to surge or pitch is of order R as R— 0, whereas the potential due to heave is 0(R¢). Similar differences will oc- cur in considering the components of each potential which are in phase and out of phase with the respective velocities of the body. In order to circumvent these difficulties without unnecessary higher order perturba- tion analysis, we decompose the velocity potential in the following form: Os y725 6) = o- (x,y,z; t) + by (x,y, 25 t) + by, (% yt) [6] +Al[g/w eKZ cos (Kx — wt) + d, (x, y, Zt)! Os y725 6) = o- (x,y,z; t) + by (x,y, 25 t) + by, (% yt) [6] +Al[g/w eKZ cos (Kx — wt) + d, (x, y, Zt)! Os y725 6) = o- (x,y,z; t) + by (x,y, 25 t) + by, (% yt) [6] where be ; by , and $y, are linear in the displacements (€,€,) and their time derivatives, respectively. where K = w/e. The potential A g/w eKZ cos (Kx - wt) represents the incident wave system and the potential Ad, (x,y,z; t) re- presents the diffracted wave potential, corresponding to waves incident on a restrained body. Each potential » in Equation [6] must satisfy the free surface boundary condition and the radiation condition; the complete potential © must satisfy the boundary condition on the body. This condi- tion, Equation [3], is reduced as follows: _ te) Ost \| Ore Oy! a OTE OZ LEG RanOZE ) ££ 9Oe Pio Nia) se as (se mt (a dx' dt dy' dt az' ot dz’ ot OR) BS) de sae, _ te) Ost \| Ore Oy! a OTE OZ LEG RanOZE ) ££ 9Oe Pio Nia) se as (se mt (a dx' dt dy' dt az' ot dz’ ot OR) BS) de sae, or' oz' dz? OR) BS) de sae, or' oz' dz? or neglecting second-order terms in A, —€,{€, and y, or neglecting second-order terms in A, —€,{€, and y, — -— —-[&+(z-2zG)b] cos 6 +(6- xb) dR/dz = 0 Gr, 72 & IBY (52), — -— —-[&+(z-2zG)b] cos 6 +(6- xb) dR/dz = 0 [7] Gr, 72 & IBY (52), where a dot denotes differentiation with respect to time. Substituting Equation [6] into Equation [7] and separating terms according to their dependence on different displacements, we obtain the following boundary conditions on the body: do : ob 2 = Ecos + 0(R =) or Zz [8] do i fea) — = Uz - 2q)cos0 + 0(R =“) + 0(R2) [9] dp toR do 2 = -= + 0(r—) [10] or Oz Oz do i fea) — = Uz - 2q)cos0 + 0(R =“) + 0(R2) [9] dp toR do 2 = -= + 0(r—) or Oz Oz [10] Oba =- w eKzZ [cose sin wt - (KR cos’ 6+ <) cos at] or dz o(R a 0(R) Oba =- w eKzZ [cose sin wt - (KR cos’ 6+ <) cos at] or dz o(R a 0(R) [11] [11] = wekz [ cos @ sin wt +(3 KR + = +3 + = KR cos 26) cos ut] CLON 3 5. 0(R + 0(R2) Oz CLON 3 5. 0(R + 0(R2) Oz To satisfy the above boundary conditions, we employ slender-body theory.° For example, the potential satisfying Equation [8] is an axial line of horizontal dipoles, of moment density 3& [R(z)]@ per unit length. where K = w/e. Thus in an infinite fluid, ') Ve ) 20 oe FE [ [R(z})] ips [ie4 + (z - 2i)° i, ZiT [12] [12] To satisfy the free ake and radiation conditions, we substitute for the source potential [r2 ar (4 > Fay 217 2 , the potential of an oscillating source under a free surface.” With this substitution we obtain, in place of Equation [12]: i ne 7 2 9 Deine Op S Be ie [R(z)] 2 {22+ -2) ee i ne 7 2 9 Deine Op S Be ie [R(z)] 2 {22+ -2) ee (oo) A f Isso Je) Js(0497 491) Jp (kr) ak} dz hy) pets 0 + roke\| Eee cee aye : ~ [Jy(Kr)] (oo) A f Isso Je) Js(0497 491) Jp (kr) ak} dz [13] hy) pets [13] 0 + roke\| Eee cee aye : ~ [Jy(Kr)] dz, and, ina similar fashion, : 2 y) 2 Daz { 2 dy=2¥ ii [R(z,)] (4 - eq) {Iz ig ((4 =F) ] kt+tK ok + + f 28 —= (aay) y o (kr) ak } dz, [14 0 + roky [ [Rein (z= ze) ele tay) 2 J o(Kr)] dz, aH 1 : 2 y) 2 Daz { 2 dy=2¥ ii [R(z,)] (4 - eq) {Iz ig ((4 =F) ] kt+tK ok + + f 28 —= (aay) y o (kr) ak } dz, [1 0 + roky [ [Rein (z= ze) ele tay) 2 J o(Kr)] dz, aH kt+tK ok + + f 28 —= (aay) y o (kr) ak } dz, 0 kt+tK ok + + f 28 —= (aay) y o (kr) ak } dz, [14] 0 + roky [ [Rein (z= ze) ele tay) 2 J o(Kr)] dz, aH Be 1 $= 2t if R (2) [p24 (z-2)°] * ee) k+K k(z+z,) i i ze 1 Jp (xr) ak dz, [15] 0 + roKt 2) SES oS 9 Sul Wf (eae) ae ns 0 1 -H Zi 0 da=-te [ eal le sad Beco! -H dz 8 92 278 + R@ sinwt — + 2 TRS coswt —— [r* + (2-2) ee o [16] k+K k(zt A . where K = w/e. k+K .k(z+ 2) ster) a} dz, k-K 0) = 7TwK \| pee tall( KR +22 )R sin wt Zi 2 - R* cosut Saag KR* a te Jo (Kr) dz, Ox 9x2 [14] 0 + roky [ [Rein (z= ze) ele tay) 2 J o(Kr)] dz, aH Be 1 $= 2t if R (2) [p24 (z-2)°] * ee) k+K k(z+z,) i i ze 1 Jp (xr) ak dz, [15] 0 + roKt 2) SES oS 9 Sul Wf (eae) ae ns 0 1 -H Zi [15] 0 da=-te [ eal le sad Beco! -H dz 8 92 da=-te [ eal le sad Beco! -H dz 8 92 278 + R@ sinwt — + 2 TRS coswt —— [r* + (2-2) ee o [16] k+K k(zt A . k+K .k(z+ 2) ster) a} dz, k-K 0) = 7TwK \| pee tall( KR +22 )R sin wt Zi 2 - R* cosut Saag KR* a te Jo (Kr) dz, Ox 9x2 [16] where f denotes the Cauchy principal value. From the Appendix we see that the potentials [13] to [16] satisfy the boundary conditions [8] to [11], respectively, with a maximum fractional error of order R. Unfortunate- ly, this error is not so small as in the classical slender-body theory for an infinite fluid, where the error is of order R2 log R; for this reason the present theory may not hold for as wide a range of slenderness as in t the aerodynamic case. However, for the slender floating bodies which are envisaged at present (viz., a rocket vehicle or one support of a stable platform), this is not expected to cause practical problems. The values of the potentials [13] to [16] on the body may be found by setting r = R(z) and retaining the leading terms for small R. To lead- ing order, only the singular term moe (za 2,)2]72 contributes to the in- tegrals over z), and the integrals may be evaluated directly since for any continuous bounded function f(z,) and small values of r, 0 1 \| MeV 4 (esas) 2 dz, = —2f(z)logr + 0(1) Br \| MeV 4 (esas) 2 dz, = —2f(z)logr + 0(1) Br 0) al { fla) = [e? where K = w/e. 2 (2 oa © Gia = 2 1) cose + 0(1) Ox -H 0 2 Zul \| g [s2 4 (esate) ° dz = p 22) cos 26 + 0(1) 2 1 \| 2 -H Ox ree tO Clal<w< 0, wiK< Jal. 0) al { fla) = [e? 2 (2 oa © Gia = 2 1) cose + 0(1) Ox -H 0 2 Zul \| g [s2 4 (esate) ° dz = p 22) cos 26 + 0(1) 2 1 \| 2 -H Ox ree 0) al { fla) = [e? 2 (2 oa © Gia = 2 1) cose + 0(1) Ox -H 0) al { fla) = [e? 2 (2 oa © Gia = 2 1) cose + 0(1) Ox -H 0 2 Zul \| g [s2 4 (esate) ° dz = p 22) cos 26 + 0(1) 2 1 \| 2 -H Ox ree 0 2 Zul \| g [s2 4 (esate) ° dz = p 22) cos 26 + 0(1) 2 1 \| 2 -H Ox ree tO Clal<w< 0, wiK< Jal. where K = w/e. Thus on the body, , = E R(z) cos + 0(R2) [17] by = - \| R(z)(z - zG) cos + 0(R) [18] 6, = ~6 RF log R + 0(R?) [19] A (hii 4 SS )R log R cos wt + Rcos® sin wt] + 0(R2) [20] e&Z R cos@ sin wt + 0(Ré log R) , = E R(z) cos + 0(R2) [17] by = - \| R(z)(z - zG) cos + 0(R) [18] 6, = ~6 RF log R + 0(R?) [19] , = E R(z) cos + 0(R2) , = E R(z) cos + 0(R2) , = E R(z) cos + 0(R2) [17] by = - \| R(z)(z - zG) cos + 0(R) [18] 6, = ~6 RF log R + 0(R?) [19] by = - \| R(z)(z - zG) cos + 0(R) [18] 6, = ~6 RF log R + 0(R?) [19] 6, = ~6 RF log R + 0(R?) [19] A (hii 4 SS )R log R cos wt + Rcos® sin wt] + 0(R2) [20] e&Z R cos@ sin wt + 0(Ré log R) A (hii 4 SS )R log R cos wt + Rcos® sin wt] + 0(R2) [20] [20] [20] e&Z R cos@ sin wt + 0(Ré log R) e&Z R cos@ sin wt + 0(Ré log R) THE FIRST-ORDER FORCES AND EQUATIONS OF MOTION From Bernoulli's equation, the linearized pressure on the body is Ra oo Sa on oes 8b = - pez - pe pp pe - pal TE + ge? sin(KR cos 0 - at) = pgz + pER(z)cos® + eWR(z)(z - Zc) cos 0 = pAweX2R cos® cos wt From Bernoulli's equation, the linearized pressure on the body is on oes 8b = - pez - pe pp pe - pal TE + ge? sin(KR cos 0 - at) = pgz + pER(z)cos® + eWR(z)(z - Zc) cos 0 = pAweX2R cos® cos wt on oes 8b pe pp pe - pal TE + ge? sin(KR cos 0 - at) = - pez - pe pp pe - pal TE + ge? where K = w/e. sin(KR cos 0 - at) = pgz + pER(z)cos® + eWR(z)(z - Zc) cos 0 = pAweX2R cos® cos wt = pgz + pER(z)cos® + eWR(z)(z - Zc) cos 0 = pAweX2R cos® cos wt te pgAeKz sin wt - ogAKe2R cos® cos wt + O(R2 log R) te pgAeKz sin wt - ogAKe2R cos® cos wt + O(R2 log R) = pgz + p€R(z)cos0 + oR (z)(z = #@) COS 8) wv pgAeX2 sin wt = pgz + p€R(z)cos0 + oR (z)(z = #@) COS 8) wv pgAeX2 sin wt - 2pwAe®4R cos® cos wt + 0(R% log R) [20] - 2pwAe®4R cos® cos wt + 0(R% log R) [20] [20] The force and moment exerted on the body by the fluid are obtained by integrating the pressure over the surface. In the absence of any other external forces, the force or moment must equal the respective accelera- tion times the mass or moment of inertia of the body. Thus, with % the unit normal vector into the body, the equations of motion are mé = ff pcos(n,x)dS [22] m(t + g) = ff p cos(n, z)ds [23] = ff pl(z2- ZG)cos(n,x) - x cos(n,z)] dS [24] mé = ff pcos(n,x)dS [22] m(t + g) = ff p cos(n, z)ds [23] v = ff pl(z2- ZG)cos(n,x) - x cos(n,z)] dS [24] mé = ff pcos(n,x)dS mé = ff pcos(n,x)dS Iv = ff pl(z2- ZG)cos(n,x) - x cos(n,z)] dS [24] Iv = ff pl(z2- ZG)cos(n,x) - x cos(n,z)] dS [24] where m is the body's mass, I its moment of inertia about the center of gravity, and the surface integrals are over the submerged surface of the body. In computing the pressure integrals over the body surface, it is ex- pedient to employ the (x', y',z') system, fixed in the body. where K = w/e. The direction cosines are cos(n,x') = — cos 6 + 0(R2) dR 2 i = = a) R cos(n, z') ag (R“) dR 2 i = = a) R cos(n, z') ag (R“) and the forces along the (x,z) axis are related to the forces along the (x',z') axis by and the forces along the (x,z) axis are related to the forces along the (x',z') axis by E 2 Fy cos b+ FPF, sin=F + F_, + 0(u%) F, =F, cos $- Fy: sin= Fi: -h Fy: + 0(u7) Thus the equations of motion may be written in the form a 27 ce C+x'w dR m€ = - cos 0+ J —]} pRdz'dé' Id ( =) 0 -H i" Big Ge ge ap real (Gar ()) = J (SS + ¥ cos 0] pRdz'de' 0 4-H fl a 27 ce C+x'w dR m€ = - cos 0+ J —]} pRdz'dé' Id ( =) 0 -H i" Big Ge ge ap real (Gar ()) = J (SS + ¥ cos 0] pRdz'de' 0 4-H fl : 2n pth Ct+x' i = J \| [(2'- 2G) cos(n,x') - x! cos(n,z')] pRdz' do! 0 -H : 2n pth Ct+x' i = J \| [(2'- 2G) cos(n,x') - x! cos(n,z')] pRdz' do! 0 -H 2m pCF-C+x' is if \| (z'- z¢)cos 8 pRdz'do' + 0(R>) 0 0 where (* is the free surface elevation at the body. Substituting Equation [21] for the pressure and neglecting second-order terms in the oscilla- tory displacements €,€,, and A, we obtain : Zant 9 dR mé -- vg [ J (- cos a+ y SS) (z'+ C - WR cos 6')Rdz' do! 0 bar dz 21 0) co - =p \|\| J cos 0'[€R cos 0' + JR(z-— ZG) cos 0! 0) -H : Zant 9 dR mé -- vg [ J (- cos a+ y SS) (z'+ C - WR cos 6')Rdz' do! 0 bar dz 21 0) co - =p \|\| J cos 0'[€R cos 0' + JR(z-— ZG) cos 0! 0) -H 21 0) co - =p \|\| J cos 0'[€R cos 0' + JR(z-— ZG) cos 0! 0) -H 1 + gAe®” sin wt — 2w“Ae’” R cos 0' cos wt] Rdz' do! 1 + gAe®” sin wt — 2w“Ae’” R cos 0' cos wt] Rdz' do! where K = w/e. 10 0 = -— Tog f (yR+2y2 oe) R ae -H 0 oy oo Kz' > (Our i] le + w(zt— ZG) DY OCI cos wt] R¢ dz' -H 0 = -— Tog f (yR+2y2 oe) R ae -H 0 oy oo Kz' > (Our i] le + w(zt— ZG) DY OCI cos wt] R¢ dz' -H or, since 0 { (yR + 2y2" oe) R az! = [i 1 (R22!) dz! = 0 -H H dz it follows that 0 ee ee 2 K 3 i [é + f(z - 26) - 20 Ae” cos wt] S(z) dz + 0(R~ log R) [25] [25] [25] where S(z) = 7 [R(z)]* is the sectional area function. is the sectional area function. is the sectional area function. In a similar manner we obtain In a similar manner we obtain : 0 e ds m(¢+g) =-pgOS(0)+ pg \| ' S(z)dz + pgA sin wt f eKkz ©? az -H -H dz 4 + O(R~ log R) [26] 4 + O(R~ log R) 4 + O(R~ log R) 4 + O(R~ log R) [26] [26] Ip =-pgy (z - 2) S(z)dz IL, eee Ip =-pgy (z - 2) S(z)dz IL, eee 0 - J [E +h (2 - 2¢)- 2u%Ae KZ cos wt] (z- Zc) S(z)dz + 0(R° log R) + 0(R° log R) [27] [27] 11 From Archimedes! principle, or equivalently, satisfying Equation [26] to zero order in ©, 0) gaa \| Oe i] S(z)dz -H and thus [28] 0) gaa \| Oe i] -H [28] and thus and thus - 0 mC =-pgtS(0)+ pgA sin wot \| -H eKz = dz + 0(R* log R) Z [29] while, from Equations [28] and [25], 0 Dre 2S p I [G(z - 26) - 2u2Ae® cos ut] $(z)dz + 0(R? log R) [30] while, from Equations [28] and [25], 0 Dre 2S p I [G(z - 26) - 2u2Ae® cos ut] $(z)dz + 0(R? log R) [30] while, from Equations [28] and [25], while, from Equations [28] and [25], 0 Dre 2S p I [G(z - 26) - 2u2Ae® cos ut] $(z)dz + 0(R? log R) [30] 0 Dre 2S p I [G(z - 26) - 2u2Ae® cos ut] $(z)dz + 0(R? where K = w/e. log R) [30] Let us denote: Let us denote: Tete mké > Vertical Prismatic Coefficient pHS(0) p v n 12 = — J (2-2) S(z)dz (n = 1,2) n m G -H v n Q,(K)= £ [ cK@(z-2g)"S(z)dz (m= 0,1) n ma J) ns and note that €, ©, and \ must be sinusoidal with frequency w. equations of motion then become The fats, op 129 Wh 2AQ) cos wt (1 -XKH)¢ = A(l- XKHQ)) sin wt 200] (P2+ky-=)4+ ES = = GINO} cos wt [31] [32] [33] fats, op 129 Wh 2AQ) cos wt fats, op 129 Wh 2AQ) cos wt (1 -XKH)¢ = A(l- XKHQ)) sin wt 200] (P2+ky-=)4+ ES = = GINO} cos wt [33] 12 Note also that surge and pitch are coupled, unless P, = 0 or unless the centers of gravity and buoyancy coincide. The above equations of motion are not unexpected. The restoring forces on the left-hand side consist of hydrostatic and inertial forces plus entrained mass terms which double the inertial force at each section. This might have been deduced as a consequence of slender-body theory and the fact that the entrained mass of a circular cylinder in an infinite fluid is just equal to the displaced mass. In other words, the hydrodynam - ic forces on the left-hand side of Equations [31] to [33] could have been obtained by neglecting the presence of the free surface. Moreover, the exciting forces on the right-hand side of these equations are those which follow from the "Froude-Krylov" hypothesis that the pressure in the wave system is not affected by the presence of the body. These results are, of course, a consequence of the fact that the body is slender. where K = w/e. The solutions of Equations [31] to [33] are 1 - XQ) KH = A sinwt \| ————_ 34 : ( 1 -XKH ee 34 ee 2 P,Q, -Q,(P5+k = 12%) 5S) b= 2Acos otf E22 yt \| [35] 2(P, + LS - P,/K) - PY P,Q, -Q,(P5+k = 12%) 5S) b= 2Acos otf E22 yt \| [35] 2(P, + LS - P,/K) - PY P{Qy9- 2Q) wb = 2A cos wt as a ween ee [36] 2(P, + IS - P,/K) - PY P{Qy9- 2Q) wb = 2A cos wt as a ween ee [36] 2(P, + IS - P,/K) - PY Ke [37] XH [37] there is resonance in heave, and when Pe Is) there is resonance in pitch and surge. To determine the oscillation am- plitudes in the vicinity of these resonance frequencies, it is necessary to consider the damping mechanism due to energy dissipation in outgoing waves. Thus, for these frequencies, we must consider the free-surface effects on the restoring forces. For this purpose we must retain some terms which are of second order in the radius of the body. THE DAMPING FORCES The damping forces will follow by considering the last terms in Equations [13] to [16] and will consequently be of higher order in R than those terms which we retained in the previous analysis. This procedure is nevertheless consistent, since at resonance the lower order restoring forces vanish. In other words, we are retaining the lowest order force or moment of each phase separately. Fora further discussion of this point, see Reference 4. We proceed, therefore, to study the damping forces, or the forces in phase with each velocity. The only contribution from Equations [13] to [16] is the potential K v dR 2, @ Kz ec! Zz o* = twKe [ye (eG t [E + (z) - 2G)]R me 1 Jo (Kr) dz, [39] K v dR 2, @ Kz ec! Zz o* = twKe [ye (eG t [E + (z) - 2G)]R me 1 Jo (Kr) dz, [39] [39] Since Jo (Kr) =l- i (Kr)@ + ..., it follows that on the surface r = R(z), the leading terms are l- i (Kr)@ + ..., it follows that on the surface r = R(z), s are Since Jo (Kr) =l- i (Kr)@ + ..., it follows that on the surface r = R(z), the leading terms are II Jo (Kr) 0 1 al Jp (Kr) = 24 Kea = -1K2R cos 0 Ox to second order in R the damping potential on the body is Thus, to second order in R the damping potential on the body is 14 i 3 lé+y (z) = Zc)] K* R(z)R(z)) cos o} eh41 dzy i 3 lé+y (z) = Zc)] K* R(z)R(z)) cos o} eh41 dzy 0 =1yKeK2zz J oKZ1 ds_ dz [40] -H dz, : 0 =1yKeK2zz J oKZ1 ds_ dz -H dz, : [40] 3 K : K - + wK’R(z) cos 0e"™” J [€ + b(z, - zc)Je al S(z}) dz) -H The damping pressure on the body is * : 0 pe Sop 20% = -4 wp Kel? THE DAMPING FORCES ¢ \| eee ee dz, ot _H dz, [41] [41] 0 + + op K?R(z) cos 0 eX [6 + U(z, - Zc)] eal S(z)) dz) 0 + + op K?R(z) cos 0 eX [6 + U(z, - Zc)] eal S(z)) dz) Then the heave damping force is Then the heave damping force is 0 27 0 2 Pein = J J Drak Rvdeldz = — S wp Kt eKz 9S a, [42] Z -H (0) dz BE dz [42] Similarly, the surge damping force and the pitch damping moment are 0 27 Fe oul J p* cos @RdO dz -H Jo [43] 0 OR i: Kz, 2 - top> f s(z) eK az) \| [é + p(Z)- zc)le “1 8(2,)42] -H -H and 0 27 Fe oul J p* cos @RdO dz -H Jo 0 27 Fe oul J p* cos @RdO dz -H Jo [43] 0 OR i: Kz, 2 - top> f s(z) eK az) \| [é + p(Z)- zc)le “1 8(2,)42] -H -H 0 OR i: Kz, f s(z) eK az) \| [é + p(Z)- zc)le “1 8(2,)42] -H -H 0 OR 2 - top> f s(z) eK az) \| [é -H -H and I) 0 27 M* =! p(z - 2G) cos 6 R dé dz -H “0 (z- 2q) S(2) eK? az ) [ 44] 0 27 M =! p(z - 2G) cos 6 R dé dz -H “0 0 27 M =! p(z - 2G) cos 6 R dé dz -H “0 (z- 2q) S(2) eK? az ) [ 44] (z- 2q) S(2) eK? az ) [ [ 44] or in terms of the integrals Pj; Po, Qo: and Q): or in terms of the integrals Pj; Po, Qo: and Q): or in terms of the integrals Pj; Po, Qo: and Q): erms of the integrals Pj; Po, Qo: and Q): Ee = -i wp Kt[K = Q(K) - 5(0)] 45 _ i eK 2 "al or in terms of the integrals Pj; Po, Qo: and Q): 45 "al o 1 wm? 3 : ‘ pe Bos K~ Qo(K)[€ Qo(K) + Qy(K)] [ao]) eet Moca ; My = - 3 —— K°Q)(K)[6 Q9(K) + $Q)(K)] [47] o 1 wm? 3 : ‘ pe Bos K~ Qo(K)[€ Qo(K) + Qy(K)] [ao]) o 1 wm? THE DAMPING FORCES 3 : ‘ pe Bos K~ Qo(K)[€ Qo(K) + Qy(K)] [ao]) eet Moca ; My = - 3 —— K°Q)(K)[6 Q9(K) + $Q)(K)] eet Moca ; My = - 3 —— K°Q)(K)[6 Q9(K) + $Q)(K)] [47] eet Moca ; My = - 3 —— K°Q)(K)[6 Q9(K) + $Q)(K)] [47] [47] In place of Equations [31], [32], and [33], we obtain the damped equa- tions of motion (a {2 9p Pi¥ =- 24 Q)(K) cos wt ne \| } [48] +5 op K Q)(K)[€ Qo(K) a pQ,(K)] (a {2 9p Pi¥ =- 24 Q)(K) cos wt ne (a {2 9p Pi¥ =- 24 Q)(K) cos wt ne \| } [48 [48] ne \| } [48] +5 op K Q)(K)[€ Qo(K) a pQ,(K)] (1-xKH)¢ = A sin wot[1 -XKH Q,(K)] m KC 2 [ (1-xKH)¢ = A sin wot[1 -XKH Q,(K)] [ m KC 2 [ 2A Q,(K) cos ot m KC 2 [ U(P - Py/K+ Ke) +P) & = - 2A Q,(K) cos ot U(P - Py/K+ Ke) +P) & = - 2A Q,(K) cos ot [50] pspiadh K? Q,(K) [€ Qo(K) + JQ,(K)] U(P - Py/K+ Ke) +P) & = - 2A Q,(K) cos ot U(P - Py/K+ Ke) +P) & = - 2A Q,(K) cos ot Ke) +P) & = - 2A Q,(K) cos ot [50] [50] pspiadh K? Q,(K) [€ Qo(K) + JQ,(K)] [50] pspiadh K? Q,(K) [€ Qo(K) + JQ,(K)] The damping terms of these equations of motion are given by the terms linear in the velocities é, t , and wb. It should be noted that for a slender body m— 0, and thus the damping coefficients will be small, as was to be expected. To solve these equations for the three unknown displacements and their phases is a straightforward but tedious matter. For applications in ranges not including a resonance frequency, it is much simpler to em- ploy the undamped equations of motion, [31] to [33], and the resulting displacements, [34] to [36]. CALCULATIONS FOR THE CIRCULAR CYLINDER Then 2 Sa, lie sellin 2 & 12 0; P5 Game QS) ae ; 17 cn Figure 2 - Plot of the Surge Amplitude-Wave Amplitude Ratio for the Circular Cylinder agg eS ie ea eas\| Ea ne Bs LI Be Hl Ei ics ie 3 4 5 La] Figure 3 - Plot of the Pitch Amplitude-Wave Slope Ratio for the Circular Cylinder cn Figure 2 - Plot of the Surge Amplitude-Wave Amplitude Ratio for the Circular Cylinder Figure 2 - Plot of the Surge Amplitude-Wave Amplitude Ratio for the Circular Cylinder agg eS ie ea eas\| Ea ne Bs LI Be Hl Ei ics ie 3 4 5 La] Figure 3 - Plot of the Pitch Amplitude-Wave Slope Ratio for the Circular Cylinder agg eS ie ea eas\| Ea ne Bs LI Be Hl Ei ics ie 3 4 5 La] Figure 3 - Plot of the Pitch Amplitude-Wave Slope Ratio for the Circular Cylinder Figure 3 - Plot of the Pitch Amplitude-Wave Slope Ratio for the Circular Cylinder and it follows that E = - 532 (1 - e KH) cos ut [22] ae vote 5 ; [a SER) Shr bs (1 - KH)@ +[4 KH le) ae H [53] [53] Plots of the above amplitudes and the heave phase angle are shown in Figures 2 to 6 as functions of KH. Figure 2 shows the ratio of surge amplitude to wave amplitude. For zero frequency this ratio is one and for increasing frequencies it decreases monotonically to zero. Figure 3 shows the ratio of pitch angle to the maximum wave slope KA, multiplied by the coefficient C = 4 + 6 (ky /H). This coefficient is equal to one if the mass in the cylinder is uniformly distributed throughout its submerged length. The ratio starts at one for zero frequency and decreases mono- tonically to zero. Thus the pitch amplitude is always less than the wave slope. Figure 4 shows the ratio of heave amplitude to wave height for frequencies away from the vicinity of resonance. Near resonance, the amplitude is shown in Figure 5 and the phase angle in Figure 6 for the particular case R/H=.0.1. The ratio of heave amplitude to wave ampli- tude is unity for zero frequency, rises to a maximum of 2 2 £(2) ~ 0.865 (7) ™\R R at the resonance frequency KH = 1, and then decreases monotonically to zero. CALCULATIONS FOR THE CIRCULAR CYLINDER As a special case, we shall consider the circular cylinder R(z)=R = constant. Then ¢ 2 1.0 1 0 Bos \|\| (z-2zG)dz = -FH- Ze -H 1 y 2 2 2 Bp ig lL, (oe dz = 3H + Hzq4+ 26 1 v K 1 KH ee Z Ss eu eae oreo iia a a edz sax Vi e ) 1 Kz 1 -KH 1 -KH Op (SS) Se i o(4 2 aq) G4 B Se Sera iS Yl + Kzc) Ho Jy K K°H ¢ 2 1.0 1 0 Bos \|\| (z-2zG)dz = -FH- Ze -H 1 y 2 2 2 Bp ig lL, (oe dz = 3H + Hzq4+ 26 1 v K 1 KH ee Z Ss eu eae oreo iia a a edz sax Vi e ) 1 Kz 1 -KH 1 -KH Op (SS) Se i o(4 2 aq) G4 B Se Sera iS Yl + Kzc) Ho Jy K K°H 1 0 Bos \|\| (z-2zG)dz = -FH- Ze -H We shall assume, moreover, that the centers of buoyancy and gravity co- incide, or ZG ais H/2, so that the equations of motion are uncoupled and there is no resonance in pitch or surge. CALCULATIONS FOR THE CIRCULAR CYLINDER The phase angle is similar to conventional one-degree-of-freedom LY) 2.8 PEERS 24 O \| : \| me ; \| me \| Fa a \| - Rese \| Ds ee ~ 2 3 4 ls) 6 v KH Figure 4 - Plot of the Heave Amplitude-Wave Amplitude Ratio for the Undamped Circular Cylinder 0.5 0.6 0.7 0.8 0.9 10 11 I. KH Figure 5 - Plot of the Heave Amplitude-Wave Amplitude Ratio for the Damped Circular Cylinder with R/H= 0.1 2.8 PEERS 24 O \| : \| me ; \| me \| Fa a \| - Rese \| Ds ee ~ 2 3 4 ls) 6 v KH Figure 4 - Plot of the Heave Amplitude-Wave Amplitude Ratio for the Undamped Circular Cylinder Figure 4 - Plot of the Heave Amplitude-Wave Amplitude Ratio for the Undamped Circular Cylinder 0.5 0.6 0.7 0.8 0.9 10 11 I. KH Figure 5 - Plot of the Heave Amplitude-Wave Amplitude Ratio for the Damped Circular Cylinder with R/H= 0.1 20 200 ae é IN DEGREES ” i 80 - 40 wll es 0.6 07 0.8 x : : L if ie) Figure 6 - Plot of the Heave Phase Lag for the Damped Circular Cylinder with R/H = 0.1 Figure 6 - Plot of the Heave Phase Lag for the Damped Circular Cylinder with R/H = 0.1 harmonic oscillators with linear damping; for low frequencies the heave displacement and wave height are in phase, at resonance they are in quad- rature, and at high frequencies they are 180 deg out of phase. DISCUSSION AND CONCLUSIONS The damped equations of motion as given by Equations [48] to [50] may be solved for an arbitrary body of revolution to obtain the oscillation amplitudes and phases. Except in the vicinity of the resonance frequencies defined by Equations [37] and [38], it should be sufficient to use the sim- pler undamped equations; the resulting oscillations are given by Equations [34] to [36]. Plots of these oscillations are shown in Figures 2 to 6 for a circular cylinder, with the important restriction that the centers of buoy- ancy and gravity coincide. If this restriction is relaxed, a resonance will be introduced into the equations for pitch and surge, but the frequency of this resonance may be kept small by ballasting. The amplitudes at reso- nance are extreme, but the resonance frequency for heave is quite small and can be kept out of the practical range of ocean waves by making the 21 draft sufficiently large. It would seem wise to do this in practice and to provide appropriate ballast so that the pitch resonance occurs at or below the heave resonance frequency. From Equations [37] and [38] this re- quires that draft sufficiently large. It would seem wise to do this in practice and to provide appropriate ballast so that the pitch resonance occurs at or below the heave resonance frequency. From Equations [37] and [38] this re- quires that The advantage of spar-buoy-type bodies lies in their very small motions in the higher frequency range. By proper design this advantage may be utilized; thus very calm motions can be expected in waves. ACKNOWLEDGMENT The author is grateful to Mrs. Helen W. Henderson for computing the results shown in Figures 2 to 6 and to Dr. W. E. Cummins for his critical review of the manuscript. 22 APPENDIX APPENDIX Here the potentials E> y ' Pi. and da» defined by Equations [13] to [16], are shown to satisfy the boundary conditions [8] to [11], respec- tively, to leading order in R. For this purpose, let us consider the po- tential 0) of(z,,t) - pat J a fr te - 271 -H oe) k+K + + olay ai) Jp (kr) dk } dz, [54] o k-K tle K(z+t 2) 0 + mwK I f(z,,t)e Jo (Kr) dz, -H 0) of(z,,t) - pat J a fr te - 271 -H tle oe) k+K + + olay ai) Jp (kr) dk } dz, [54] o k-K oe) k+K + + olay ai) Jp (kr) dk } dz, o k-K [54] K(z+t 2) 0 + mwK I f(z,,t)e Jo (Kr) dz, -H where f(z),t) has sinusoidal time dependence with circular frequency w. By appropriate choice of the function f, the potentials Pe» by ; yp and oa can all be obtained from and dy/dx. Thus it is sufficient to estab- lish that the following conditions are satisfied on the body surface r = R: © R= Ile f(z, t) [55] a — f(z,t 56 arIoe my ia Sena o! [55] a — f(z,t 56 arIoe my ia Sena o! 56 o! Employing an alternative form of the source potential,” we write in the form 1 ok (44 2)) [57] 23 Jo(Kr)dz, = Shy hea b) = $ ip ~- ir ap ((v4p, S £4) 2 +[r~+ (z, + zy} -H 0 TONS J ea pO) Co ae Jy (Kr) dz, a b) = $ ip ~- ir ap ((v4p, S £4) 2 +[r~+ (z, + zy} } az, -H b) = $ ip ~- ir ap ((v4p, S £4) 2 +[r~+ (z, + zy} } az, -H The potential vy corresponds to an axial distribution of simple sources together with an image distribution above the free surface z= 0. To emphasize this fact we write yy) in the form pl 2 “2 = £(-\|z,\|.t) [> + (z= 2,)7] > okay pl 2 “2 = £(-\|z,\|.t) [> + (z= 2,)7] > okay [58] [58] From the conventional slender-body theory of aerodynamics, we may ex- pect this potential to satisfy the boundary conditions [55] and [56] on the body to leading order in R. [57] continued continued APPENDIX In fact, differentiating with aspect to r and neglecting terms which are of order R or Ros @ inthe neighborhood of the body r = R, we have a or + (z= 2,)°] dz) [59] a or + (z= 2,)°] dz) [59] [59] = es peek Ret SROs ans, Lense RS Sr continued Sh Ciera, aye r go bs (FA 2) -H mS il he a at and similarly 2 Oh mn COED OF [60] Or Ox a Be continued mS il he a at and similarly and similarly 2 Oh mn COED OF [60] Or Ox a Be [60] Thus on the body the potential satisfies the conditions [55] and [56] to leading order in R. To establish that the same is true of J, we now show that the contributions from > and db>/ Ox are of higher order in R. Since fs) Be Jo (kr) = -kJ) (kr) it follows that dY> 0 © Buisie ot Ub eh eel ay (teriidled 2 or Er ot ig) Mc aiK 1 3 [61] + Tw Ke { f (z,,t) or 2 eel TK) dzn We wish to show that Ny) = O(f a (f) and 2 ay t= off Or Ox R 25 fs) Be Jo (kr) = -kJ) (kr) it follows that it follows that dY> 0 © Buisie ot Ub eh eel ay (teriidled 2 or Er ot ig) Mc aiK 1 3 [61] + Tw Ke { f (z,,t) or 2 eel TK) dzn dY> 0 © Buisie ot Ub eh eel ay (teriidled 2 or Er ot ig) Mc aiK 1 3 + Tw Ke { f (z,,t) or 2 eel TK) dzn dY> 0 © Buisie ot Ub eh eel ay (teriidled 2 or Er ot ig) Mc aiK 1 3 [61] 3 + Tw Ke { f (z,,t) or 2 eel TK) dzn We wish to show that Ny) = O(f a (f) and 2 ay t= off Or Ox R 25 as R-— 0, and thus that as R-— 0, and thus that 2 2 OOM Be Oh << or or Or Ox Or Ox for R/H<< 1. From the series expansion of the Bessel function, for R/H<< 1. From the series expansion of the Bessel function, J, (kr) = kr + 0(k?r?) and thus, where this expansion is permissible in Equation [61], the re- sulting terms are clearly of order fK. APPENDIX However, in the neighborhood of z= 0, the power series expansion is not permissible in the integral over k. It follows that, in the neighborhood of r=R, ow af(0,t Qos GS ge CENCE) J f is e871 3, (kr) dkdz, Or at jan d@ ie IX l A WE OOo) f u J, (kr) dk im at ) Rom OE af(0,t) fe J, (kr) Sif K eae SE we ak at 0 k oOo ae 0(£) at Similarly, 2 a” t+ of) Or Ox R Thus, onthe body, A WE OOo) f u J, (kr) dk im at ) Rom OE af(0,t) fe J, (kr) Sif K eae SE we ak at 0 k oOo ae 0(£) at A WE OOo) f u J, (kr) dk im at ) Rom OE af(0,t) fe J, (kr) Sif K eae SE we ak at 0 k Thus, onthe body, Thus, onthe body, 26 26 and a bo / a(n ai) or Ox Or Ox Therefore, the potential \ satisfies the conditions [55] and [56] witha fractional error of order R. 4. Newman, J.N., "A Linearized Theory for the Motions of a Thin Ship in Regular Waves,'' Journal of Ship Research, Vol. 5, No. 1 (1961). a bo / a(n ai) or Ox Or Ox 3. Lighthill, M.J., "Mathematics and Aeronautics,'' Journal of the Royal Aeronautical Society, Vol. 64, No. 595 (Jul 1960), pp. 375-394. REFERENCES 1. Barakat, Richard, ''A Summary of the Theoretical Analysis of a Vertical Cylinder in a Regular and an Irregular Seaway,'' Reference No. 57-41, Woods Hole Oceanographic Institution (Jul 1957), Unpublished 1. Barakat, Richard, ''A Summary of the Theoretical Analysis of a Vertical Cylinder in a Regular and an Irregular Seaway,'' Reference No. 1. Barakat, Richard, ''A Summary of the Theoretical Analysis of a Vertical Cylinder in a Regular and an Irregular Seaway,'' Reference No. 57-41, Woods Hole Oceanographic Institution (Jul 1957), Unpublished Manuscript. Vertical Cylinder in a Regular and an Irregular Seaway,'' Reference No. 57-41, Woods Hole Oceanographic Institution (Jul 1957), Unpublished Manuscript. 57-41, Woods Hole Oceanographic Institution (Jul 1957), Unpublished Manuscript. 2. Wehausen, J.V., "Surface Waves, '' Handbuch der Physik, Springer Verlag, Section 13 (1961). 2. Wehausen, J.V., "Surface Waves, '' Handbuch der Physik, Springer Verlag, Section 13 (1961). 3. Lighthill, M.J., "Mathematics and Aeronautics,'' Journal of the Royal Aeronautical Society, Vol. 64, No. 595 (Jul 1960), pp. 375-394. 3. Lighthill, M.J., "Mathematics and Aeronautics,'' Journal of the Royal Aeronautical Society, Vol. 64, No. 595 (Jul 1960), pp. 375-394. 27 INITIAL DISTRIBUTION Copies Copies 10 10 CHBUSHIPS Tech Lib (Code 210L) Appl Res (Code 340) Des, Shipbldg, & Fleet Maint (Code 400) Prelim Des (Code 420) Sub Br (Code 525) LCDR B.I. Edelson (Code 3614) Oceanography (Code 342C) — i Oo CHBUWEPS 1 Aero & Hydro Br (Code RAAD-3) 1 Capt. Freitag (Code 45) 1 Mr. Murri (Code RTSV-13) 1 Dyn Sec (Code RAAD-222) CNO (Op-76), Attn: LCDR Duncan CHONR 1 Nav Applications (Code 406) 1 Math Br (Code 432) 2 Fluid Dyn (Code 438) ONR, New York ONR, Pasadena ONR, Chicago ONR, Boston ONR, London CDR, USNOL, White Oak DIR, USNRL 1 Mr. Faires (Code 5520) CDR, USNOTS, China Lake CDR, USNOTS, Pasadena CDR, USNAMISTESTCEN Attn: Mr. Eberspacher (Code 5610) CDR, PACMISRAN, Point Mugu, California Attn: Mr. W.L. Mackie, Consultant (Code 4110-1) CDR, DDS, Attn: TIPDR DIR, Natl BuStand Attn: Dr. G.B. REFERENCES Schubauer DIR, APL, JHUniv DIR, Fluid Mech Lab, Columbia Univ, New York Copies 1 DIR, Fluid Mech Lab, Univ of California Berkeley 5 DIR, Davidson Lab, SIT, Hoboken 1 DIR, Exptl Nav Tank, Univ of Michigan Ann Arbor 1 DIR, Inst for Fluid Dyn & Appl Math Univ of Maryland, College Park 1 DIR, Hydraulic Lab, Univ of Colorado Boulder 1 _—DIR, Scripps Inst of Oceanography, Univ of California, La Jolla 1 DIR, ORL Penn State 1 DIR, WHOI 3 OinC, PGSCOL, Webb 1 Prof. Lewis 1 Prof. Ward 2 DIR, lowa Inst of Hydraulic Res, State Univ of lowa, lowa City 1 Dr. Landweber 1 DIR, St. Anthony Falls Hydraulic Lab, Univ of Minnesota, Minneapolis 3 Head, NAME, MIT. 1 Prof. Abkowitz 1 Prof. Kerwin 1 Inst for Math & Mech, New York Univ 2 Dept of Engin, Nav Arch, Univ of California Berkeley 1 Prof. Wehausen 2 Hydronautics, Inc, Pindell School Rd Laurel, Maryland 1 Dr. Willard J. Pierson, Jr., Coll of Engin New York Univ 1 Mr. Robert Taussig, Grad Math Dept Columbia Univ, New York 1 Or. 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Fifth Ave. Columbus, Ohio 30 “9OUBUOSEl] 3B INDIO Bsutxew disys AJ0A oJeym eduBi Aouenbedy Mo] ey} 10j ydeoxe suoljow e]quys ATowesyxe Mos Jopur]AD IepNoITO IBpNoyIed B 103 OpBU SUOT}B[ND[BH ‘eoUBUOSes Jurpnjour sorouenbeay [][¥ 7B PI[BA SUOTIN[OS eprtAoid 0} PeAlJep e18 SedI0J Surdwuep 1epio-puodes qnq ‘1ojewsIp Apog ey} UI JopsO 4S11j 07 poduwpun ose Yyorym PpeAliep 018 UOT}OW Jo SUOTZeNbY ‘seABM Is[Ndel Jo edueseid ey) UT SuUNVOT] ST YOTYM ‘SIX’ [BOIZIOA YYIM ‘TOTNTOAeI Jo Apoq Jepue\|s B FO SUOCT}OW ey} 10) pedojeaep st Al0ey) peziswoUl] SBIOYDIN “f ‘UBUIMEN ‘J Ar00y L.--uotop--([891912 A) UOMNJOAeI JO SeIpog °% SISA\|BUB [BOT]BWOEYISA CaIMISSV'TIONN ‘sjoa ‘snqt “dog ‘at “gg6T ABW “UeUIMEN “NE --wolop--sXonq reds : “T Aq ‘SAVM AV INDY NI AONE UVdS V AO SNOILOW AHL "66PL Hodey . CHBUWEPS 1 Aero & H ‘ulsog jepow 40;40] piaog 90uB uosel q8 Ind90 ewurxew disys AoA oJeym osuszi AOuenbely Moy ey} 10} ydeoxe suonjou e1qu3s AJowe%xe Moys JoputfAd JeNdIId AB[NOTVIVd B 10J eps SUOTZB[NO[BD ‘eoUBUOSeEI SUIpN{oUr SeroueNbeg] []¥ 7B PI]BA SUOTIN[OS EptAoid Oj peAtJep 018 SsedJ0}) Jutduep 1epio-puodes qnq ‘1ejouBIp Apoq ey} UI JopsO 4S11j 07 pedwepun ese YoryM PeAliop o18 UOT}OUI JO sUOT}eNbyY “seABM JB[Ndel Jo edueseid OY) UL SUNBOT] ST YOTYM ‘SIX’ [BOIZIOA YQTM ‘TOTNTOAeI Jo Apoq Jepue\|[s B JO SUOTJOW ey} 10) pedoyeaep st A10ey) peziseeUl] V SBIOYOIN ‘f ‘UBUIMEN ‘] Ax00y I, --wornop--({821910 A) UOTNOAeI JO SeIpog °Z sIsh[eue [BoIBMEYyeW GIAISSVTONN ‘sjes ‘snqt “dog ‘at “eg6T ABW “UBUIMON “N’L --wo1jopj--sXong seds : ‘T Aq ‘SAVM AVINDOAA NI AONE UVdS V AO SNOILOW AIL “66PL HOdey ‘ulsog japow 40)40] praog SBIOYDIN ‘f ‘UBUIMEN ‘[ Ax00y L.--uornop--(1801910 A) UONIJOASI JO SeIpog °G SIsA[BUB [BOIFBUIO NIB --wonop--sfonq reds : “T SB[OYDIN f ‘UBMIMEN ‘\| Ar00y [.--wornop--([801910 A) UOTJNIOAGI jo seIpog °Z SISA[BUB [BOIVBUIE YB --worow--sXonq seds : ‘T BUOSEl suonjoul e]qz3s ATewes3xe MOYys JopurAD IB[NoITO Opeul SUOTZB[ND[BD ‘eoUBUOSeEI SuIpn[our s SUOTJN[OS EptAoid 0} peAtiop e148 se010j yng ‘1ejeulstp Apoq ey UI Jopio ysi1j PoeAlJep e18 UOT}OW Jo suoTywnbyY ‘seA ©Y} UL BuNBOTJ St YOTYA ‘SIXB [BOIZI0A Y Jepye\|s B JO SUOTJOW ey} 10} pedojedep s Aq ‘SHAVM AV TANGA NI AONA UVdS V “66pl woday -ulsog sutxeu disys AJoA o1eym oSuvs Aouenbey SUOT]N[OS eprAoid Oj peAtJop 018 se0J0j GAIMISSVTONNA ‘sjei ‘“‘snqyt “dog ‘at ‘gg6T Aq ‘SHAVM UV INDUY NI AON UVdS V OW SBOYDIN f ‘UBUIMEN ‘] Aso0y L--Wornop--(1891770 A) WONNJOAeI JO SeIpog S stsh[eus [BOIQBMOYIwY --wonow--sfong reds : ‘T SB[OYSIN “f¢ ‘UBMIMeN ‘\| Ar100y, [.--Worop--([891710 A) UOTINIOAGI JO SeIpog Z sisk[wus [BOIBMEYwW --wolop--sXong reds : “T @ouBUOSeI 4B INDDO sutxeu disys Alon esoym oduvi Aouenbaay mo] ey 10) ydeoxe suoKNoU e]qB3s AJouIe+xe MoYys Joput]AD Ie[NOIIO JB[NoI7ZIEd B 1OJ eps SUOIZB[ND[BD ‘edUBUOSe JuIpNjouL selouenbed [[¥ 48 PI]eA SUOI}NJOS Optaoid 07 peAlJop e18 Se010j Julduisp Jop1o-puoves qnq ‘10jewe1p Apog ey UI Jepso 4S811j 07 pedwepun oe YOIyA peatiep es UOTjJOW Jo SUCTwNbY “seABmM IB[Ndel jo edueseid oY} Ul SUNBOT] ST YOY ‘SIxXB [BOTZIEA YT ‘TOINTOAeI Jo Apoq Jepue\|s B JO suoT}OU ey} 10} pedojeaep st A100y3 peztBeUl] V CAIMISSV'TIONN ‘sjea ‘snqtt “dog ‘at “eg6T Av “weuMON “N“P hq ‘SHAVM AVTINDAY NI AONE UVdS V AO SNOLLOW FHL “66pL Hoday “uysog japow 40/40, p1aog @DUBUOSOE 3B INDDO Bsuixew disys Aiea oroym odusBs Aouenbedy Mo] ey} 10} ydeoxe suotoUW 01q83s AToule;yxe MoYs JopurTAD IBpNOITO IJB[NoIyIed B I0y EpBul SUOTIB[ND[BD ‘eouBUOSeI Zurpnjour serouenbed [[B 9B PI[BA SUOTIN[OS eptAoid oj peatiep e18 SsedI0} Juidwep Jopso-puoves qnq ‘1ojowsrp Apog eyj UI Jopi0 4SI1j 07 podurspun ese YoryM PpeAtiep o18 UOT}OU Jo SUOTyBNbY ‘“seABmM JB[Ndel Jo edueseld OY} UL TUNVOT ST YOTYM ‘SIXB [BOTZIOA YIIM ‘UONJOAeI Jo Apoqg JOpue\|s B JO SUOTJOW oY} J0J pedojedep st Alooy) peztieoUul] V GAIMISSVIONN “sjes ‘snqyt “dog ‘at “gg6T ABW “aeulMeN “N’C hq ‘SHAVM AV INDAA NI AON AVdS V AO SNOLLOW GH “66pL Hoday -usog japow 40j40) pang SBIOYOIN “f ‘UBUIMEN ‘] Ayooy, L--wonop--([891)20 A) UONNIOAeI Jo seIpog ° sIsh[ Bus [BOTBUEYIwW --uorop--sXkonq sed : “T SB[OYDIN ‘f ‘UBUIMEN ‘T Arooy J,--uonop--([891910 A) WONNJOAeI Jo seIpog * sIsh[Bus [BOI}BWOYIBA --uonop--sXonq iedg : ‘T “@DUBU suixsw disys AJoA eloym oduss Aouonbedy MO] suooU e]qB4s ATowe+Nxe MOYs Joput[AD JB[NOIIO I OpBul SUOTZB[NO[VD ‘eouBUOSeI ZuIpnjour setouenbed SUOTIN[OS eptAoid 0) peAlJep e18 60010) Jutduwp 4nq ‘1ojewetp Apog ey) Ut Jopso 4sS11j 0} pedww PeAliep e128 UOT}OU Jo SUCTWBNb\|Y “seABM IJB[Nd 04) UL BUIQBOTJ SI YOTYM ‘SIXB [BOTZIOA YWTM ‘MOMN Jopye\|s B JO SUOTJOU! 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https://openalex.org/W4378799423	https://mathjournal.unram.ac.id/index.php/Rengganis/article/download/310/315	Indonesian	null	Pelatihan pembuatan google form sebagai media evaluasi pembelajaran bagi Guru SD Gugus V Lingsar	Rengganis Jurnal Pengabdian Masyarakat	2,023	cc-by-sa	1,526	Abstrak Observasi dan wawancara yang dilakukan pada guru-guru sekolah dasar gugus V Lingsar memperoleh hasil bahwa guru masih melakukan evaluasi secara manual menggunakan kertas. Guru-guru memerlukan media evaluasi yang praktis dan efektif untuk menghemat waktu pengoreksian jawaban. Merefleksi dari kebutuhan tersebut, kegiatan pengabdian dilakukan untuk memberikan pelatihan penggunaan google form sebagai media evaluasi yang praktis dan efektif. Peserta kegiatan terdiri dari 20 orang guru-guru sekolah dasar di gugus V Lingsar. Kegiatan terdiri dari penyampaian materi dan praktek. Seluruh peserta memberikan respon positif terhadap kegiatan yang dilaksakan karena kegiatan tersebut sesuai dengan kebutuhan peserta. Kata Kunci: Google form; evaluasi; pelatihan guru; praktek terbimbing Abstract Ob i Observations and interviews conducted with elementary school teachers in cluster V Lingsar obtained the result that teachers were still evaluating manually using paper. Teachers need practical and effective evaluation media to save time on correcting answers. Reflecting on this need, community service activities are carried out to provide training on using the google form as a practical and effective evaluation medium. The activity participants consisted of 20 elementary school teachers in cluster V Lingsar. Activities consist of delivery of material and practice. All participants gave a positive response to the activities carried out because these activities were in accordance with the needs of the participants. p p Keywords: Google form; evaluation; teacher training; guided practice 1,2,3,4,5 Pendidikan Matematika, FKIP, Universitas Mataram, Mataram intan@unram.ac.id Amrullah1, Nani Kurniati2, Muhammad Turmuzi3, Ni Made Intan Kertiyani4* dan Wahidaturrahmi5 1,2,3,4,5 Pendidikan Matematika, FKIP, Universitas Mataram, Mataram 2. METODE PELAKSANAAN Sasaran kegiatan adalah guru-guru sekolah dasar di Gugus V Lingsar. Kegiatan ini dilakukan secara luring di salah satu sekolah yang tergabung dalam Gugus V Lingsar. Kegiatan ini berupa pelatihan yang terdiri dari sesi pemaparan materi dan praktek terbimbing bersama pemateri. Selama kegiatan, ada dua observer yang mengobservasi kegiatan peserta. Di akhir pelaksanaan, terdapat angket evaluasi yang bertujuan untuk mengetahui pendapat peserta mengenai kegiatan yang berlangsung. 1. PENDAHULUAN Penggunaan kurikulum merdeka pada jenjang sekolah dasar menyebabkan guru harus mampu memanfaatkan teknologi, informasi dan komunikasi (TIK). Dalam kurikulum ini, guru dapat menggunakan TIK dalam hal pembuatan bahan ajar, penggunaan multimedia, evaluasi pembelajaran dan manajemen kelas (Aka, 2017). Dengan memanfaatkan TIK, beberapa tugas guru dapat dijalankan dengan lebih cepat. Berdasarkan observasi dan wawancara yang dilakukan dengan guru-guru sekolah dasar (SD) di gugus V Lingsar, evaluasi pembelajaran masih dilakukan secara manual, yakni menggunakan kertas. Evaluasi manual dilaksanakan untuk ulangan harian, penilaian tengah semester dan penilaian akhir semester. Evaluasi dengan kertas memiliki beberapa kekurangan, yakni adanya biaya cetak dan lamanya durasi untuk mengoreksi Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Mei 2023 Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Mei 2023 \|1 \|103 Pelatihan pembuatan Gooogle Form … Amrullah et al jawaban siswa. Adanya media penilaian yang dapat mempercepat durasi pengoreksian dan mengurangi biaya cetak soal menjadi hal yang diharapkan oleh guru-guru. Google form merupakan salah satu media evaluasi yang efektif dan efisien (Azis dan Shalihah, 2020). Media ini dapat memangkas waktu yang diperlukan guru untuk mengoreksi jawaban siswa (Wulandari, et. al., 2019). Beberapa studi juga menunjukan bahwa siswa merespon positif penggunaan google form sebagai alat evaluasi maupun latihan soal di kelas (Hakim dan Safi’i, 2021). Lebih jauh, siswa sekolah dasar juga merasa teknis menjawab pertanyaan pada google form cukup mudah dipahami dan dilakukan (Namira, 2021). Melihat adanya kebutuhan guru SD di gugus V Lingsar terhadap media evaluasi yang praktis, diperlukan pelatihan untuk dapat membantu guru-guru. Sebelumnya guru-guru di gugus V Lingsar belum pernah mendapatkan pelatihan untuk membuat media evaluasi menggunakan google form. Untuk itu, program pelatihan dilakukan terhadap guru-guru SD di gugus V Lingsar dengan tujuan untuk memberikan teori dan praktek kepada guru-guru mengenai cara menggunakan google form untuk melakukan penilaian. 3. HASIL DAN PEMBAHASAN Peserta kegiatan pengabdian ini terdiri dari sekolah dasar di Gugus V Lingsar. Guru- guru tersebut berasal dari SDN 1 Langko, SDN 1 Giri Madra, SDN 1 Duman dan SD Peduli Anak. Total peserta sebanyak 20 orang. Kegiatan pelatihan dibagi menjadi dua bagian, yakni pemaparan materi dan praktek penggunaan google form. Dokumentasi pada sesi penyampaian materi disajikan pada Gambar 1. gganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 Gambar 1. Dokumentasi Kegiatan pada Sesi Penyampaian Materi Gambar 1. Dokumentasi Kegiatan pada Sesi Penyampaian Materi \|104 Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 Pelatihan pembuatan Gooogle Form … Amrullah et al Amrullah et al Dalam sesi praktek terbimbing, pemateri mendemonstrasikan cara menggunakan google form. Peserta kemudian bersama-sama mempraktekan hal tersebut pada perangkat masing-masing. Praktek tersebut memerlukan koneksi internet. Tim pengabdian membantu penyediaan internet untuk menunjang proses praktek peserta. Tim pengabdian juga berkeliling membantu peserta yang melakukan praktek. Pada sesi ini, peserta juga bertanya mengenai hal-hal ayng masih belum dipahami. Berdasarkan observasi yang dilakukan selama pelaksanaan kegiatan oleh observer, peserta terlihat memperhatikan penjelasan narasumber dan turut melakukan praktek dengan baik. Adapun kendala yang ditemui selama pelakasanaan yaitu kurang stabilnya listrik di sekolah tempat pelaksanaan selama 30 menit karena terdapat kegiatan lain juga yang berjalan di sekolah yang memerlukan listrik dalam jumlah besar. Hal ini menyebabkan matinya listrik untuk menunjang jaringan internet. Namun, kendala dapat teratasi ketika kegiatan lain tersebut sudah selesai dilakukan. Pada akhir kegiatan, tim pengabdian memberikan modul cetak dari materi yang disampaikan untuk dapat dipelajari kembali oleh peserta. Dokumentasi penyerahan modul dalam Gambar 4. Gambar 4. Dokumentasi Penyerahan Modul Materi Pembuatan google form Gambar 4. Dokumentasi Penyerahan Modul Materi Pembuatan google form Di akhir kegiatan, peserta mengisi lembar evaluasi yang melalui google form. Berdasarkan hasil respon peserta, seluruh peserta merasa kegiatan yang dilaksanakan sangat bermanfaat dan sesuai dengan kebutuhan. Modul yang diberikan juga dapat membantu peserta untuk melakukan praktek setelah kegiatan berlangsung. Adapun masukan dari peserta terkait kegiatan yakni, pelatihan sebaiknya dilakukan secara berkala dan dimulai dari praktek yang paling dasar. Amrullah et al Pemaparan materi dilakukan oleh salah satu dosen pada tim pengabdian selama 30 menit. Materi yang dipaparkan meliputi 1) manfaat google form, 2) cara membuat soal pilihan ganda dengan google form, 3) cara memberikan link soal pada siswa dan 4) cara melihat hasil evaluasi yang diberikan. Materi yang disampaikan juga dirangkum dalam bentuk modul yang diberikan kepada peserta. Adapun tampilan modul disajikan pada Gambar 2. Pemaparan materi dilakukan oleh salah satu dosen pada tim pengabdian selama 30 menit. Materi yang dipaparkan meliputi 1) manfaat google form, 2) cara membuat soal pilihan ganda dengan google form, 3) cara memberikan link soal pada siswa dan 4) cara melihat hasil evaluasi yang diberikan. Materi yang disampaikan juga dirangkum dalam bentuk modul yang diberikan kepada peserta. Adapun tampilan modul disajikan pada Gambar 2. Gambar 2. Tampilan Modul Pengabdian pada Sesi Penyampaian Materi Gambar 2. Tampilan Modul Pengabdian pada Sesi Penyampaian Materi Sesi kedua adalah sesi praktek terbimbing. Sesi ini berlangsung selama 120 menit. Dokumentasi kegiatan ini dapat dilihat pada Gambar 2. Sesi kedua adalah sesi praktek terbimbing. Sesi ini berlangsung selama 120 menit. Dokumentasi kegiatan ini dapat dilihat pada Gambar 2. Gambar 3. Dokumentasi Kegiatan pada Sesi Praktek Terbimbing Gambar 3. Dokumentasi Kegiatan pada Sesi Praktek Terbimbing Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 \|105 Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 \|105 Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 \|105 Pelatihan pembuatan Gooogle Form … Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 6. REKOMENDASI Kegiatan pengabdian pelatihan penggunaan google form membutuhkan listrik dan koneksi internet yang stabil. Kepada pihak yang akan melakukan kegiatan serupa, disarankan untuk memperhatikan kestabilan listrik dan koneksi internet. Selain itu, banyak peserta juga perlu diperhatikan agar listrik dan koneksi internet yang tersedia dapat mencukupi kebuthan semua peserta. 4. SIMPULAN Pengabdian kepada masyarakat berupa pelatihan pembuatan google form dilaksanakan dalam dua sesi yakni sesi materi dan sesi praktek. Seluruh peserta memberikan respon \|106 Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 Pelatihan pembuatan Gooogle Form … Pelatihan pembuatan Gooogle Form … Amrullah et al positif terhadap kegiatan yang dilaksakan. Kegiatan ini menjawab kebutuhan peserta dalam menggunakan media evaluasi yang praktis dan efektif. positif terhadap kegiatan yang dilaksakan. Kegiatan ini menjawab kebutuhan peserta dalam menggunakan media evaluasi yang praktis dan efektif. 5. UCAPAN TERIMA KASIH Terimakasih untuk FKIP Universitas Mataram atas dukungan yang diberikan Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 7. REFERENSI Aka, Kukuh A. (2017). Pemanfaatan Teknologi Informasi dan Komunikasi (TIK) Sebagai Wujud Inovasi Sumber Belajar Di Sekolah Dasar, Else, Elementary School Education Journal): Jurnal Pendidikan Dan Pembelajaran Sekolah Dasar, 1(2a), Doi: http://dx.doi.org/10.30651/Else.V1i2a.1041 p g Azis, T. N., & Shalihah, N. M. (2020). Pengembangan Evaluasi Pembelajaran Berbasis Google form. Tawazun: Jurnal Pendidikan Islam, 13(1), 54–65. https://doi.org/10.32832/tawazun.v13i1.3028 Hakim’s, L., & Safi`iI. (2021). Efektivitas Evaluasi Hasil Belajar Bahasa Indonesia Melalui Aplikasi Google form. Bahtera: Jurnal Pendidikan Bahasa Dan Sastra, 20(2), 151-156. https://doi.org/10.21009/bahtera.202.03 Namira, D. (2021). Alternatif Media Evaluasi Pembelajaran Tematik Berbasis Android Menggunakan Google formulir di Sekolah Dasar Plus Tahfizhul Quran (PTQ) Annida. Faktor: Jurnal Ilmiah Kependidikan, 8(1), 114-123. Wulandari, P., Maswani, M., & Khotimah, H. (2019, May). Google form sebagai alternatif evaluasi pembelajaran di SMAN 2 Kota Tangerang. In Prosiding Seminar Nasional Pendidikan FKIP (Vol. 2, No. 1, pp. 421-425). \|107 Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023 Rengganis Jurnal Pengabdian Masyarakat Volume 3 Nomor 1, Bulan 2023
https://openalex.org/W2990364955	https://europepmc.org/articles/pmc6877902?pdf=render	English	null	The Impact of Grammar on Mentalizing: A Training Study Including Children With Autism Spectrum Disorder and Developmental Language Disorder	Frontiers in psychology	2,019	cc-by	13,137	ORIGINAL RESEARCH published: 19 November 2019 doi: 10.3389/fpsyg.2019.02478 The Impact of Grammar on Mentalizing: A Training Study Including Children With Autism Spectrum Disorder and Developmental Language Disorder Stephanie Durrleman1,2, Morgane Burnel3, Jill Gibson De Villiers4, Evelyne Thommen5, Rachel Yan4 and Hélène Delage1 1 Department of Psycholinguistics, University of Geneva, Geneva, Switzerland, 2 Department of Linguistics, University of Geneva, Geneva, Switzerland, 3 Laboratoire de Psychologie et Neurocognition, Department of Psychology, Université Grenoble Alpes, Saint-Martin-d’Hères, France, 4 Department of Psychology, Smith College, Northampton, MA, United States, 5 EESP, University of Applied Sciences and Arts, Western Switzerland, Lausanne, Switzerland Training on complements in English, German, and Mandarin has been reported to trigger improvements on both complements and Theory of Mind (ToM), with typically developing (TD) pre-schoolers on the verge of developing these skills (Hale and Tager-Flusberg, 2003; Lohmann and Tomasello, 2003; Shuliang et al., 2014). In the current study, we build on the idea that increasing mastery of complementation holds the promise of enhancing ToM, and seek (i) to replicate the positive effects observed in previous work for this effect in French-speaking TD children, and (ii) to pilot extending this to clinical children, more speciﬁcally those with Autism Spectrum Disorder (ASD) and Developmental Language Disorder (DLD), through exploring whether improvement in the latter, clinical groups follows that of the TD group. Sixty children with ToM difﬁculties, 16 with ASD (aged 5;6–11;8), 20 with DLD (aged 4;8–9;0) and 24 typically developing children aged (2;9–5;3 years), participated in a 4-week training program. Half received training targeting sentential complements and half received a control training targeting lexical skills. Complementation training, but not lexical training, led to a signiﬁcant direct increase in complements, and also had the indirect effect of signiﬁcantly boosting belief reasoning. TD and clinical groups followed the same patterns of performance. These results conﬁrm previous ﬁndings in other languages for TD, and further suggest promising new directions for therapeutic programs addressing ToM delays in populations of different aetiologies, namely the incorporation of a motivating training on complementation. Citation: Durrleman S, Burnel M, De Villiers JG, Thommen E, Yan R and Delage H (2019) The Impact of Grammar on Mentalizing: A Training Study Including Children With Autism Spectrum Disorder and Developmental Language Disorder. Front. Psychol. 10:2478. doi: 10.3389/fpsyg.2019.02478 Keywords: Autism Spectrum Disorder (ASD), Developmental Language Disorder (DLD), Theory of Mind (ToM), sentential complements, training program Edited by: Ilaria Grazzani, University of Milano-Bicocca, Italy Reviewed by: Francesca Panzeri, University of Milano-Bicocca, Italy Steven Stagg, Anglia Ruskin University, United Kingdom Correspondence: Stephanie Durrleman stephanie.durrleman@unige.ch *Correspondence: Stephanie Durrleman stephanie.durrleman@unige.ch Specialty section: This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology Received: 02 May 2019 Accepted: 21 October 2019 Published: 19 November 2019 (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] (1) Sally places a ball in a basket, then leaves. While she is absent, Anne arrives and moves the ball from the basket to a box. Sally returns, and the children are asked: (a) Where Sally will look for her marble? (The critical “belief” question), (b) Where it is really? (The “reality” question), (c) Where it was at the beginning? (The “memory” question). This linguistic tool would serve to eﬃciently represent subjective truths because the content of the complement (in brackets) has an independent truth-value, and consequently can be false while the entire sentence remains true. These semantic and syntactic properties render complements ideal tools for grasping propositional attitudes and thus eﬃciently representing subjective truths (Perner, 1988; de Villiers et al., 2014), albeit with some cross-linguistic variation (Perner et al., 2003; Cheung et al., 2004; Tardif et al., 2007). (2) After being presented with a Smarties tube, the children are asked what they think is inside, to which they typically reply: ‘Smarties.’ It is then revealed that in fact there are pencils inside, at which point the child is asked if s/he can remember the contents of the tube, as well as the critical test question: What would another person would think is inside? In support of the view that complementation assists complex ToM reasoning, authors have reported links between mastery of this structure and success at FB in young TD children (de Villiers, 2000; de Villiers and de Villiers, 2000) as well as in children with ASD (Tager-Flusberg, 2000; Tager-Flusberg and Joseph, 2005; Lind and Bowler, 2009) and language-delayed deaf children (Schick et al., 2007). Interestingly, these links are also found when the complements do not occur with mental state verbs but rather with verbs of communication, which themselves do not refer explicitly to mental states (de Villiers and Pyers, 2002; Durrleman and Franck, 2015). Indeed the latter verbs, being less abstract than mental state verbs, have even been argued to be most crucial for ToM success in children with ASD (Tager-Flusberg and Joseph, 2005). Knowledge of sentential complements, rather than of mental state lexicon, would therefore allow children to bootstrap their meta-representational grasp of beliefs. Accurate responses during tasks such as those above are only attested around the age of 4–5 years in TD (Wellman et al., 2001; Milligan et al., 2007). (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] It is important to emphasize the conceptual diﬃculty involved during FB attribution: the child must reconcile the contradiction between what s/he knows and what the other believes. This is a sophisticated step, preceded by simpler mental states attributions. The attribution of diverse desires and beliefs, for instance, does not require the child to reconcile a perspective in contradiction with what they know to be true, and these FB- precursors emerge earlier in development than the attribution of FB, i.e., before age 4 (Wellman and Liu, 2004). The emergence of successful mentalizing including FB reasoning is important for the development of social cognition on various levels, e.g., ﬂuid conversational skills, conﬂict resolution, popularity amongst peers, etc. (Astington and Jenkins, 1999; Astington, 2003; Astington and Pelletier, 2005; Astington and Edward, 2010; Mazza et al., 2017; Derksen et al., 2018). In certain clinical populations, such as children with Autism Spectrum Disorder, diﬃculties with FB reasoning often persist later in development, aﬀecting performance on FB tasks even at a mental age of 9 years (Baron-Cohen et al., 1985). This marked delay has been interpreted to indicate a core mind- reading deﬁcit (Baron-Cohen, 1990), which would explain weaknesses in communicative and social skills characteristic of the autistic condition (Diagnostic and Statistical Manual of Mental Disorders, 5th edition). However, a subset of children with ASD, from 20 to 50% (Baron-Cohen et al., 1985; Prior et al., 1990), systematically succeeds at FB attribution and thus arguably can surmount their fundamental ToM diﬃculty (Tager-Flusberg and Joseph, 2005). It has been claimed that in order to accomplish this, they apply verbal strategies. Put diﬀerently, children on the spectrum could use language to support their reasoning about others’ beliefs, with some studies suggesting that lexical abilities play a role in ToM (e.g., Happé, 1995), and others pointing rather to the importance of grammatical skills (Fisher et al., 2005; Milligan et al., p g p If language skills, in particular with complementation, serve for belief reasoning, then populations with language diﬃculties that include complementation would also be expected to struggle with this aspect of ToM. This seems to be the case for children with Developmental Language Disorder (DLD). INTRODUCTION The ability to grasp that people’s mental representations of the world may or may not correspond to reality is an important milestone in the development of ‘theory of mind’ (ToM) (Dennett, 1978; Premack and Woodruﬀ, 1978; Flavell, 1999). Typically developing (TD) preschool children struggle at tasks requiring them to predict another’s actions based on their false belief (FB) November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org The Impact of Grammar on Mentalizing Durrleman et al. (Wellman et al., 2001). Such tasks usually involve a protagonist whose mistaken belief about an object has arisen because (1) the object was displaced (Change of Location Task, Wimmer and Perner, 1983; Baron-Cohen et al., 1985) or (2) the object has the appearance that it might contain something else than its actual contents (Unexpected Contents, Gopnik and Astington, 1988). 2007). The linguistic determinism approach (de Villiers, 2007) maintains that a speciﬁc grammatical structure is most crucially solicited during mentalizing, namely complement clauses such as (3), where a proposition is embedded under a verb of mental-state (e.g., think, believe) or communication (e.g., say, mention): (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] Frontiers in Psychology \| www.frontiersin.org (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] The current work is thus concerned with elucidating whether training sentential complements can be beneﬁcial for the remediation of belief reasoning in children with ASD and those with DLD, along the lines of TD. It is also an open question whether enhancing complementation can also be useful for other aspects of ToM beyond false belief reasoning, such as grasping diversity of desires. and ASD (Durrleman et al., 2016a). Taking as a point of departure that language is not only fundamentally related to mentalizing, but also inﬂuences its development rather than vice versa as revealed by longitudinal studies (TD: Astington and Jenkins, 1999; ASD: Tager-Flusberg and Joseph, 2005), researchers have aimed to trigger ToM via the training of complements in preschool TD children. Results have revealed that this training is indeed eﬀective at boosting ToM, even when training involved complements of verbs of communication alone (Hale and Tager-Flusberg, 2003; Shuliang et al., 2014) and when deceptive scenarios (i.e., involving appearance-reality dissociations) were not included to train complements (Lohmann and Tomasello, 2003; Shuliang et al., 2014), although capitalizing on both complements and deceptive scenarios together appears to be especially useful for consolidating ToM. Still, none of these studies on complementation training included participants delayed for either language or ToM, and instead focussed on children on the cusp of developing these skills anyway. It thus remains to be determined whether populations where ToM and/or language is aﬀected would show similar boosts in belief reasoning to that already observed in TD children due to complementation training. The current work is thus concerned with elucidating whether training sentential complements can be beneﬁcial for the remediation of belief reasoning in children with ASD and those with DLD, along the lines of TD. It is also an open question whether enhancing complementation can also be useful for other aspects of ToM beyond false belief reasoning, such as grasping diversity of desires. regressions or ANOVAS, especially when variables are highly intercorrelated, as they are in this study. It models the relationships among multiple independent and dependent variables simultaneously, unlike linear regression, which can only analyze one layer of linkages at a time. Because SEM can test multiple pathways, it allows the investigation of both direct and indirect eﬀects in one hypothesized model (Gefen et al., 2000). This is important in determining the particular role that the complement training plays in advancing false belief understanding. Frontiers in Psychology \| www.frontiersin.org Participants ll f h All of the participants in this study were native French-speakers, recruited in Geneva and Lausanne, Switzerland and Paris, France. The project received approval from the Ethics Committee of the Faculty of Psychology and Educational Sciences of the University of Geneva as well as from the Geneva Cantonal Ethics Commission, and was also declared at ‘La Commission Nationale de l’Informatique et des Libertés (CNIL)’ in France. Children’s parents all provided written, informed consent for their child to participate. Sixty children participated in the study: 16 children with ASD aged 5;6–11;8 (M = 8;3), 20 children with DLD aged 4;8–9;0 (M = 6;9) and 24 TD children aged 2;9–5;3 years (M = 4;3). Diﬀerences in age were due to the fact that diﬃculties on ToM have been attested at diﬀerent phases of development in these three populations. Matching was done on the groups’ linguistic and cognitive characteristics as explained below. Children with ASD were recruited from specialized schools, those with DLD from speech-language centers which they attended after school, and TD children from kindergartens and day-care facilities. We targeted children of the age range when complements and ToM are reportedly not yet mastered, hence for TD children this meant choosing children between the ages of 3 and 6 years (Wellman et al., 2001), for children with DLD the upper cut-oﬀwas age 9 (Andrés-Roqueta et al., 2013; Nilsson and de López, 2016) and for children with ASD this cut-oﬀwas extended to 12 years (Baron-Cohen et al., 1985; Yirmiya et al., 1998). Then, for these children to be included in the study, they had to meet several criteria: (i) TD children had to have no history of language impairment and needed to be included in normal classrooms without support. (ii) In contrast, clinical groups had to have been previously given the relevant diagnosis by a qualiﬁed professional. More speciﬁcally, children with DLD needed to have obtained language scores of at least 2 SDs below age- speciﬁc norms according to standardized tests used by speech and language pathologists in Switzerland and France (CIM 10; De La Santé, 1993), while children with ASD had to have met the criteria for this condition according to the DSM-IV-TR (American Psychiatric Association [APA], 2000), the Autism Diagnostic Observation Schedule, ADOS (Lord et al., 2003) and/or the ADI-R (Rutter et al., 2003). (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] Children with this condition display primary diﬃculties in formal language (Leonard, 2014) including complementation (Tuller et al., 2012; Steel et al., 2016) and are also reportedly delayed in ToM, even if these delays appear to be more subtle than those attested in ASD (Holmes, 2002; Tucker, 2004; Andrés-Roqueta et al., 2013). Moreover, mastery of complements by children with DLD also relates to their success at ToM as measured by false-belief tasks (de Villiers et al., 2003; Miller, 2004). Interestingly, the verbal demands of the ToM tests administered in the studies conducted with this population may impact their performance (Miller, 2001), but these alone do not suﬃce to clearly explain their ToM performance, as even tasks that rely minimally on language pose problems, suggesting that the diﬃculty is at the level of ToM reasoning (Nilsson and de López, 2016). In favor of the view that language inﬂuences ToM reasoning and not only verbal ToM task performance, relations between complements and low verbal ToM tasks have been reported for both DLD (Durrleman et al., 2017a) November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org Frontiers in Psychology \| www.frontiersin.org 2 The Impact of Grammar on Mentalizing Durrleman et al. and ASD (Durrleman et al., 2016a). Taking as a point of departure that language is not only fundamentally related to mentalizing, but also inﬂuences its development rather than vice versa as revealed by longitudinal studies (TD: Astington and Jenkins, 1999; ASD: Tager-Flusberg and Joseph, 2005), researchers have aimed to trigger ToM via the training of complements in preschool TD children. Results have revealed that this training is indeed eﬀective at boosting ToM, even when training involved complements of verbs of communication alone (Hale and Tager-Flusberg, 2003; Shuliang et al., 2014) and when deceptive scenarios (i.e., involving appearance-reality dissociations) were not included to train complements (Lohmann and Tomasello, 2003; Shuliang et al., 2014), although capitalizing on both complements and deceptive scenarios together appears to be especially useful for consolidating ToM. Still, none of these studies on complementation training included participants delayed for either language or ToM, and instead focussed on children on the cusp of developing these skills anyway. It thus remains to be determined whether populations where ToM and/or language is aﬀected would show similar boosts in belief reasoning to that already observed in TD children due to complementation training. 1A total of 11 children were eliminated after the pre-tests from the initial cohort of 71 participants: 3 TD, 4 ASD and 4 DLD. For 10 children, this was because of ceiling performance, and for 1 child with ASD this was because of hypersensitivity to the sounds of the testing material. Participants ll f h (iii) Scores on pre- (training)-tests assessing ToM and complements also had to In the current study, we build on the idea that increasing mastery of complementation holds the promise of enhancing ToM, and seek (i) to replicate the positive eﬀects observed in work on other languages for this eﬀect in French-speaking TD children, and (ii) to pilot extending this to clinical children, more speciﬁcally those with ASD and DLD, through exploring whether improvement in the latter, clinical groups follows that of the TD group. If this proves to be the case, our results would suggest a novel, evidence-based, clinical intervention, addressing both language and ToM in these populations. We explore several other questions as well with our rich data set. We verify that complementation training is more eﬀective for complements and ToM than a more general, lexical training. We test whether the eﬀects of complement training are particular to false beliefs assessed verbally, or encompass low-verbal false beliefs too. We ask whether the contribution of complementation is speciﬁc to false belief reasoning, or whether it can be observed to assist other, earlier-mastered aspects of ToM, like diverse desires and true beliefs. Importantly for clinical purposes, we ask whether the hypothesized ToM gains persist through time by retesting after a delay. Finally, we ask whether the control group, who received lexical training, diﬀerentially improved to the target, complementation training group, on the lexical tasks. In addition to individual analyses on the outcome measures, we undertake a Structural Equation Model (SEM) to look more closely at the pathways of change, for example, asking whether the success of complement training depended on other abilities, such as non-verbal reasoning or language skills at outset. SEM allows several advantages over simple November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 3 The Impact of Grammar on Mentalizing Durrleman et al. 20 boys and 10 girls. The two groups of 30 were matched on a variety of global cognitive and linguistic standardized measures (all t < 1, see Table 1 for precise p-values), namely non-verbal reasoning (Raven et al., 1998), as well as general morphosyntax and lexicon (Exalang 3–6; Helloin and Thibault, 2006). For more speciﬁc measures, we created tests assessing: (i) complementation understanding (based on de Villiers and Pyers, 2002) and (ii) ToM abilities. Participants ll f h The latter included a verbal measure of false-belief (based on Baron-Cohen et al., 1985), a minimally (low-)verbal measure of false-belief (based on Woolfe et al., 2002) as well as a test assessing skills emerging just before false-belief reasoning (FB precursors), namely diverse desires and diverse beliefs (based on Burnel et al., 2017). We refer to the latter as low-verbal ToM and FB precursors. Table 1 presents the descriptive measures of the children included in each of the training groups. Details on the descriptive characteristics of the cognitive groups (TD, ASD and DLD) are reported in Table 2. While these groups diﬀer for age [F(2,52) = 37.57, p < 0.001], for reasons explained above, they do not diﬀer on standardized measured of non- verbal reasoning (p = 0.09), and morphosyntax (p = 0.23) or lexicon (p = 0.27). leave enough of a margin for progress to be achieved, thus only children performing equivalent to or below 70% were included (equivalent to a maximum of 8 successful items out of 12 on FB and 4 successful items out of 6 on false complements). (iv) In addition, parents had to report that their child’s language comprehension was of the level to understand simple subject- verb-object sentences, which was subsequently conﬁrmed by experimenters upon the ﬁrst meeting during language tasks (Exalang et al., 2006), such that leading them up to complex sentences in a relatively short space of time could be feasible. (v) Finally, only children who could attend to pre-tests could proceed to training.1 Within each population, one half was assigned to the target-training program involving the teaching of sentential complements, while the other half was assigned to an alternative training program focussing on lexical enrichment. The latter group allowed us to conﬁrm that any eﬀects arising with complementation training were not due to general linguistic stimulation. This preliminary study involved small groups of participants for each population of children. Because our hypotheses were identical for all of these populations, we analyzed their data grouped together and focused on the type of training, and then conducted analyses to see whether the overall results were driven by any subgroup/speciﬁc population(s), i.e., whether progress in the TD and clinical groups were similar. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org Material and Procedure TABLE 2 \| Descriptive characteristics of participants. TD (n = 24) ASD (n = 16) DLD (n = 20) Chronological Age 4.32 (0.67) 8.28 (2.02) 6.92 (1.53) Raven 13.71 (4.72) 15.94 (15.94) 17.90 (4.54) Global morphosyntax 10.29 (2.29) 10.43 (2.06) 11.65 (2.16) Global lexicon 33.75 (3.17) 32.38 (8.31) 34.05 (2.72) TABLE 2 \| Descriptive characteristics of participants. material, so that children had to adjust their predictions depending on the changing epistemic state of the agent (Forgeot d’Arc and Ramus, 2011). We created three sets of tests, meaning that children who participated in the entire study saw a total of 108 diﬀerent items (54 FB items and 54 TB items) over the course of three testing sessions, and never saw the same item twice. The order of the items which made up each test was randomized and counterbalanced across participants. program lasted 4–6 weeks. One to two weeks after training ceased, immediate post-(training)-tests were administered to determine potential gains on abilities targeted by the programs, namely complements, ToM and lexicon. Again 4–6 weeks went by, this time without training, and another set of tests was administered, i.e., ‘follow-up’ or ‘delayed’ tests. These ‘follow-up’ tests were only conducted with children who had made progress of at least 10% between pre-tests and immediate post-tests in order to determine whether gains on complements and ToM could be maintained. Figure 1 outlines the overall experimental design of the study. program lasted 4–6 weeks. One to two weeks after training ceased, immediate post-(training)-tests were administered to determine potential gains on abilities targeted by the programs, namely complements, ToM and lexicon. Again 4–6 weeks went by, this time without training, and another set of tests was administered, i.e., ‘follow-up’ or ‘delayed’ tests. These ‘follow-up’ tests were only conducted with children who had made progress of at least 10% between pre-tests and immediate post-tests in order to determine whether gains on complements and ToM could be maintained. Figure 1 outlines the overall experimental design of the study. Theory of Mind Tests ToM was evaluated via a total of 12 FB items. These were interspersed with 12 TB items. Of the 12 FB items, 6 formed a verbal ToM task and 6 others a low-verbal one (again 6 true and 6 false beliefs). For each task, the child’s response always implied selecting one element amongst three, two involved in the scenario presented (corresponding to a true vs. Material and Procedure Pre-tests assessed a series of relevant measures, namely ToM (via verbal and low-verbal FB tasks as well as a mini-test of FB precursors), complements, lexicon, morphosyntax and non-verbal reasoning (see below for more details on these measures). One to two weeks after being tested for the ﬁrst time, participants were randomly assigned to one of the two training programs, i.e., either that of complements or lexicon. Each Amongst the target-training group, there were 21 boys and 9 girls and amongst the control-training group there were TABLE 1 \| Means (standard deviations) on paired variables at the moment of pre-test for the two groups (syntactic training, lexical training) and the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). TABLE 1 \| Means (standard deviations) on paired variables at the moment of pre-test for the two groups (syntactic training, lexical training) and the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). Syntactic training Lexical training T-tests results n = 30 n = 30 TD ASD DLD TD ASD DLD n = 12 n = 8 n = 10 n = 12 n = 8 n = 10 Chronological Age 5.92 (1.97) 6.57 (2.36) t(58) = 1.16, p = 0.25 4.29 7.52 6.58 4.35 9.04 7.25 Raven 15.53 (5.26) 15.87 (4.90) t(58) = 0.25, p = 0.80 12.92 16.25 18.10 14.50 15.63 17.70 Global morphosyntax 10.62 (2.54) 10.97 (1.94) t(56) = 0.58, p = 0.56 10.00 10.57 11.40 10.58 10.29 11.90 Global lexicon 34.2 (2.8) 32.8 (6.4) t(58) = 1.13, p = 0.27 33.8 34.4 34.5 33.7 30.4 33.6 False Complements/6 1.47 (1.50) 1.57 (1.48) t(58) = −0.26, p = 0.79 0.92 1.50 2.10 1.17 2.13 1.60 Verbal FB/6 0.63 (0.76) 0.93 (1.17) t(58) = −1.18, p = 0.25 0.50 0.75 0.70 0.90 1.50 0.70 Low-verbal FB/6 2.60 (1.90) 2.73 (2.18) t(58) = −0.25, p = 0.80 2.58 3.13 2.20 2.50 4.13 1.90 FB Precursors 4.77 (1.48) 4.69 (1.65) t(57) = 0.19, p = 0.85 4.25 5.38 4.90 3.50 5.88 5.22 ) on paired variables at the moment of pre-test for the two groups (syntactic training, lexical training) and the three populations of D, Autism Spectrum Disorder; DLD, Developmental Language Disorder). November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 4 The Impact of Grammar on Mentalizing Durrleman et al. Material and Procedure a false belief), while the third was unrelated to the story. The verbal ToM task was directly inspired by the Sally- Anne Task (Baron-Cohen et al., 1985). As explained above, in the FB scenarios of this task, the child is confronted with an object being moved from location 1 to location 2 in the main protagonist’s ignorance and has to capitalize on this protagonist’s false belief to predict that s/he will look for the desired object in location 1 (where it is no longer present). For example, one of the proposed scenes in our assessment was: “This is Bob. This is the mother. Bob has a yellow pot. The mother has a green pot. Bob has a ball, he puts the ball in his yellow pot. Bob is going out to play. The mother takes the ball out of the yellow pot and puts it in the green pot. Now Bob comes back, he wants to play with the ball. Where’s Bob going to get his ball?”. The child must then choose from three answers: the initial position (here the basket), the place where the object is actually located (here the box) and the position of the middle representing an object not involved in the story (here a bag). To succeed, children must take into account Bob’s misrepresentation while putting aside their own knowledge of reality. In the true belief, ﬁller scenarios, the displacement occurred in front of Bob (see Figure 2), or while he was absent the object was manipulated and returned to its original position. Pre- and Post-tests All tests, both pre and post, were conducted on laptop computers, to contrast with the materials used for the training itself, which was administered via iPads. This distinction between testing and training modalities allowed us to ascertain that any gains between pre and post-tests could not simply be attributed to increased familiarity with the material used during tests. Tests assessing ToM and sentential complements were speciﬁcally designed for the study, but followed the same basic format of tests which have been used in various previous studies, as explained in more details below. Administering ToM tests on a screen as opposed to having them administered by an experimenter held the promise of being the least penalizing option in particular for children with ASD (Chevallier et al., 2014). Each test of our target variables (i.e., complements and ToM) contained 36 items, which were all animated scenarios. Eighteen of these items involved FB, i.e., they corresponded to the test condition, while the other items involved true beliefs (TB). TB items cannot be taken as unambiguous ToM measures, as accurate responses coincide with reality responses (Dennett, 1978), however, they allowed varying the FIGURE 1 \| Experimental design. Frontiers in Psychology \| www.frontiersin.org 5 November 2019 \| Volume 10 \| Article 2478 FIGURE 1 \| E i t l d i November 2019 \| Volume 10 \| Article 2478 5 The Impact of Grammar on Mentalizing Durrleman et al. The low-verbal ToM evaluation was inspired by Woolfe et al. (2002), who claim that the task “minimize(s) verbal task- performance requirements.” This is because the pictures alone are informative enough for the child to both interpret the scene and to respond. As an illustration, images would appear on the screen clearly depicting someone blindfolded who was trying to obtain an object. This information was then also provided verbally in the form of commentary, which was thus not crucial for task success. For example (see Figure 3), in one scene there was a blindfolded man with a ﬁshing rod and seaweed covering the object at the end of his rod and the commentary went: “Look! The man is ﬁshing! He can’t see anything. Let’s see what is behind the seaweed - Click here!” All children understood and clicked, which made the seaweed move aside. In one scenario there was a ﬁsh, in another test there was a boot. FB Precursors If the participant met the inclusionary criteria deﬁned above after the main tests, we administered a mini-test evaluating the skills emerging before the ability to assign false beliefs, namely the understanding that people diﬀer from each other in their desires and beliefs (Wellman and Liu, 2004). There were a total of 6 items seen in each mini-test, 3 diverse desires and 3 diverse beliefs, such that children participating in the three testing sessions saw a total of 18 items assessing FB precursors. As an illustration of an assessment of diverse desires, the child saw an animated story while hearing the following narration: “What do you prefer: a carrot or a biscuit?” The child would then select one (usually the biscuit) and then see a small scene in which another character chooses the opposite, e.g., “Here is Theo. Theo prefers carrots. Theo is hungry. What do you think Theo will eat?”. For an assessment of diverse beliefs, the child heard: “This is Thomas’ book. Sometimes Thomas’ mother puts his book on the table, sometimes Thomas’ mother puts his book on the shelf. This is Thomas. Thomas is looking for his book. Where do you think the book is? On the shelf or on the table?” The child would then click on one, say the shelf, in which case the story would Pre- and Post-tests Then children were then presented with three objects and asked to click on the object the man was thinking about, in this instance selecting between a ﬁsh, a boot and a wheel. three options to select from, the voice said: “Look here: what is Dad saying that Jean is doing?” (see Figure 4). We pointed back to the picture and maintained the present tense in light of observations that past-tense can be diﬃcult for children with DLD (Rice and Wexler, 1996; Bishop, 2013) and ASD (Tager- Flusberg, 1989; Roberts et al., 2004). In the ﬁllers, the report and the event coincided (true complement) such that it suﬃced for children just to touch the only event evoked (e.g., the father says that Jean is eating ﬁsh and Jean is shown to indeed be eating ﬁsh). Control Training: Lexicon The control, lexical training was based on diﬀerent applications teaching the lexicon, namely Bitsboard, Flashcards, French FEL, Apprends-moi les mots (‘Teach me words”) and Animaux (“Animals”). Several themes are covered during the proposed exercises, such as colors, food, means of transport, animals, etc. At each session, we recorded the words learned and thus in subsequent sessions only checked these again before addressing the novel words. This training involved the same sort of demands as the target training, namely image designation, repetition, truth-value judgment, but also carefully steered away from mental state terms. Non-verbal Reasoning Raven’s matrices (Raven et al., 1998), were also administered during the pre-test to assess the child’s level of non-verbal reasoning. During this task, the child must complete 36 series of increasing diﬃculty. Each series is presented with a piece missing, which the child must select amidst six pieces. FIGURE 4 \| Illustration of the complements test. detail in Supplementary Appendix A. The ﬁrst activity, inspired by Wilson and Fox (2013), dealt with inﬁnitival complements such as: “Sophie sees a baby crying” which are the ﬁrst kind to be mastered (Bloom et al., 1989; Diessel, 2004). All other activities focused on tensed complements of communication verbs, such as: “The little girl screams that there is a spider in the bathtub” which are the kind speciﬁcally hypothesized to support ToM (de Villiers, 2007). Six sessions contained new material, composed of approximately 100 diﬀerent items. All children were presented with the entire material at least once, and some saw it a second time if the experimenters noticed they were still not excelling after 3 weeks. In this case, material from the beginning would start over. Target Training: Complementation For the training of complements, we administered a novel iPad application (Durrleman et al., 2016b), called DIRE, which means ‘to say’ in French. This name indicates that the program focuses mainly on the training of complements of verbs of communication (as well as some complements of verbs of perception or desire), thus abstracting away from verbs of mental state such as ‘think’ or ‘believe.’ DIRE also stands for ‘Diﬀerentiating Ideas from Reality via Exercises,’ since the purpose of the training oﬀered is to assist children with ToM diﬃculties to acquire these complements so that they may in turn apply them during ToM reasoning. We opted for iPad training, as such methods have already proven to be eﬀective with clinical populations (Alzrayer et al., 2014). Our training involved ﬁve types of activities, various using pictorial representations of speech, as previous work has found that visual cues are eﬀective in remediation programs with ASD (e.g., Wellman et al., 2002; Paynter and Peterson, 2013). Frontiers in Psychology \| www.frontiersin.org Language Finally, we evaluated the child’s language level using a test normed for children aged 3–6 years: EXALANG 3-6 tests (Helloin and Thibault, 2006). We opted for this task to assess receptive lexical skills (via the designation of images) and morphosyntactic (via the morphosyntax subtest) because its general format was very similar to our other tests for ToM and complements, namely they contained simple, computerized animations. Also, as mentioned earlier, belief attribution emerges generally around 4–5 years of age, i.e., along with general language skills corresponding to this age range, thus we reasoned that a language task for this age range would be appropriate for our sample, who were still struggling with FB. Training Training programs either focused speciﬁcally on complements (for the target training) or more generally on the lexicon (for the control training). Each involved ﬁve types of activities conducted on iPads, two to three times per week for maximum duration of 6 weeks, and a minimum duration of 3 weeks in the event that children already performed at ceiling at this point of the training program. Complements Test The evaluation of sentential complements was inspired by de Villiers and Pyers (2002). The general format involved one protagonist reporting an event to another, after which the actual event was shown. There were a total of 12 items, 6 test FB items and 6 TB items. In the test items, the complement reported an event inaccurately (false complement). The child had to simply recall the content of the erroneous complement uttered in the ﬁrst scene in order to score a point. An illustration would be: “The mother asks the father what Jean is doing. And the father answers that Jean is eating ﬁsh. Look! Jean is giving ﬁsh to the cat!” Then, pointing back to the picture of the parents now with FIGURE 2 \| Illustration of the verbal ToM task. FIGURE 3 \| Illustration of the low-verbal ToM task. FIGURE 2 \| Illustration of the verbal ToM task. FIGURE 2 \| Illustration of the verbal ToM task. FIGURE 3 \| Illustration of the low-verbal ToM task. FIGURE 3 \| Illustration of the low-verbal ToM task. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 6 Durrleman et al. The Impact of Grammar on Mentalizing continue: “For Thomas, the book is on the table. Where will Thomas look for his book?”. continue: “For Thomas, the book is on the table. Where will Thomas look for his book?”. FIGURE 4 \| Illustration of the complements test. Question 1: Did the Target and Control Training Have Differential Effects? Our ﬁrst research question concerns whether the trainings had diﬀerential eﬀects, namely an improvement from pre-test to post-test speciﬁc to the type of intervention (descriptive data for the syntactic and lexical training are provided in Figure 5). Speciﬁcally, it is necessary to show that the syntactic training resulted in improvement on false complements, but the lexical training did not. Next, it is necessary to show that the syntactic training has eﬀects on False Belief performance, and that the lexical training did not. Third, it is necessary to show that the training was not restricted to the verbal false belief tasks, but applied equally to the verbal and low-verbal tasks. The ﬁve activities of DIRE were administered during each training session. The order of appearance of the activities was the same for all children, beginning with activity 1 and ending with activity 5. Each activity addressed a particular aspect of complementation via brief exercises, which are explained in Recall that the children were selected to have poor performance on complements and false belief, with the result that the data were non-normally distributed, as the distributions were truncated. To do ANOVAs, we tried using the Box-Cox transformation but homogenous variance and normal error November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 7 The Impact of Grammar on Mentalizing Durrleman et al. FIGURE 5 \| Scores on ToM and complements tasks at pre-test and post-test for the syntactic and lexical groups. FIGURE 5 \| Scores on ToM and complements tasks at pre-test and post-test for the syntactic and lexical groups. There was no prediction that these would be aﬀected by syntactic or lexical training, so a second set of analyses looked at the change in these variables compared to the variables targeted in Question 1. distribution could not be achieved due to this truncation at one end of the distributions. Therefore, on the variables that constituted selection criteria, namely false belief (verbal and low-verbal) and false complements, non-parametric tests were necessary. Wilcoxon signed rank tests were used in the analysis of pre- and post-training eﬀects on the false belief and complement measures for the diﬀerent training groups. Eﬀects sizes (using r, Rosenthal, 1994) are reported for the non-parametric test and regular Cohen’s d (Cohen, 1988) are reported for the parametric test. Question 1: Did the Target and Control Training Have Differential Effects? Using Cohen’s guidelines for r, a large eﬀect is 0.5, a medium eﬀect is 0.3, and a small eﬀect is 0.1 (Cohen, 1988). The Box-Cox transformations were applied ﬁrst to variables including precursors to false belief, true belief (verbal and low-verbal) and true complements, after which assumption of homogeneity of variance and normal distribution of residuals were met according to Levene’s tests and Shapiro’s tests. In order to assess if the target training had a speciﬁc eﬀect as compared to the control training on false complements and false belief attribution, both crucial to ToM, we ran factorial ANOVAs with the training group (syntax vs. lexical) and the moment of test (pre-test vs. post-test) as independent variables on the following dependent variables: true complements, verbal TB, low-verbal TB, and precursors to FB. The interaction eﬀect between the training group (syntactic vs. lexical training) and the moment of test (pre-test vs. post-test) was not signiﬁcant eﬀect for true complements [F(1,55) = 0.58, p = 0.45], verbal TB [F(1,58) = 2.47, p = 0.12], low-verbal TB [F(1,58) = 2.03, p = 0.16], and precursors to FB [F(1,57) = 0.18, p = 0.67]. In the syntactic training group, Wilcoxon’s signed rank test showed a statistically signiﬁcant diﬀerence between pre-test and post-test on Verbal False Belief (Pre-test: median = 0.50; post- test : median = 4.0; Z = −4.07, p < 0.001, r = 0.74), low-verbal False Belief (Pre-test: median = 2.0; post-test: median = 5.0; Z = −3.42, p < 0.001, r = 0.63), and False Complements (Pre- test: median = 1.0; post-test: median = 6.0; Z = −4.53, p < 0.001, r = 0.83) with large eﬀect sizes. However, one-way ANOVAs showed that there was no signiﬁcant progression between pre- test and post-test on FB precursors [F(1,58) = 2.89, p = 0.09], Verbal True Belief [F(1,58) = 1.08, p = 0.30], low-verbal true belief [F(1,58) = 2.60, p = 0.11], and true complements [F(1,58) = 3.20, p = 0.08], possibly due to already high scores on these variables (see Supplementary Appendix B). 2One child with ASD in the lexical training group displayed particularly poor performance in lexical designation in the pre-test, which could bias group results in the comparison between pre- and post-test. However, the progression of the “lexical training group” is still signiﬁcant even when removing this child from the analyses (Z = 2.4, p < 0.05). Note also that we proceeded to non-parametrical (Wilcoxon signed-rank) tests for these comparisons, due to ceiling eﬀects on this measure. 3A 10:1 ratio of cases to free parameters is suggested as a minimum sample size for SEM by Bentler and Chou (1987) and echoed by many others as a rule of thumb. Question 3: Are There Population Differences in the Effects of Training? post-test) on verbal false belief, low-verbal false belief and false complements for the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). and group as the independent variable. The untransformed data met the condition on homogeneity of variance by Levene’s test, and there was no signiﬁcant diﬀerence between the three groups on total post-FB performance after complement training [F(2,27) = 0.546, p = 0.586]. (pre-test vs. follow-up and post-test versus follow-up) for the dependent variables, on 22 children who had showed gains of at least 10% on the post-test in the syntactic training group. The progression between pre-test and follow-up test was statistically signiﬁcant for verbal FB (p = 0.001, r = 0.81), low-verbal FB (p = 0.003, r = 0.64), and false complements (p = 0.001, r = 0.88), with higher scores in follow-up. The mean scores were higher on immediate post-tests compared to follow-up post-tests, but the diﬀerence between these two post-tests was not signiﬁcant for verbal FB (p = 0.265, r = 0.035), and did not reach signiﬁcance for low-verbal FB (p = 0.066, r = 0.39) or false complements (p = 0.096, r = 0.36). Thus there was generally only a small drop between post-test and follow-up 4–6 weeks after the ﬁrst post-test (see Figure 7). Question 4: Did the Lexical Training Group Show Differential Results on the Lexicon? As for results on our standardized test of receptive lexicon, the syntactic training group did not show a diﬀerence between pre- and post-test scores (Z = 1.3, p = 0.2), whereas the lexical training group did (Z = 2.6, p < 0.01).2 For detailed information about participants and their individual results, see Supplementary Appendices C,D. Question 3: Are There Population Differences in the Effects of Training? Differences in the Effects of Training? In order to compare the eﬀects of the two trainings in the three populations, additional factorial ANOVAs were run (see Figure 6). This third ANOVA (time × training × clinical group) is exploratory, given the small sample sizes (approximately 10 children per condition). Results showed no interaction eﬀect between training group (syntax vs. lexical), moment of test (pre-test vs. post-test) and population (TD vs. ASD vs. DLD) for false complement [F(2,105) = 0.54, p = 0.59], Verbal FB [F(2,103) = 0.56, p = 0.57], low-verbal FB [F(2,105) = 0.02, p = 0.98], true complements [F(2,105) = 1.12, p = 0.33], Verbal TB [F(2,108) = 0.60, p = 0.55], non- verbal TB [F(2,108) = 0.77, p = 0.46), and precursors to FB [F(2,106) = 0.68, p = 0.51]. In the lexical group, one-way ANOVAs showed no signiﬁcant progression between pre-test and post-test on precursors of FB [F(1,56) = 1.46, p = 0.23], verbal True Belief [F(1,54) = 0.60, p = 0.44], low-verbal true belief (F < 1), or true complements [F(1,55) = 1.36, p = 0.25]. Non-parametric tests indicate no signiﬁcant changes occurred from pre-test to post-test in false complements (Z = −1.18, p = 0.24, r = 0.21), verbal False Belief (Z = −1.52, p = 0.13, r = 0.28), or low-verbal false belief (Z = −1.31, p = 0.50, r = 0.24). Question 2: Are There Training Effects on the ToM Skills Other Than False Belief? Question 2: Are There Training Effects on the ToM Skills Other Than False Belief? The children were tested also on True Complements, True Beliefs, and Precursors to False belief both pre and post training. The children were tested also on True Complements, True Beliefs, and Precursors to False belief both pre and post training. A one-way ANOVA was conducted on the complement training condition only, with total post FB as the outcome November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 8 The Impact of Grammar on Mentalizing Durrleman et al. FIGURE 6 \| Interaction between training (syntax vs. lexicon) and moment of test (pre-test vs. post-test) on verbal false belief, low-verbal false belief and false complements for the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). FIGURE 6 \| Interaction between training (syntax vs. lexicon) and moment of test (pre-test vs. We test six parameters with a sample of 60 participants. Though the 10:1 ratio is often considered safe, there are simulations (e.g., Nevitt and Hancock, 2004; Wolf et al., 2013) that suggest higher numbers are desirable in certain conditions. Latent variables would render the sample size insuﬃcient compared to the simple path model explored here. Missing data also would drastically increase the necessary sample size. An insuﬃcient sample size can result in a failure to converge on a model, or have poor ﬁt estimates. The current model does not show these problems, and is supported by the regressions, so we judge it to be a worthwhile addition despite the small sample. Question 5: Did the Training Result Persist Beyond Immediate Post-test? TABLE 3 \| Regression results predicting children’s total post-false belief (N = 60). A signiﬁcant B-weight indicates the beta-weight and semi-partial correlation are also signiﬁcant. B represents unstandardized regression weights. β indicates the standardized regression weights. sr2 represents the semi-partial correlation squared. r represents the zero-order correlation. LL and UL indicate the lower and upper limits of a conﬁdence interval, respectively. ∗p < 0.05. ∗∗p < 0.01. ∗∗∗p < 0.001. False Belief understanding contribute to the training eﬀect? What about non-verbal intelligence, as measured by Ravens, or the level of general language skill (using EXALANG)? And did the child’s trained mastery of false complements contribute to the false belief post-test score, or did some children succeed on post-test even if they did not improve on complements? groups. For that reason, the groups and populations could be collapsed to explore regressions with the outcome variable of false beliefs (combining low-verbal and verbal tasks into one 12- point score). The ﬁnal regressions contained just the variables that contributed unique variance to this outcome. As shown in Table 3, ﬁrst age was entered and then Raven’s matrices, the total of pre-training score on False belief (non-verbal and verbal combined), then Training condition, then the total post- training score on False complements, since both of these variables contributed to the outcome. groups. For that reason, the groups and populations could be collapsed to explore regressions with the outcome variable of false beliefs (combining low-verbal and verbal tasks into one 12- point score). The ﬁnal regressions contained just the variables that contributed unique variance to this outcome. As shown in Table 3, ﬁrst age was entered and then Raven’s matrices, the total of pre-training score on False belief (non-verbal and verbal combined), then Training condition, then the total post- training score on False complements, since both of these variables contributed to the outcome. The ANOVAs across training and control groups showed no diﬀerence across the TD, ASD and DLD populations in the outcome, nor any interactions between populations and training Having established the signiﬁcant variables in the regression for prediction of the outcome, various SEM models were tried to ﬁnd the model with the best ﬁt. Although the number of subjects is on the low side for a SEM, the ﬁt indices can give an indication of whether the sample has suﬃcient power to justify the model. Question 5: Did the Training Result Persist Beyond Immediate Post-test? More powerful statistics were used to explore the contributing eﬀects of the background variables and training conditions. Although certain criteria had to be met to be included in the study, it would be impossible with such a small sample to match the groups on every variable. Using multiple regressions ﬁrst to discover which variables share variance with the outcome, structural equation modeling (SEM) provided a powerful tool with which to look at the paths of inﬂuence on the outcome of false belief understanding.3 For example, did the initial level of In order to assess if the progression observed at post-test was still present between pre-test and follow-up test, we ran non- parametric comparisons using Wilcoxon matched-pairs signed- ranks tests with the moment of test as the repeated variable 3A 10:1 ratio of cases to free parameters is suggested as a minimum sample size for SEM by Bentler and Chou (1987) and echoed by many others as a rule of thumb. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 9 The Impact of Grammar on Mentalizing Durrleman et al. FIGURE 7 \| Scores on verbal FB, low-verbal FB and false complements at the moment of pre-test, post-test, and follow-up. TABLE 3 \| Regression results predicting children’s total post-false belief (N = 60). Predictor B B 95% CI [LL, UL] SE B β sr2 r Fit Difference (Intercept) −0.78 [−3.74, 2.18] 1.48 Age −0.36 [−0.75, 0.04] 0.20 −0.19 0.03 −0.04 Raven’s Total 0.26∗∗ [0.09, 0.42] 0.08 0.31 0.08 0.26∗ Total Pre-False Belief 0.61∗∗ [0.28, 0.93] 0.16 0.35 0.12 0.30∗ Training 4.88∗∗∗ [3.38, 6.39] 0.75 0.60 0.35 0.58∗∗ R2 = 0.547∗∗ (Intercept) −1.08 [−3.65, 1.48] 1.28 Age −0.32 [−0.67, 0.02] 0.17 −0.17 0.02 −0.04 Raven’s Total 0.19∗ [0.04, 0.34] 0.07 0.23 0.04 0.26∗ Total Pre-False Belief 0.51∗∗∗ [0.22, 0.79] 0.14 0.30 0.08 0.30∗ Training 2.50∗∗ [0.81, 4.19] 0.84 0.31 0.05 0.58∗∗ Total Post-False Complements 0.79∗∗∗ [0.43, 1.15] 0.18 0.46 0.12 0.72∗∗ R2 = 0.667∗∗ 1R2 = 0.120∗∗ A signiﬁcant B-weight indicates the beta-weight and semi-partial correlation are also signiﬁcant. B represents unstandardized regression weights. β indicates the standardized regression weights. sr2 represents the semi-partial correlation squared. r represents the zero-order correlation. LL and UL indicate the lower and upper limits of a conﬁdence interval, respectively. ∗p < 0.05. ∗∗p < 0.01. ∗∗∗p < 0.001. TABLE 3 \| Regression results predicting children’s total post-false belief (N = 60). DISCUSSION Theory of Mind plays a fundamental role in social cognition (Harris, 2006), and an important step of ToM development occurs around the age of 4–5 years, when TD children begin to understand that others have beliefs that diﬀer from their own and may be in conﬂict with reality (Wellman et al., 2001). In contrast, marked delays in consolidating this step in mental reasoning can be observed in children with ASD (Yirmiya et al., 1998), and more subtle delays in children with DLD (Nilsson and de López, 2016). The overarching objective of this work was to identify an eﬃcient way to address ToM diﬃculties in ASD and DLD. y Amidst these clinical populations, the subset succeeding at tasks assessing false beliefs has been shown to display a better level of language, and in particular grammatical skills (ASD: Fisher et al., 2005; Paynter and Peterson, 2010; DLD: Farrar et al., 2009; Andrés-Roqueta et al., 2013). Some authors claim that mastery of ‘complement clauses’ such as ‘X thinks/ says that Y,’ would be the grammatical component par excellence facilitating belief reasoning (de Villiers, 2000, 2007), including in ASD and DLD (Tager-Flusberg and Joseph, 2005; Durrleman et al., 2016a, 2017a), because the content of the embedded sentence may refer to a subjective truth. In light of the fact the privileged links have been found between complements and belief reasoning, not only in TD (de Villiers and Pyers, 2002) but also in clinical populations including ASD and DLD (Farrar et al., 2017), our main objective with this work was to see if complementation training could yield similar gains across these populations. As such, our target program aimed to enhance complementation so as to boost ToM performance not only in TD, which has already been found, but also in ASD and DLD, which has never before been investigated. This training was compared to a control training, which promoted lexical enrichment. Our ﬁndings replicate the results for TD children in other languages to French indicating that syntactic training focussing on sentential complements improves both these structures as well as performance on false belief attribution in this population (Hale and Tager-Flusberg, 2003; Lohmann and Tomasello, 2003; Shuliang et al., 2014). Question 5: Did the Training Result Persist Beyond Immediate Post-test? Table 4 shows the results, and Figure 8 shows the optimum model result, with excellent ﬁt indices. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 10 The Impact of Grammar on Mentalizing Durrleman et al. TABLE 4 \| Standardized parameter estimates for the hypothesized model (N = 60). B SE β p Path Analysis Raven’s Total →Total Post-FB 0.13∗ 0.06 0.16 0.040 Total Pre-False Belief →Total Post-FB 0.45∗∗∗ 0.13 0.27 0.001 Training →Total Post-FB (c) 2.63∗∗∗ 0.80 0.33 0.001 Total Post-False Complements →Total Post-FB (b) 0.80∗∗∗ 0.17 0.48 0.000 Age →Raven’s Total 1.05∗∗ 0.27 0.46 0.000 Age →Total Pre-False Belief 0.28∗ 0.14 0.26 0.038 Training →Total Post-False Complements (a) 3.00∗∗∗ 0.48 0.62 0.000 Indirect Effect a × b 2.38∗∗∗ 0.63 0.30 0.001 Total Effect c 5.01∗∗∗ 0.74 0.63 0.000 Standardized parameter estimation for the hypothesized model. All reported estimates are the maximum likelihood standardized point estimates. □2(7, N = 60) = 8.934, p = 0.257; comparative ﬁt index = 0.982; Tucker-Lewis index = 0.963, root mean square error of approximation = 0.068, standardized root mean square residual = 0.067. ∗p < 0.05. ∗∗p < 0.01. ∗∗∗p < 0.001. TABLE 4 \| Standardized parameter estimates for the hypothesized model (N = 60). DISCUSSION Moreover, children with DLD and ASD, who can display delays in both syntax and ToM (Yirmiya et al., 1998; Nilsson and de López, 2016), did not show any diﬀerence from the TD group regarding As background variables, the Raven’s score and the pre- test False belief score contributed to the ﬁnal outcome, but age did not have a direct eﬀect. Importantly, the training condition had a signiﬁcant eﬀect on both the False Belief outcome, and also on the children’s skill on the False complement post-task. That skill then contributed signiﬁcantly also to False belief. A further analysis asked whether the Training on False complements was instrumental in the outcome on False Belief via two paths: one direct, and the other with ﬁnal performance on False Complements as the mediating variable. The analysis revealed that there was indeed an additional mediating eﬀect of the false complements, as revealed in Table 4. Training has a highly signiﬁcant eﬀect on complements (a), which then has an eﬀect on False Beliefs (b). So the indirect eﬀect is a x b. In addition, Training has a direct eﬀect (c) on false beliefs, also highly signiﬁcant. FIGURE 8 \| Standardized parameter estimation for the hypothesized model. FIGURE 8 \| Standardized parameter estimation for the hypothesized model. 11 November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org The Impact of Grammar on Mentalizing Durrleman et al. these positive outcomes. This ﬁrst attempt at explicit syntactic training with a group including these clinical populations is thus encouraging regarding the potential direct linguistic gains as well as the indirect cognitive beneﬁts, as measured by ToM tasks. argument structure of cognitive verbs, have children who develop false belief skills earlier (Tompkins et al., 2018). Much debate has arisen over whether the causal eﬀect is on the child’s own language, or directly in providing evidence for the theory the child is building about other minds (de Villiers and de Villiers, 2014). The model in this experimental study hints at a role for each eﬀect. The ToM beneﬁts associated with enhanced complementation skills were observed speciﬁcally for false belief reasoning, and not, e.g., for precursors of this ability such as the comprehension of diverse beliefs and desires (Wellman and Liu, 2004) or true belief items which can be resolved via reality responses. The interest of complementation appears indeed to support a speciﬁc component of ToM. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. DISCUSSION It is important to underline that improvement on FB was observed whether measured via verbal or low-verbal tasks. Performance on both of these measures was indeed correlated to complementation skills, suggesting that language supports not only FB-task performance, but also the reasoning implied in belief attribution (de Villiers, 2007). It is also interesting to note that both general grammatical skills and speciﬁc mastery of complementation relate to mentalizing abilities in typical development on post- tests, while atypical development appears to speciﬁcally capitalize on complementation, in line with previous reports (Farrar et al., 2017). This may suggest diﬀerent pathways to FB understanding, potentially related to diﬀerences between these populations to beneﬁt from social interactions (Farrar et al., 2017). Future work on the eﬀects of complementation training on ToM should seek to include larger cohorts of children with DLD and ASD, as well as other populations such as deaf children, who also show diﬃculties with both embedding (Tuller and Delage, 2014) and ToM (Peterson and Siegal, 2000). Subsequent studies should also seek to determine whether or not the gains are limited in scope (e.g., giving rise merely to verbal strategies for solving ToM tasks, see e.g., Leslie and Roth, 1993; Happé, 1995; Tager-Flusberg etal., 1997; Tager- Flusberg, 2000; Senju et al., 2010) or short-term, as these are important concerns, especially for children on the autism spectrum. It would thus be worthwhile to include a dimension of testing involving more ecological tasks, such as hide and seek, as well as parent questionnaires assessing the quality of the children’s social interactions, testing to be also conducted well after the intervention has ceased. This would allow a deeper understanding of whether children’s enhanced grasp of belief reasoning resulting from complementation training can give rise to more successful social skills, including in the long term. Such results could provide clearer arguments in favor of the beneﬁts of syntactic remediation in ToM programs across aetiologies. Encouragingly, improvements in complements and ToM were observed not only during immediate tests but also in follow-up post-tests, revealing that the training eﬀects were still detectible over time as revealed by higher performance on follow-up post-tests compared to pre-tests. FUNDING The training on complements had two eﬀects, one direct and one indirect via the improvement on children’s own complementation skills. That is, some component of the outcome variance was contributed by being exposed to an enhanced and carefully designed verbal input on complementation, and the other, by the child’s skill in producing correct answers to complementation questions. Evidence for both eﬀects is found in the developmental literature on false beliefs. Research has shown that families that engage in much discourse about mental states, which frequently is coupled with complements given the This work was supported by Swiss National Science Foundation (SNSF), grant number 100014_159606. This work was supported by Swiss National Science Foundation (SNSF), grant number 100014_159606. DISCUSSION Still, despite this durability in improvements between pre-tests and post-tests conducted roughly a month after training ceased, there was nevertheless a very modest drop in performance between immediate post- tests and follow-up post-tests, which could indicate that more training would be required for the results to be sustainable. None of the previous studies examining the eﬀects of complementation training in TD children included follow-up post-tests (Hale and Tager-Flusberg, 2003; Lohmann and Tomasello, 2003; Shuliang et al., 2014), thus more work is needed to determine whether or not this is speciﬁc to our cohort which included clinical children. AUTHOR CONTRIBUTIONS SD designed the study in collaboration with HD as well as JD and ET. SD, HD, and ET orchestrated recruiting and training. MB and RY analyzed data, with RY more speciﬁcally building structural equation models and ﬁne-tuning results. SD wrote the manuscript with the input from HD, JD, ET, and MB. While the children of all populations (TD, DLD, and ASD) who received the control training improved their lexical abilities, they did not improve skills on complements or ToM. The increase in performance observed in the target-training group on both complements and FB is therefore not achievable via just any linguistic stimulation, but rather stems from a speciﬁc training on complementation, here administered via DIRE. Frontiers in Psychology \| www.frontiersin.org REFERENCES The language-cognition interface in ASD: complement sentences and false belief reasoning. Res. Autism Spectrum Disord. 21, 109–120. doi: 10.1016/ j.rasd.2015.10.003 Astington, J. W., and Pelletier, J. (2005). “Theory of mind, language, and learning in the early years: developmental origins of school readiness,” in The development of social cognition and communication, eds B. D. Homer, and C. S. Tamis- Lemonda, (Mahwah, NJ: Erlbaum), 205–230. Durrleman, S., Da Costa, J., and Delage, H. (2016b). 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https://openalex.org/W2586611869	https://periodicos.ufba.br/index.php/nit/article/download/17943/pdf_199	Portuguese	null	ANÁLISE DOS SISTEMAS INFORMACIONAIS DE TARJAS ELETRÔNICAS DE PRATELEIRAS	Cadernos de Prospecção	2,016	cc-by	6,734	RESUMO Com a evolução tecnológica estabelecimentos comerciais procuram por inovações em seus serviços. Diante das variedades existentes, entende-las em sua origem e descobrir seu funcionamento é de suma importância para todas as partes interessadas. Muitos já sabem das implicâncias relacionadas com filas excessivas e mal atendimento aos clientes, principalmente em um sistema demasiado caro e ineficaz. Direcionar corretamente os colaboradores, deixando de lado funções básicas como precificação para outras de gerenciamento e atendimento é necessário. Portanto, os sistemas desenvolvidos convergem com tais interesses, minimizando erros humanos e proporcionando efetividade nas lojas de conveniência atuais. Palavras-chave: Prateleira eletrônica. Tag Digital. Prateleiras digitais. Compra automática. Sistema de prateleiras. D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 ANÁLISE DOS SISTEMAS INFORMACIONAIS DE TARJAS ELETRÔNICAS DE PRATELEIRAS Paulo Henrique Rosseto de Melo1; Gilberto José da Cunha2 1 2 FATEC SEBRAE. Rec.:18.09.2016. Ace.:22.09.2016. Rec.:18.09.2016. Ace.:22.09.2016. Autor para correspondência: E-mail paulo_rosseto@hotmail.com Keywords: Self-checkout. Electronic Shelf Label. Electronic Price label. Electronic Price System. Digital Price System. INTRODUÇÃO Durante a Feira APAS 2016, realizada na cidade de São Paulo uma série de palestras foram ministradas para o público que lida com varejo e atacado de diversos setores. Dentre os variados assuntos expostos, um dos que mais chamou a atenção para a contextualização deste trabalho foi o painel de Big Data e Analytics: Os benefícios por traz da análise de dados pelo fato da estrutura apresentada pelo sr. Silvestri e pelo sr. Araújo demonstrar algumas situações pertinentes. Em uma determinada fase, o Sr. Silvestri utiliza como argumentação o porquê de se utilizar Analytics, mostrando os problemas intrínsecos em se coletar dados manuais em diversas lojas diferentes, e em outra demonstra que “o ganho está no detalhe” (SILVESTRI, APAS 2016) – Ou seja, médias atrapalham na tomada de decisão, portanto a precisão dos dados é de fundamental importância. Outra estratégia da gestão é a de otimização da promoção para clientes de diferentes perfis, mas agrupados em dois diferentes grupos: Foco em qualidade versus foco em preço (ARAÚJO, APAS 2016). Nesta mesma apresentação, Araújo afirma que 60% das promoções “não funcionam”. Além destas constatações, pode-se perceber que existem diversas inovações surgindo para o setor a algum tempo. Por exemplo, pode-se pesquisar e encontrar facilmente na internet por carrinhos inteligentes criados por diversos inventores, sistemas de checkout automáticos, automações em diversos níveis operacionais etc. Tais invenções podem resolver problemas gerenciais ou operacionais, no entanto, elas nem sempre são totalmente aplicáveis já que externalizam outros problemas intrínsecos do limite delas mesmas – Um carrinho inteligente, independente da forma como será utilizado, é somente um elo da cadeia operacional e, da mesma forma que as outras invenções, não poderá fornecer todas as informações necessárias para a tomada de decisão de controle de estoque, ou a sistematização do marketing para a promoção de produtos. Diante de tal cenário, onde existem diversas aplicações e serviços sendo ofertados para o setor, a dificuldade para se conseguir informação segura para a tomada de decisão em diversas atividades gerenciais aumenta da mesma forma que a dificuldade para inovar nos processos. E, visando pacificar este universo, o presente trabalho utiliza a prospecção tecnológica como forma de compreender a raiz do pensamento que desenvolve tecnologia para o setor. Por consequência, no início deste estudo, propôs a hipótese de que muitos dos problemas vividos pelas lojas de conveniência, supermercados, atacadistas etc., já são conhecidos de longa data. ELECTRONIC SHELF LABEL’S SYSTEMS ANALYSIS ABSTRACT Stores in general are evolving and they need to solve some problems related to services through innovation. In front of many varieties understanding such technologies through their sources are of great importance. Many knows the inconveniences brought by bad customer services or long lines, thus directing people to the right needs of a business establishment seems more important than price changes. Therefore, the systems cited in this work can help change this scenario, converging costs and equipment minimizing human mistakes through technology. Área Tecnológica: * Autor para correspondência: E-mail paulo_rosseto@hotmail.com 466 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 1 OBJETIVOS PRIMÁRIOS  Prospectar tecnologicamente os sistemas informacionais que determinam o funcionamento geral deste, através de seus documentos de patente;  Prospectar tecnologicamente os sistemas informacionais que determinam o funcionamento geral deste, através de seus documentos de patente;  Conduzir análise das 4 primeiras empresas que possuem maior número de documentos de patentes envolvidos no tema.  Conduzir análise das 4 primeiras empresas que possuem maior número de documentos de patentes envolvidos no tema. 2 OBJETIVOS SECUNDÁRIOS  Identificar a evolução temporal deste produto;  Analisar outras patentes envolvidas de forma a complementar este sistema. PROCESSOS METODOLÓGICOS INTRODUÇÃO Precisamente um deles é muito comum, qual seja, a troca de preços dos itens em prateleiras. Este problema operacional, conduziu à descoberta de uma invenção chamada Electronic Shelf Label, ou Tarja Eletrônica de Prateleira – Normalmente acoplada na prateleira logo abaixo do produto ao qual ela deve disponibilizar o preço possuindo a possibilidade de reprogramação da forma como o gerente da loja desejar. Na figura abaixo é possível observar como este tipo de invento se desenvolve durante o tempo. ELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 467 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx z.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Figura 1 – Evolução histórica das Electronic Shelf Labels Fonte: Próprio autor. Figura 1 – Evolução histórica das Electronic Shelf Labels Fonte: Próprio autor. Fonte: Próprio autor. Pela Figura 1 é possível perceber que esta invenção não pode ser somente uma forma de trocar preços dada a quantidade de documentos envolvendo a invenção. Observou-se neste ponto que isto não é a invenção de um dispositivo, mas sim de um sistema informacional, com periféricos, software, transmissão de dados e informações, operacional e gerenciável por diversas pessoas que integra todo o sistema desde lojas pequenas de diversos setores até supermercados e rede de supermercados. Conduz-se por esta descoberta, portanto, os objetivos deste trabalho. PROCESSOS METODOLÓGICOS Os processos metodológicos foram compostos pelos seguintes passos: MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 468 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 1. Definição do escopo da busca: Sistemas de automação para comércios em geral, utilizando prateleiras com dispositivos eletrônicos, que, minimamente deveriam mostrar informações do produto através de dispositivo(s) digitais e auxiliar no gerenciamento e operação destes comércios. 1. Definição do escopo da busca: Sistemas de automação para comércios em geral, utilizando prateleiras com dispositivos eletrônicos, que, minimamente deveriam mostrar informações do produto através de dispositivo(s) digitais e auxiliar no gerenciamento e operação destes comércios. 2. Identificação das palavras-chave para a realização da pesquisa preliminar, utilizando-se do Google Patents e Questel Orbit. 2. Identificação das palavras-chave para a realização da pesquisa preliminar, utilizando-se do Google Patents e Questel Orbit. 3. Refinamento da busca: 3. Refinamento da busca: 3.1. Identificação dos principais termos-chave. 3.2. Identificação das classes. 4. Análise e classificação dos resultados por efeito técnico. 4.1. Analisaram-se as famílias de patentes principais, ou seja, que versam sobre o funcionamento do S.I; 4.1. Analisaram-se as famílias de patentes principais, ou seja, que versam sobre o funcionamento do S.I; 4.2. Classificaram--se por ordem de efeito técnico, subjetivo a cada sistema de cada empresa, de modo a proporcionar uma linha de raciocínio única. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 3.1 PESQUISA PRELIMINAR, ESCOPO E ROTA DE BUSCA Após a definição do escopo da busca, qual seja, prateleiras com dispositivos eletrônicos, que, minimamente deveriam mostrar detalhes do produto através de aparatos digitais, procuramos por palavras-chave para a realização da pesquisa preliminar dentro das plataformas Questel Orbit e Google Patents (G.P.) e a composição destas pode ser verificada através da Tabela 1 abaixo: Tabela 1 – Termos Chave Preliminares. Chave 1 Chave 2 Chave 3 Classes Shelf Shelv+ Digital Electronic Display Label G06F (Processamentos elétricos de dados digitais) G06Q (Sistemas de processamento de dados) G06K (Identificação/Apresentação/Suporte dos dados) A47F (Móveis ou guarnições para lojas) Termos-Chave Encontrados (Ele?tronic) w (Shelf or Price) w (Label+ or Tag); Shelf Tag; Price Tag; Fonte: A partir desta tabela, algumas ferramentas dentro do software Questel Orbit foram utilizadas para a definição de várias sintaxes lógicas que, no final do processo, resultam no roteiro exposto na Tabela 2: A partir desta tabela, algumas ferramentas dentro do software Questel Orbit foram utilizadas para a definição de várias sintaxes lógicas que, no final do processo, resultam no roteiro exposto na Tabela MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 469 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 Tabela 2 – Rota de Busca. 3.1 PESQUISA PRELIMINAR, ESCOPO E ROTA DE BUSCA Passo Resultados Sintaxe lógica 1 606 ( (ELECTRONIC PRICE LABEL OR ELECTRONIC PRICE LABEL SOFTWARE OR ELECTRONIC PRICE LABEL DATA OR MERCHANDISE OR ITEM PRICE OR ENTRY ITEM ID OR ITEM IDENTIFICATION ENTRY)/KEYW/TI/AB/IW AND (ELECTRONIC SHELF LABEL OR ELECTRONIC LABEL)/KEYW/TI/AB/IW ) 2 475 SS 1 AND (STATE/ACT=DEAD NOT STATE/ACT=ALIVE) 3 41 SS 2 AND (STATUS/ACT=REVOKED NOT (STATUS/ACT=PENDING OR STATUS/ACT=GRANTED)) 4 606 1 NOT 3 5 606 ( (ELECTRONIC PRICE LABEL OR ELECTRONIC PRICE LABEL SOFTWARE OR ELECTRONIC PRICE LABEL DATA OR MERCHANDISE OR ITEM PRICE OR ENTRY ITEM ID OR ITEM IDENTIFICATION ENTRY)/TI/AB/IW/KEYW AND (ELECTRONIC SHELF LABEL OR ELECTRONIC LABEL)/TI/AB/IW/KEYW ) 6 94 (( (SHELF OR SHELV+)/TI/AB/IW/DESC/ODES D (DISPLAY OR LABEL )/TI/AB/IW/DESC/ODES D (DIGITAL OR ELECTRONIC)/TI/AB/IW/DESC/ODES ) AND (A47F+)/IPC) NOT (G07G+ OR B65G+ OR F25D+ OR A47B+)/IPC 7 366 (( (SHELF OR SHELV+)/TI/AB/IW/DESC/ODES D (DISPLAY OR LABEL )/TI/AB/IW/DESC/ODES D (DIGITAL OR ELECTRONIC)/TI/AB/IW/DESC/ODES ) NOT (A47F+ OR G06Q+ OR G06F+)/IPC) 8 1001 1 OR 6 OR 7 9 1001 8 NOT 3 10 32 SS 9 AND (STATE/ACT=DEAD NOT STATE/ACT=ALIVE) 11 3 SS 10 AND (STATUS/ACT=REVOKED NOT (STATUS/ACT=PENDING OR STATUS/ACT=GRANTED)) 12 1001 9 NOT 11 13 1274 (ELECTRONIC SHELF LABEL OR ELECTRONIC PRICE OR ELECTRONIC SHELF LABELS OR ELECTRONIC PRICE LABEL)/KEYW/TI/AB/IW 14 1822 13 OR 12 15 543 SS 14 AND (STATE/ACT=DEAD NOT STATE/ACT=ALIVE) 16 89 SS 15 AND (STATUS/ACT=REVOKED NOT (STATUS/ACT=PENDING OR STATUS/ACT=GRANTED)) 17 1733 14 NOT 16 Fonte: Estes dados estão atualizados na data de18/06/2015 e passaram para a fase de refinamento na mesma data. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras 3.2 REFINAMENTO DA PESQUISA Os passos 2, 3, 4, 9, 10, 11, 12, 15, 16 e 17 da Tabela 2 foram feitos com o intuito de retirar patentes indeferidas e evitar possíveis problemas que o Questel Orbit pode ter durante a pesquisa. Foi constatado também, que classificações sugeridas na Tabela 1 não poderiam ser utilizadas pelo fato da finalidade das patentes não ter relação direta com a descrição da classe, ou seja, as invenções utilizam da tecnologia exposta pela classe, que passa a ser um meio e não um fim para MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 470 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 realizar seus objetivos. Após este processo, os resultados foram agrupados para os titulares apresentados na Figura 2. realizar seus objetivos. Após este processo, os resultados foram agrupados para os titulares apresentados na Figura 2. Figura 2 – Resultados da busca agrupados por Titular. Fonte: Próprio autor. Destes titulares, os quatro primeiros com maior número de patentes foram analisados por possuírem depósitos bem colocados durante os anos, possibilitando a evolução deste sistema. Figura 2 – Resultados da busca agrupados por Titular. Figura 2 – Resultados da busca agrupados por Titular. Fonte: Próprio autor. Destes titulares, os quatro primeiros com maior número de patentes foram analisados por possuírem Fonte: Próprio autor. Destes titulares, os quatro primeiros com maior número de patentes foram analisados por possuírem depósitos bem colocados durante os anos, possibilitando a evolução deste sistema. Destes titulares, os quatro primeiros com maior número de patentes foram analisados por possuírem depósitos bem colocados durante os anos, possibilitando a evolução deste sistema. Figura 3 – Evolução temporal dos resultados agrupados por titular. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 471 Figura 3 – Evolução temporal dos resultados agrupados por titular. Fonte: Próprio autor. Figura 3 Evolução temporal dos resultados agrupados por titular. Fonte: Próprio autor. ELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 471 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 Estando agrupados os conjuntos de pesquisa, cada empresa foi analisada segundo o conteúdo destes documentos e seus resultados discutidos adiante. RESULTADOS E DISCUSSÃO 4.1 Sistema Informacional NCR Figura 4 – Sistema NCR analisado. Figura 4 – Sistema NCR analisado. Fonte: Us 6,496,121 B2. Fonte: Us 6,496,121 B2. Fonte: Us 6,496,121 B2. ELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 3.2 REFINAMENTO DA PESQUISA Pela especificação disposta por este sistema, é preferível que a ESL module os sinais recebidos, ou seja, em uma parte reservada do display, ou todo o display, outro sinal é transmitido através do piscar de luz, em uma velocidade que o olho humano não pode perceber (30 ciclos por segundo ou mais), transmitindo uma mensagem de resposta para o relay. Em suma, o relay (120) não precisa estar dentro do raio de visão da ESL já que a luz pode ser refletida pelas superfícies ou pode se usar uma câmera. comunicar, pode haver a necessidade de separação deste dispositivo em outras partes. As possíveis formas de resposta incluem modulação e reflexão dos sinais recebidos pela ESL. Pela especificação disposta por este sistema, é preferível que a ESL module os sinais recebidos, ou seja, em uma parte reservada do display, ou todo o display, outro sinal é transmitido através do piscar de luz, em uma velocidade que o olho humano não pode perceber (30 ciclos por segundo ou mais), transmitindo uma mensagem de resposta para o relay. Em suma, o relay (120) não precisa estar dentro do raio de visão da ESL já que a luz pode ser refletida pelas superfícies ou pode se usar uma câmera. A tarja (122) é constituída por um circuito, uma bateria, uma antena de recebimento/transmissão de informações, uma unidade de memória (Não se especifica qual), um display (Preferencialmente LCD) e um conector. Através deste conector pode-se inserir um display adicional, cujas formas de acoplagem são variadas, mas que possibilitam a troca de informações e possui uma bateria, um circuito auxiliar e duas formas de display. Este aparato todo é desenvolvido para uma melhor visualização do preço, principalmente em promoções, onde eles são mostrados de várias maneiras: de 2,49 por 1,99, por exemplo. Desta forma, pode-se promover melhor os produtos vendidos cuja visualização dos preços não era tão otimizada se usada somente com o display do EPL. Tabela 3 – Reclassificação setorial Área Número de Patentes Descrição Comunicação 18 Métodos e aparatos que possibilitam as mensagens serem repassadas entre os dispositivos do sistema. Aparato de Tarja 24 Componentes e funções contidas nas tarjas, bem métodos de produção das mesmas. Métodos Técnicos 88 São procedimentos que primariamente resolvem problemas técnicos inerentes à tecnologia utilizada. Serviços 24 São os métodos que criados para determinado serviço, por exemplo sistemas de compras personalizados. 3.2 REFINAMENTO DA PESQUISA A patente analisada (Otto, 2002) mostra um sistema (100) possuinte de um servidor (102), que pode estar em conjunto com o sistema de vendas (104) da loja, com o banco de dados contendo as informações necessárias sobre cada produto (PLU, ou Price Look up (107)), tal banco de dados está inserido em outro arquivo (109) contendo as identificações de cada ESL e a respectiva correspondência com cada produto, ambos localizados em uma unidade de armazenamento. Um software para a execução e coordenação de todo o sistema está contido nessa mesma unidade, da mesma forma, existe outra unidade de entrada, podendo se inserir teclado, mouse etc. O sistema de vendas (104) é constituído por um dispositivo de leitura de código de barras (118) e um terminal dedicado (116), que pode estar inserido dentro do servidor (102) ou conectado externamente por vários métodos de comunicação. O software (108) controla todas as mensagens (124) enviadas para as ESLs através das unidades de relay (120) que servem como uma ponte comunicativa entre servidor e ESL. Esses relays (120) são distribuídos periodicamente através do piso de produtos da loja. Esta comunicação, entre relay e servidor (124), e relay e ESL (126, Ida e 127, volta) pode ser uma combinação de vários tipos, sem fio e IR (Infra Red, ou infravermelho), ou até mesmo por fio. Após o recebimento da informação, a ESL (122) envia um sinal de confirmação. O relay (120) é composto de uma unidade de comunicação direta com o servidor principal (102), uma unidade que gerencia as mensagens enviadas às ESLs, uma unidade que gera sinais IR e uma quantidade variável de receptores IR ( Os modelos de circuitos, tanto dos relays quanto das ESLs encontram-se na mesma patente). Uma das formas de comunicação, prevê que o relay dispare sinais IR esperando que cada ESL responda da mesma forma, consequentemente um receptor é endereçado para cada ESL. Portanto, dependendo da quantidade de ESL que cada relay deve se MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 472 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 comunicar, pode haver a necessidade de separação deste dispositivo em outras partes. As possíveis formas de resposta incluem modulação e reflexão dos sinais recebidos pela ESL. 3.2 REFINAMENTO DA PESQUISA Sistemas Informacionais 12 São os sistemas que organizam os dispositivos ou reorganizam serviços e métodos técnicos. Fonte: Próprio autor 4.2 Sistema Informacional Ishida Seisakusho 4.2 Sistema Informacional Ishida Seisakusho 4.2 Sistema Informacional Ishida Seisakusho Figura 5 – Sistema Ishida. Figura 5 – Sistema Ishida. Fonte: US 2010/0225444 A1. Figura 5 – Sistema Ishida. Fonte: US 2010/0225444 A1. Fonte: US 2010/0225444 A1. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 473 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 De acordo com este documento, 9 aspectos gerais compõe o modelo de utilidade, quais sejam: De acordo com este documento, 9 aspectos gerais compõe o modelo de utilidade, quais sejam: 1. Descrever o Sistema; 2. Dispositivo portátil para a criação e destruição de ligações entre tarjas e produtos (Handy Terminal 7); y 3. Um método de detecção de carga remanescente da bateria; 4. Sistema onde o display não necessita de eletricidade direta para a contínua amostragem das informações de um produto; 5. Um método para a contagem de mudanças ocorridas em um display; 6. Um dispositivo capaz de transmitir mensagens para as tarjas, onde tal dispositivo determina a ordem em que as mensagens devem ser amostradas pelo display (Remote controller 43); 7. A tarja utilizada pelo sistema em seu meio comunicativo; 8. A tarja e seus componentes necessários para cumprir com todos os processos necessários para este sistema; p 9. Método para a criação e destruição de hiperligações entre tarjas e produtos. A tabela abaixo consegue resumir os processos gerenciais básicos oferecidos por este sistema, que visa manter o controle dos preços e o gerenciamento deles através de controles e dispositivos de mão. A tabela abaixo consegue resumir os processos gerenciais básicos oferecidos por este sistema, que visa manter o controle dos preços e o gerenciamento deles através de controles e dispositivos de mão. Tabela 4 – Dispositivos do Sistema Ishida. Store Controller Computador típico Gerencia POS server e ESL server POS Computador Típico Product Master com Informação do Produto Gerencia Caixas Registradoras ESL Server Computador Típico ROM Disco Rígido Unidade de Comunicação Interface RAM, CPU, Inputs, Display Device Programa de rotinas básicas Programas das funções ESL Data file idêntico ao product master Links entre tarja e produtos. Comunica-se com POS, Store controller e outros dispositivos Comunica-se com Base station. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. Fonte: US 2011/0186633 A1. Fonte: US 2011/0186633 A1. Tal servidor (101) compreende todos os sistemas de gerenciamento necessários para um estabelecimento comercial (Inventário, Estoque, Preços, POS etc.), e transmite as informações relevantes em um pacote, chamado Master Data, diretamente para o controle de comunicações (102). Tal servidor (101) compreende todos os sistemas de gerenciamento necessários para um estabelecimento comercial (Inventário, Estoque, Preços, POS etc.), e transmite as informações relevantes em um pacote, chamado Master Data, diretamente para o controle de comunicações (102). Por sua vez, o controle de comunicações (102), executa um programa de sua ROM e utiliza a RAM como espaço para computação das informações recebidas e transmitidas (Master Data), alocadas na unidade de armazenamento. Da mesma forma, este dispositivo recebe informações de identificação dos terminais de mão (106) e das ESLs (105) para estabelecer correspondência entre esses dispositivos, necessárias durante alguns processos. Uma unidade de entrada pode ser utilizada para modificações necessárias dentro do Master Data e uma unidade de comunicações conecta-se com o servidor principal para recebimento de informações deste pacote, bem como suas atualizações. Continuando o sistema, todas as comunicações realizadas com os dispositivos seguintes da hierarquia são através da estação de relays (103). O Pacote de informações Master Data, possui toda a identificação necessária do produto, ao mesmo tempo que contém as informações necessárias para a rota do sinal, ou seja, o dispositivo de comunicação (102) sabe para quais relays e, consequentemente, para quais ESLs (104) cada informação relevante a cada produto deve ir. Cada relay (103) possui, como a unidade de controle de comunicação (102), uma ROM, contendo um programa, executado pela unidade de controle, que utiliza a RAM como espaço de trabalho e uma unidade de armazenamento com uma tabela de identificações das ESLs (104) subordinadas e seu esquema de comunicações. O programa executado pela unidade de controle estabelece comunicações com o dispositivo de comunicação, os monitores (106) e os terminais de mãos (105) e as ESLs (104), através das unidades de comunicação contidas em seu hardware. Pelo menos duas unidades de comunicação estão acopladas, a primeira comunica-se diretamente com o dispositivo de controle, os monitores (106) e os terminais de mão (105), enquanto a segunda é direcionada para as ESLs (104), ambas podem utilizar tecnologias como WLAN, ou IR, ou Zigbee etc. ELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 4.2 Sistema Informacional Ishida Seisakusho Base Station Recebe Informações das tarjas e Envia Informações Transceptores agindo como relays Tarjas Unidade de Controle Unidade de Memória Unidade de Comunicação Display Distingue e gerencia as informações recebidas Não volátil acoplada ao circuito integrado de controle Recebe e envia dados do ESL Server e do Remote Controller ITO (Não-volátil), Dot matrix bidimensional ortogonal (possui tarjas que não necessitam de carga contínua) Handy terminal Cria e destrói Hyperligações entre produtos e tarjas Unidade de comunicação por radiofrequência com o servidor ESL. Remote Controller Controla a forma de exibição das mensagens pelas ESL através de comunicação por raios infravermelhos. Fonte: Próprio autor Tabela 4 – Dispositivos do Sistema Ishida. 4.3 Sistema Seiko Instruments MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 474 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Figura 6 – Sistema Seiko. Fonte: US 2011/0186633 A1. Figura 6 – Sistema Seiko. F t US 2011/0186633 A1 Figura 6 – Sistema Seiko. Figura 6 – Sistema Seiko. Figura 6 – Sistema Seiko. Fonte: US 2011/0186633 A1. Tal servidor (101) compreende todos os sistemas de gerenciamento necessários para um estabelecimento comercial (Inventário, Estoque, Preços, POS etc.), e transmite as informações relevantes em um pacote, chamado Master Data, diretamente para o controle de comunicações (102). Por sua vez, o controle de comunicações (102), executa um programa de sua ROM e utiliza a RAM t ã d i f õ bid t itid (M t D t ) l d Fonte: US 2011/0186633 A1. Também possuindo o mesmo método computacional do relay (103) e do controle de comunicação (102), o terminal de mão (105) possui um ROM, uma RAM, uma unidade de controle e outra de armazenamento. As informações armazenadas na unidade de armazenamento são providenciadas pela unidade de controle de comunicação (102). Também possui duas unidades de comunicação, uma diretamente com o relay (103) e outra com as ESLs (104), ambas podendo utilizar tecnologias da mesma forma que os relays – WLAN, IR etc. Tal unidade é utilizada na modificação/transmissão 475 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 de preços e quantidade de estoque, por exemplo, bem como estabelecer vínculos entre ESLs e Relays. Uma unidade de leitura observa o código contido dentro do IC ou no código de barras de cada ESL e uma unidade de impressão pode ser usada somente quando o produto possui descontos, imprimindo tarjas adesivas. de preços e quantidade de estoque, por exemplo, bem como estabelecer vínculos entre ESLs e Relays. Uma unidade de leitura observa o código contido dentro do IC ou no código de barras de cada ESL e uma unidade de impressão pode ser usada somente quando o produto possui descontos, imprimindo tarjas adesivas. Por fim, o servidor (101) deve ser um computador típico, formado por hardware, S.O. e periféricos, com a possibilidade de ser instalado um programa gerenciador com as rotinas necessárias para o funcionamento correto do próprio servidor, podendo se inserir rotinas em caso de sistemas de gerenciamento que não estejam interligados. Nota-se também, que essa empresa possui patentes relacionadas a sincronia de dados por causa das variadas formas de precificação existentes, da mesma forma que nos documentos posteriores pode- se constatar que cada ESL pode conter dados gráficos muito mais evoluídos que os da Ishida ou da NCR, sendo necessário a criação de outros documentos mostrando como estes dados devem ser transmitidos, codificados etc. Em consequência às situações expostas acima, a tabela abaixo mostra os dispositivos e seus principais componentes, em conjunto com uma breve descrição de sua principal função. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 476 Tabela 4 – Dispositivos do sistema Seiko Instruments. Fonte: US 2011/0186633 A1. Neste sistema de tarjas (51) utiliza-se a comunicação híbrida, ou seja, do servidor de gerenciamento (10), que envia ou recebe a informação da unidade de interface (20), que, por sua vez, envia ou recebe a informação do gateway (41), ela toda é transportada por fios. A partir deste ponto, a comunicação é realizada sem fio de forma bilateral e utilizando Zigbee, atingindo, por fim, o nível das tarjas. S d d t US20110240731 A1 l t bté f õ bá i d d Figura 6 – Sistema Samsung. Fonte: US20110240731 A1. Neste sistema de tarjas (51) utiliza-se a comunicação híbrida, ou seja, do servidor de gerenciamento (10), que envia ou recebe a informação da unidade de interface (20), que, por sua vez, envia ou recebe a informação do gateway (41), ela toda é transportada por fios. A partir deste ponto, a comunicação é realizada sem fio de forma bilateral e utilizando Zigbee, atingindo, por fim, o nível das tarjas. Neste sistema de tarjas (51) utiliza-se a comunicação híbrida, ou seja, do servidor de gerenciamento (10), que envia ou recebe a informação da unidade de interface (20), que, por sua vez, envia ou recebe a informação do gateway (41), ela toda é transportada por fios. A partir deste ponto, a comunicação é realizada sem fio de forma bilateral e utilizando Zigbee, atingindo, por fim, o nível das tarjas. Segundo o documento US20110240731 A1, claramente obtém-se as funções básicas de cada dispositivo dentro deste sistema, descrevendo, da forma mais abstrata possível, os processos contidos nele. O servidor de gerenciamento (10) funciona como um banco de dados, com identificação das tarjas e dos produtos. Da mesma forma, possui variadas informações para que se cumpra com as operações de gerenciamento de um estabelecimento e, também escreve e envia mensagens de comando para funções como sincronização, atualização e alteração de informações de produtos mostradas nas tarjas (5). Outro ponto importante deste dispositivo é que ele possui ambos os bancos de dados das informações dos produtos e da identificação das tarjas em vínculo mútuo, criando-os e destruindo- os conforme o desejo do usuário. Já a unidade de interface (20), serve para formar um canal de comunicação do servidor com os gateways (40), de forma que as mensagens criadas pelo servidor (10) são enviadas em tempo real para os gateways (40). Fonte: US 2011/0186633 A1. ESL (101) Computador típico Pode ser o POS do estabelecimento Controle de Comunicação (102) Unidade de controle RAM Unidade de armazenamento Rom - Software Unidade de comunicação Display Inputs Processa os dados Atualiza preços e product master do servidor 101. Cópia de Product Master, Determina e analisa solicitações de troca de preços. Comunica-se com terminal de mão, relays (ESL e Displays) e servidor. Periféricos, Mostra dados. Estação de Relay (103) Unidade de Controle RAM Unidade de Armazenamento ROM Unidade de Comunicação 1 e 2 Processa o programa contido na ROM para escolher qual unidade de comunicação usar. Armazena o esquema de comunicação com as tarjas (104) e um software na ROM Dependendo da forma de comunicação uma unidade é responsável pelas comunicações entre Display (106), terminal de mão (105) e controle de comunicação (102). Enquanto a outra se comunica com as ESL (104). Tarja (104) Unidade de Controle Unidade de Armazenamento Bateria Unidade de Recebimento de informações Display Faz o display mostrar as informações contidas na unidade de armazenamento Armazena informações de Produtos Proporciona carga para os componentes Recebe as informações Mostra as informações Feito de LCD Ou Cristal líquido de memória Terminal de Mão (105) Unidade de Controle RAM Input Unidade de Armazenamento Rom Unidade de Recebimento de informações 1 e 2 Display Unidade de Leitura Executa (RAM) um software instalado na ROM com a finalidade de controlar as unidades de comunicação Insere dados, como quantidade de estoque Armazena informações de acordo com a finalidade Programa básico (Firmware) Uma delas se comunica com a estação de relay (103), a outra com as tarjas (104). A primeira através de WLAN a outra por infravermelho, por exemplo. Mostra as informações Lê o código de barras das tarjas (104) ou do IC chip. Fonte: Próprio autor. Tabela 4 – Dispositivos do sistema Seiko Instruments. ELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. 476 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 4.4 Sistema ESL Samsung Figura 6 – Sistema Samsung. Fonte: US20110240731 A1. 4.4 Sistema ESL Samsung 4.4 Sistema ESL Samsung 4.4 Sistema ESL Samsung Figura 6 – Sistema Samsung. Fonte: US20110240731 A1. MELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. Fonte: US 2011/0186633 A1. A unidade de interface (20) recebe as mensagens do servidor (10), as configura em pacotes pré-estabelecidos para a forma de comunicação dos Gateways (40), envia estes pacotes e recebe, por consequência, uma mensagem dos Gateways (40) de confirmação do recebimento deste pacote, que também é reconfigurado e transferido para o servidor (10), habilitando os processos subsequentes. Neste processo pode-se usar conexões com fio ou sem fio. O grupo gateway (40), pode ser formado por vários gateways (41), e sua função é reconfigurar as mensagens recebidas do servidor (10) em outro tipo de pacote de forma a serem enviadas para as tarjas (50), localizadas nas prateleiras que contêm os produtos. Da mesma forma recebe uma mensagem de resposta de reconhecimento é enviada da tarja (50) para o servidor. Esta comunicação 477 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 é realizada utilizando a forma Zigbee de comunicação sem fio, habilitando um gateway (40) para se comunicar com várias tarjas. é realizada utilizando a forma Zigbee de comunicação sem fio, habilitando um gateway (40) para se comunicar com várias tarjas. A tarja (51), arranjada no setor de tarjas (50), opera em dois canais de comunicação um de Wake-up e outro de transferência de dados dos produtos. A maior parte do tempo, apesar de sua bateria acoplada durar por anos, a tarja opera em esquemas de comunicação Wake-up, ou seja, ela possui ciclos de operação. Quando a tarja (51) recebe este sinal Wake-up ela passa confirmar a necessidade de receber informações dos produtos pelo canal de comunicação e enviar mensagens de reconhecimento em resposta, para posteriormente retornar ao estado desligado. Somente quando existe dados a serem recebidos é que a tarja passa a funcionar com integralmente. O equipamento de PDA (30), assistente pessoal digital, serve para o gerente da loja se comunicar diretamente com o servidor criando operações de sincronização entre produto e tarja, da mesma forma que pode destrui-las. Somando a este sistema principal, existe a convergência com outro sistema de compras baseado em mobile e integra todo o processo de aquisição de produtos dentro da loja, eliminando a necessidade de caixas registradoras. Tabela 5 – Dispositivos do sistema Samsung. Fonte: US 2011/0186633 A1. ESL (10) Computador típico Unidade Interface (20) Serve como um gerenciador e configurador de mensagens a serem enviadas tanto para os gateways (40), quanto para o servidor (10) Gateway (41) Adapta e sincroniza as mensagens transmitidas/recebidas das tarjas (51); Possibilita a comunicação com aparelhos mobile, habilitando sistema de compras e outras funções; Tarja (51) Possui capacidade de armazenar informações e mostrar informações através de sua unidade de memória e seu display; Transmite e recebe informações com o servidor através do Gateway (41) após sincronia; Possui unidade de reconhecimento que transmite informações do produto para aparelhos mobile; Possui versões com função simplificada (Somente para transmissão de informações entre tarja e gateway) e completa; PDA (30) Possibilita comunicação com o servidor para a criação ou destruição de sincronia entre produtos e tarjas; Possibilita a atualização de informação de produtos. Sistemas Convergência de sistemas diferentes para somar serviços ao cliente. Fonte: Próprio autor. Tabela 5 – Dispositivos do sistema Samsung. ELO, P.H.R.; CUNHA, G.J.C.. Análise dos sistemas informacionais de tarjas eletrônicas de prateleiras. CONCLUSÃO Com a evolução da tecnologia e da forma de pensar como um estabelecimento comercial deve funcionar, estes sistemas foram moldados para resolver a maior parte das questões operacionais e gerenciais de lojas e supermercados. Com isso, os problemas levantados na introdução deste trabalho se sanariam, já que a coleta de informações necessárias para a tomada de decisão não dependeria mais de processos manuais; Decisões em tempo real podem ser tomadas levando em conta a inteligência que se pode gerar através da automatização destes processos. Outro ponto importante, é o estabelecimento de uma linha de gerenciamento da cadeia de valor onde a análise de tecnologias antes de elas serem inseridas no mercado propicia a simulação de possíveis transformações a serem aplicadas pelos gestores de estabelecimentos – Fato que resolve 478 Cad. Prospec., Salvador, v. 9, n. 4, p.466-480, out./dez.2016 D.O.I.: dx.doi.org/10.9771/S.CPROSP.2016.009.046 outro problema: A possível inserção de tecnologias periféricas desenvolvidas por outras empresas ou pessoas. O constante monitoramento das invenções protegidas em patente permite uma miríade de possibilidades de negócios, que podem ser pretendidos segundo as estratégias das empresas. outro problema: A possível inserção de tecnologias periféricas desenvolvidas por outras empresas ou pessoas. O constante monitoramento das invenções protegidas em patente permite uma miríade de possibilidades de negócios, que podem ser pretendidos segundo as estratégias das empresas. É possível afirmar também que outra dor que a prospecção tecnológica se propõe a solucionar se faz plena, qual seja, a de antecipar fatores comerciais. Vídeos como o supermercado do futuro, facilmente encontrados no youtube, já foram pensados e protegidos há muito tempo – Balanças inteligentes e prateleiras inteligentes são invenções que possuem seu processo patenteado pela NCR, por exemplo. Da mesma forma, após todas as análises e exposições dos sistemas, pode-se inferir sobre um futuro próximo de como estes estabelecimentos poderão ser reorganizados afim de satisfazer cada vez mais as inconstantes necessidades dos clientes. REFERÊNCIAS SILVESTRI, Fábio. Desafio de Crescimento e Rentabilidade. O uso prático de ANALYTICS no varejo. FEIRA APAS 2016, São Paulo, 32 p., palestra ocorrida em 03/05/2016. < http://feiraapas.com.br/palestra/>, conteúdo acessado em 10/09/2016. ARAÚJO, Adriano. Big Data no Varejo. FEIRA APAS 2016. 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https://openalex.org/W3217195595	https://dirros.openscience.si/Dokument.php?lang=slv&id=18736&dn=	English	null	Stand Up to Excite the Spine: Neuromuscular, Autonomic, and Cardiometabolic Responses During Motor Imagery in Standing vs. Sitting Posture	Frontiers in physiology	2,021	cc-by	17,289	ORIGINAL RESEARCH published: 23 November 2021 doi: 10.3389/fphys.2021.762452 ORIGINAL RESEARCH published: 23 November 2021 doi: 10.3389/fphys.2021.762452 Stand Up to Excite the Spine: Neuromuscular, Autonomic, and Cardiometabolic Responses During Motor Imagery in Standing vs. Sitting Posture Sidney Grosprêtre1, Uros Marusic2,3, Philippe Gimenez1, Gael Ennequin4, Laurent Mourot5,6 and Laurie Isacco4,5 Sidney Grosprêtre1, Uros Marusic2,3, Philippe Gimenez1, Gael Ennequin4, Laurent Mourot5,6 and Laurie Isacco4,5 1 EA4660-C3S Laboratory - Culture, Sports, Health and Society, University Bourgogne Franche-Comté, Besançon, France, 2 Institute for Kinesiology Research, Science and Research Centre of Koper, Koper, Slovenia, 3 Department of Health Sciences, Alma Mater Europaea–ECM, Maribor, Slovenia, 4 Université Clermont Auvergne, CRNH, AME2P, Clermont-Ferrand, France, 5 EA3920-Prognostic Markers and Regulatory Factors of Heart and Vascular Diseases, and Exercise Performance, Health, Innovation Platform, University Bourgogne Franche-Comté, Besançon, France, 6 National Research Tomsk Polytechnic University, Tomsk, Russia Grosprêtre S, Marusic U, Gimenez P, Ennequin G, Mourot L and Isacco L (2021) Stand Up to Excite the Spine: Neuromuscular, Autonomic, and Cardiometabolic Responses During Motor Imagery in Standing vs. Sitting Posture. Front. Physiol. 12:762452. doi: 10.3389/fphys.2021.762452 Keywords: heart rate, ˙VO2, Center of Pressure (COP), H-reﬂex, electromyography Edited by: Alberto Porta, University of Milan, Italy Reviewed by: Paolo Castiglioni, Fondazione Don Carlo Gnocchi Onlus, Scientiﬁc Institute for Research, Hospitalization and Healthcare (IRCCS), Italy Antonio Roberto Zamunér, Catholic University of Maule, Chile Motor imagery (MI) for health and performance strategies has gained interest in recent decades. Nevertheless, there are still no studies that have comprehensively investigated the physiological responses during MI, and no one questions the inﬂuence of low-level contraction on these responses. Thus, the aim of the present study was to investigate the neuromuscular, autonomic nervous system (ANS), and cardiometabolic changes associated with an acute bout of MI practice in sitting and standing condition. Twelve young healthy males (26.3 ± 4.4 years) participated in two experimental sessions (control vs. MI) consisting of two postural conditions (sitting vs. standing). ANS, hemodynamic and respiratory parameters, body sway parameters, and electromyography activity were continuously recorded, while neuromuscular parameters were recorded on the right triceps surae muscles before and after performing the postural conditions. While MI showed no effect on ANS, the standing posture increased the indices of sympathetic system activity and decreased those of the parasympathetic system (p < 0.05). Moreover, MI during standing induced greater spinal excitability compared to sitting posture (p < 0.05), which was accompanied with greater oxygen consumption, energy expenditure, ventilation, and lower cardiac output (p < 0.05). Asking individuals to perform MI of an isometric contraction while standing allows them to mentally focus on the motor command, not challenge balance, and produce speciﬁc cardiometabolic responses. Therefore, these results provide further evidence of posture and MI-related modulation of spinal excitability with additional autonomic and cardiometabolic responses in healthy young men. *Correspondence: Sidney Grosprêtre sidney.grospretre@univ-fcomte.fr orcid.org/0000-0003-1023-5842 Specialty section: This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Physiology Specialty section: This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Physiology Received: 21 August 2021 Accepted: 23 October 2021 Published: 23 November 2021 Abbreviations: ABPV, arterial blood pressure variability; ANS, autonomic nervous system; Bf, breathing frequency; CTRL, control; CO, cardiac output; CoP, Center of Pressure; DAP, diastolic arterial pressure; EE, energy expenditure; EMG, electromyography; GL, gastrocnemius lateralis; GM, gastrocnemius medialis; HF, high frequencies; HMAX, maximal Hoﬀmann (H) reﬂex; HR, heart rate; H50, H-reﬂex recorded at 50% of maximal H-wave amplitude; LF, low frequencies; MAP, mean arterial pressure; MI, motor imagery; MMAX, maximal muscle compound action potential; RQ, respiratory quotient; SAP, systolic arterial pressure; SOL, soleus; SV, stroke volume; TA, tibialis anterior; TPR, total peripheral resistance; VCO2, carbon dioxide production; VE, ventilation; VL, vastus lateralis; VO2, oxygen consumption; VO2, oxygen consumption; Vt, tidal volume. INTRODUCTION The lack of modulation of other larger spinal structures, such as the motoneurons themselves, may explain the absence of global spinal excitability changes observed in some previous studies. However, it was shown that repetition of this minor stimulus, such as after an acute bout of mental practice, may increase the sensitivity of all spinal structures (Grosprêtre et al., 2019). The most common method of activating these functions at a low level and making the participant cognitively available for a MI task is to get him into a posture that requires automated low-level contractions. To this aim, performing MI in standing position allows to observe its eﬀect with a low-grade background electromyographic (EMG) activity. Furthermore, spinal excitability of the triceps surae muscle is known to decrease from sitting or lying to standing posture (Katz et al., 1988; Koceja et al., 1995; Chalmers and Knutzen, 2002; Kawashima et al., 2003), independent of the level of background EMG (Cattagni et al., 2014). Interestingly, pre- synaptic inhibitory mechanism has been suggested as one of the main contributors to this particularly lower spinal excitability in standing as compared to sitting (Baudry and Duchateau, 2012; Johannsson et al., 2015). Then, given the pre-activation of pre- synaptic circuitry in standing position, it can be hypothesized that spinal excitability can be increased to a greater extent when practicing MI in standing posture. Regarding ANS and cardiometabolic modulations from sitting to standing, while increased sympathetic and decreased parasympathetic activity associated with greater cardiorespiratory response are often observed, EE and, overall, metabolic adjustments are not consistently reported (Miles-Chan et al., 2013; Miles-Chan and Dulloo, 2017; Amaro-Gahete et al., 2019). However, it has been shown that the metabolic changes associated with the standing posture vary widely between individuals, indicating that this position is highly sensitive to slight mild perturbations, in contrast to the more stable seated position. Therefore, the standing posture may represent a condition in which neuromuscular and metabolic factors are more prone to MI- induced modiﬁcations. While activation of the voluntary motor system is no longer discussed during MI, this may not be the only physiological tract triggered. Indeed, some authors pointed out a relationship between MI (i.e., human cognitive abilities) and the autonomic nervous system (ANS) (i.e., vital function regulation via sympathetic and parasympathetic activity) (Decety et al., 1991, 1993; Wang and Morgan, 1992; Fusi et al., 2005; Di Rienzo et al., 2012; Collet et al., 2013). INTRODUCTION changes in ANS activity (e.g., sympathetic activation) to insure the necessary cardiorespiratory responses to the upcoming expected energy expenditure (EE). Some studies have reported respiratory and hemodynamic modiﬁcations in response to mental simulation exercise (Decety et al., 1991, 1993; Wang and Morgan, 1992; Fusi et al., 2005). Interestingly, Decety et al. (1991) also noted that ventilation, during imaged locomotion at increasing speed (i.e., running), was more important than the actual metabolic demand. One hypothesis to explain this observation could be a dissociation between the appropriate ANS response in anticipation of the motor action (and thus the energy mobilization and cognitive demand required to provide this expected movement), and the lower EE induced by mental imagery per se. However, despite increasing interest in MI for health and performance strategies during the past decades, studies that have comprehensively investigated neuromuscular, ANS, and cardiometabolic responses during MI remain rare or non-existent. Motor function in sport performance or rehabilitation could be improved by mental training. The most popular modality of mental training is motor imagery (MI), the mental simulation of an action without the corresponding motor output (Decety, 1996). Numerous previous studies have shown that motor brain areas are activated during MI, such as the parietal, premotor, and primary motor cortices (Decety et al., 1994; Lotze et al., 1999; Grèzes and Decety, 2001; Ehrsson et al., 2003; Guillot et al., 2009; Munzert et al., 2009; Kilintari et al., 2016). Some recent evidence highlighted that this minor activation of motor regions possibly generates a sub-threshold brain output, which could reach spinal levels. However, at the spinal level, the general picture of MI activation is less clear, with some authors showing upward modulation of spinal excitability (Bonnet et al., 1997; Hale et al., 2003; Cowley et al., 2008; Aoyama and Kaneko, 2011; Grosprêtre et al., 2016) and others showing downward or no modulation (Oishi et al., 1994; Yahagi et al., 1996; Mouthon et al., 2015). In fact, it has been suggested that such sub- threshold cortical output was susceptible to partially reach the spinal networks by aﬀecting the most sensitive structures, i.e., the spinal interneurons (Grosprêtre et al., 2014). More particularly, the spinal pre-synaptic inhibitory processes, mediated by primary aﬀerent depolarizing interneurons, were shown to be decreased during MI as compared to rest (Grosprêtre et al., 2016). Citation: Grosprêtre S, Marusic U, Gimenez P, Ennequin G, Mourot L and Isacco L (2021) Stand Up to Excite the Spine: Neuromuscular, Autonomic, and Cardiometabolic Responses During Motor Imagery in Standing vs. Sitting Posture. Front. Physiol. 12:762452. doi: 10.3389/fphys.2021.762452 Keywords: heart rate, ˙VO2, Center of Pressure (COP), H-reﬂex, electromyography November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 1 Neural Responses to Motor Imagery Grosprêtre et al. Participants Twelve healthy, moderately active young males participated in the present study (age: 26.3 ± 4.4 years, height: 1.77 ± 0.05 m, weight: 75.0 ± 10.1 kg, between 150 and 200 min of moderate to vigorous physical activity by week estimated by interview based on the Global Physical Activity Questionnaire items) and were screened before examination. Our sample size calculation is based on the primary eﬃcacy outcome that relates to changes in spinal excitability before and after an MI session. In previous studies, an improvement of H-reﬂex of +15 to +35% was found during MI in a sitting position with 9–13 participants (Grosprêtre et al., 2018, 2019; Bouguetoch et al., 2020). Considering a signiﬁcance level of 5%, a power of 90%, 12 participants are included to meet the objectives of the study. The sample size calculation was performed on PASS 13 Power Analysis and Sample Size Software (Machin et al., 2008). The exclusion criteria included: former or current smoker, current medication use, and the presence of obvious neurological, cardiovascular, or metabolic disease. Participants completed the revised version of the Movement Imagery Questionnaire (MIQ-r) to determine their MI ability (Hall and Martin, 1997). Participants reported an average MIQ- R score of 47.2 ± 6.7 over 56, indicating good imagery capacity. After explaining all risks and beneﬁts associated with the study, all participants gave their written informed consent to participate in the present study. They committed to no unusual training or exercise program throughout the duration of the study, were asked to avoid any intense physical activity 48 h prior to each experimental session, and to maintain usual dietary and sleep habits throughout the duration of the study. This study protocol was approved by the institutional review board prior to participant recruitment (CPP: 2016-A00511-50) and was conducted in accordance with international ethical standards (Harriss et al., 2017) and the guidelines of the World Medical Association Declaration of Helsinki. After skin preparation and electrode positioning, the optimal stimulation parameters for H-reﬂex and M-wave recordings were determined in each experimental session. PRE neuromuscular assessment was always performed ﬁrst, before the participant was equipped for continuous measurements. Experimental Design The study was conducted in two experimental sessions of about 2 h 30 min in the post-prandial state (i.e., ∼2 h after a standardized breakfast consisting of bread, butter, jam, yogurt, fruits and water; energy content represented 9.5–10 kcal·kg−1 of body mass), a MI, and control (CTRL) conditions, performed in random order. A 48-h washout period between experimental sessions was used to eliminate potential carryover eﬀects. Each experimental session took place at the laboratory in a temperature, pressure, and humidity-controlled and quiet room (20◦C, 765 mmHg, 50% of relative humidity, respectively). Each experimental session was composed of two conditions assessed randomly, one in sitting and one in standing position, interspaced by 20 min of rest (Figure 1). For the sitting condition, participants sat in a comfortable chair with the hip at 120◦ and the leg extended to provide a similar conﬁguration of calf muscles from the standing position: knee at 180◦and ankle at 90◦. For the standing position, participants stood in bipodal upright posture with feet spaced in a natural position (shoulder width), reproduced between sessions. In both sitting and standing, some parameters were recorded PRE and POST MI and CTRL conditions (mainly evoked responses to nerve stimulation), while other parameters were continuously recorded during the experimental conditions (beat by beat hemodynamic, respiratory parameters, and myoelectrical activities). In the standing position, displacements of the Center of Pressure (CoP) were also continuously recorded during MI and CTRL conditions. Neuromuscular parameters were recorded on the right triceps surae muscles, being one of the most solicited muscle to maintain the standing posture. INTRODUCTION The ANS anticipates the action in preparing cardiorespiratory responses and providing metabolic resources necessary for the motor control (Collet et al., 2013). In addition, ANS is not only activated in response to an energy demand but also to cognitive and/or emotional tasks (Collet et al., 2013). MI generates a motor preparation phase and shares part of the mechanisms underlying this motor preparation and execution. MI is thus likely to elicit Thus, the aim of the present study was to investigate the neuromuscular, ANS, and cardiometabolic changes associated with acute bout of MI practice in sitting and standing condition. It has been shown that acute MI practice in sitting induces spinal plasticity (Grosprêtre et al., 2019). Therefore, given the pre-activation of pre-synaptic circuitry in standing, it can be hypothesized that spinal excitability is increased to a greater extent when MI is practiced in standing posture. Finally, with November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 2 Neural Responses to Motor Imagery Grosprêtre et al. in women and considering the potential eﬀects of endogenous and exogenous sex hormones on spinal excitability, ANS, and metabolism, we decided to include only men (Abhishekh et al., 2013; Casey et al., 2016). regards to ANS and cardiometabolic modiﬁcations, the few previous and controversial results of the literature do not allow to clearly predict an eﬀect of MI. It could nevertheless be hypothesized that standing posture, due to its greater sensitivity, would be more subjected to ANS and cardiometabolic MI- induced modulations, as compared to sitting posture. It is now well-recognized that motor function and learning could be improved by MI in sport and health context. The present study may thus help in deciphering conditions (e.g., MI during standing) that potentiate spinal excitability and cardiometabolic responses for both athlete and patient practice. Frontiers in Physiology \| www.frontiersin.org Anthropometric and Body Composition Parameters After the participant voided their bladder, body mass (kg) was assessed to the nearest 0.1 kg using calibrated scale (Digital scale Seca model 873 Omega, Germany) and standing height (m) was determined to the nearest 0.01 m using a standing stadiometer (Seca model 720, Hamburg, Germany). The weight and height of participants were measured barefoot while wearing underwear, and body mass index (BMI) was thus calculated as body weight (kg) divided by height squared (m2). Waist and Respiratory Measurements and Metabolic Assessment After calibration following the recommendation of the manufacturer and prior each session, oxygen consumption ( ˙VO2), carbon dioxide production ( ˙VCO2), ventilation ( ˙VE), tidal volume (Vt), and breathing frequency (Bf) were recorded breath by breath through gas exchange measurement (MetaMax R⃝, Cortex Biophysik, Leipzig, Germany). Respiratory quotient (RQ) was calculated as ˙VCO2/ ˙VO2 and EE (kcal·min−1) as ˙VO2 × energy equivalent of oxygen as already described (Isacco et al., 2016). Participants The control condition had the same rationale with 15 min rest observed instead of the MI training session. H50: H-reﬂex recorded at 50% of maximal H-wave amplitude. MMAX: maximal muscle compound action potential. hip circumferences (WC and HC) were measured in triplicate to the nearest 0.5 cm in a standing position with a standard non- elastic tape applied horizontally midway between the last rib and the superior iliac crest for the WC, and at the widest portion of the buttocks for the HC. The waist to hip ratio (WHR) was calculated as WC divided by HC. Body composition (fat mass and fat-free mass) and hydration were assessed using the bioelectrical impedance Tanita MC-780 (Tanita Corporation, Tokyo, Japan). participants were asked to estimate their MI quality through a quotation on a Likert scale, from 1 (poor) to 7 (excellent). To avoid the dependency of postural sways on visual feedbacks, participants were asked to perform MI with their eyes open in both the sitting and standing posture. For PRE and POST tests, as well as during the training sessions, every 5 s imagined trial was preceded and ended by an auditory signal. In PRE and POST tests, during each 5 s MI trial, an electrical stimulation was delivered to elicit either H or M-waves (see below) at a random interval after the auditory signal to avoid from anticipatory mechanisms. Participants This sequence was followed to ensure (i) a minimum time with the mask for the assessment of gas exchanges and (ii) suﬃcient time after the last nerve stimulation to allow beat by beat, hemodynamic, and respiratory measurements to be taken with as little anxiety as possible about the discomfort associated with electrical stimulation. For POST measurement, neuromuscular assessments were performed immediately after the end of the CTRL or MI condition. The participants removed the facemask before the ﬁrst nerve stimulation. In the MI condition, independently of the posture, participants had to imagine a maximal isometric contraction of the right calves and to feel the sensation corresponding to this eﬀort (kinaesthetic imagery) both during PRE and POST tests and during the 15-min sitting or standing MI training session. The MI training session in sitting and standing position consisted of four series of 15 MI trials of 5 s (5 s rest in-between), interspaced by 90 s rest between series. After each series, The rationale for our sample size was based on previous results on H-reﬂex modulation after MI training (Grosprêtre et al., 2018, 2019). Considering a signiﬁcance level of 5% and a power of 80%, a minimum sample of 10 participants was required to meet the objectives of the study. Then, Hoﬀman et al. (2018) previously reported a sexual dimorphism in the H-reﬂex that likely underlies neurological diﬀerences between men and women. In addition, due to the diﬃculty of standardizing hormonal status November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 3 Grosprêtre et al. Neural Responses to Motor Imagery FIGURE 1 \| Experimental design. An experimental motor imagery (MI) condition is depicted. The control condition had the same rationale with 15 min rest observed instead of the MI training session. H50: H-reﬂex recorded at 50% of maximal H-wave amplitude. MMAX: maximal muscle compound action potential. n. An experimental motor imagery (MI) condition is depicted. The control condition had the same rationale with 15 min rest observe n. H50: H-reﬂex recorded at 50% of maximal H-wave amplitude. MMAX: maximal muscle compound action potential. FIGURE 1 \| Experimental design. An experimental motor imagery (MI) condition is depicted. The control condition had the same instead of the MI training session. H50: H-reﬂex recorded at 50% of maximal H-wave amplitude. MMAX: maximal muscle compou FIGURE 1 \| Experimental design. An experimental motor imagery (MI) condition is depicted. Cardiometabolic and Autonomic Nervous System Parameters The CTRL condition was designed to assess the time- eﬀect of 15 min of sustained standing or sitting posture on neuromuscular, ANS, and cardiometabolic parameters. Participants were asked to stay relaxed without thinking of any movement or activity. The EMG signals were monitored during the whole period to ensure that no minor contraction was induced during this resting period. Hemodynamic Analysis Systolic arterial pressure, DAP, MAP, HR, CO, SV, and TPR values were averaged over the whole MI training sessions, each series (4 × 2 min30) and each resting period (3 × 1 min30). Similarly, CTRL conditions were split into seven parts, reproducing times of MI training sessions and the corresponding averaged SAP, DAP, MAP, HR, CO, SV, and TPR values were considered for analysis. Autonomic Nervous System Activity (Heart Rate Variability and Cardiac Baroreﬂex Sensitivity Analyses) All the IBI, SAP, and DAP values were ﬁltered out by means of a moderate error correction ﬁlter and were edited initially by visual inspection to exclude all the undesirable beats (i.e., to ensure that each analysis for the segment was free of movement artifact and/or sharp transient change in the signal due to premature beats) which counted for <1% in every participant. 256 stable heart cycles were used for each analysis (i.e., each subject, each condition, and each posture). Neuromuscular Recordings Electromyographic Activity Respiratory and Metabolic Analyses During the MI training sessions, ˙VO2, ˙VCO2, EE, RQ, ˙VE, Vt, and Bf values were averaged over the whole sessions, each series (4 × 2 min30), and each resting period (3 × 1 min30). Similarly, CTRL conditions were split into seven parts, reproducing times of MI training sessions and the corresponding averaged ˙VO2, ˙VCO2, EE, RQ, ˙VE, Vt, and Bf values were considered for analysis. Electromyographic activity was recorded from ﬁve muscles of the right leg [soleus (SOL), gastrocnemius medialis (GM), gastrocnemius lateralis (GL), tibialis anterior (TA), and vastus lateralis (VL)]. The skin was ﬁrst shaved and dry-cleaned with alcohol to keep low impedance (<5 k). Wireless sensors equipped with two silver chloride recording points and its own reference (Delsys, Natick, MA, United States) were used to record EMG signals and ﬁrmly strapped to the leg. To record SOL EMG, the sensor was placed 2 cm below the insertion of the gastrocnemii muscles on the Achilles’ tendon. GM and GL sensors were placed over the muscle belly in line with their insertion. TA and VL EMG activity were recorded to CTRL synergists and antagonist activities in order to maximize the isolation of experimental measures (force, evoked potentials) on the triceps surae. TA EMG was recorded by placing the sensor at 1/3 of the distance on the line between the ﬁbula and the tip of the medial malleolus, and VL at 2/3 on the line from the anterior spina iliaca superior to the lateral side of the patella. EMG signals were ampliﬁed with a bandwidth of 15 Hz to 1 kHz (gain: 1,000) and digitized on-line (sampling frequency: 2 kHz) using LabChart software (LabChart 8, ADInstruments, Sydney, NSW, Australia). Autonomic Nervous System (Heart Rate Variability, Cardiac Baroreﬂex Sensitivity, and Hemodynamic Assessments) Inter-beat interval derived from ECG performed at 1,000 Hz (IBI; BioAmp, ADInstruments, Sydney, NSW, Australia) and beat-to- beat blood pressure (Human NIBP Nano ADInstruments, November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 4 Neural Responses to Motor Imagery Grosprêtre et al. Sydney, NSW, Australia) were measured continuously. The Human NIBP Nano measures arterial pressure using photoplethysmography of the middle phalanx of the middle ﬁnger (Wesseling et al., 1995; Imholz et al., 1998). Arterial pulse pressure (PP, mmHg) was calculated from systolic arterial pressure (SAP) minus diastolic arterial pressure (DAP). The arterial pressure signal was analyzed using LabChart 8 Pro (ADInstruments, Sydney, NSW, Australia). Stroke volume (SV) was estimated using the Windkessel Model (3-Element) (Bogert and Van Lieshout, 2005) and cardiac output (CO) was calculated as the product of HR and SV, while total peripheral resistance (TPR) was determined by dividing mean arterial pressure [MAP; (SAP+2DAP)/3] by CO. in the present study (Grosprêtre et al., 2019). Subsequently, the intensity that provides an H-reﬂex response of 50% of its maximal amplitude (H50) in the ascending part of the recruitment curve was determined. The intensity was then increased with 5 mA increment until M-wave of the three muscles no longer increased. This last stimulation-intensity was increased by 20% to ensure supramaximal stimulation and used to record maximal M-wave (MMAX). In each condition (MI and REST) and time point (PRE and POST), four stimulations were performed to record HMAX and MMAX, and twelve stimulations were performed to record H50. Frontiers in Physiology \| www.frontiersin.org Statistical Analyses All data are presented as mean ± SD. Normality of data sets was tested using the Shapiro–Wilk test, and variance homogeneity was tested using Levene’s test. When data were not normally distributed, a natural logarithm transformation (Ln) was applied to obtain a normal distribution, which allowed the parametric statistical comparisons. Regarding cardiometabolic parameters, a three-way repeated measure ANOVA was performed (time × condition × posture) for the analysis of the kinetic values and a two-way ANOVA was performed to compare mean values over the session (condition × posture). Finally, ANS variables were evaluated using a two-way ANOVA (condition × posture). Peripheral Nerve Stimulation is often assumed that LF/HF ratio reﬂects the sympathetic- parasympathetic balance (Taylor, 2006). However, it is frequently shifted due to reductions in LF power which do not reﬂect sympathetic nervous system activity at rest. Moreover, both branches of the ANS can be simultaneously active (Berntson and Cacioppo, 1999). Furthermore, the interactions between parasympathetic and sympathetic nervous systems are complex, non-linear, and frequently non-reciprocal (Billman, 2013). is often assumed that LF/HF ratio reﬂects the sympathetic- parasympathetic balance (Taylor, 2006). However, it is frequently shifted due to reductions in LF power which do not reﬂect sympathetic nervous system activity at rest. Moreover, both branches of the ANS can be simultaneously active (Berntson and Cacioppo, 1999). Furthermore, the interactions between parasympathetic and sympathetic nervous systems are complex, non-linear, and frequently non-reciprocal (Billman, 2013). The respiratory rate was not controlled. However, on an individual basis, we systematically checked that the respiratory sinus arrhythmia peak fell within the HF band. All recordings were consistent in this regard. Peripheral Nerve Stimulation Neuromuscular parameters were assessed by means of recording triceps surae electrophysiological responses evoked by posterior tibial nerve stimulation, such as H-reﬂexes and M-waves (Rozand et al., 2015). Single rectangular pulses (1-ms width) were delivered by a constant-current stimulator (Digitimer, model DS7A, Hertfordshire, United Kingdom) through self-adhesive anode (8-mm diameter, Ag-AgCL) ﬁrmly strapped to the knee in the popliteal fossa (Cattagni et al., 2018). The cathode (5 × 10 cm, Medicompex SA, Ecublens, Switzerland) was placed over the patella. The monitoring of TA EMG activity during the setting of the stimulation electrode ensured that the common peroneal nerve was not activated. For the time domain, the root mean square of successive RR interval diﬀerences (RMSSD), an indicator of parasympathetic activity, was calculated. Spectrum analysis was performed to quantify the power of spectral components in the low frequencies (LF) (0.04–0.15 Hz) and high frequencies (HF) (0.15–0.50 Hz). The very LF (0–0.04 Hz) were not addressed in the present study. However, the VLF were calculated in order to obtain the HF and LF in normalized units (nu) {HF(nu) = HF(ms2)/[(total power(ms2)-VLF(ms2)]; LF(nu) = LF(ms2)/[(total power (ms2)- VLF(ms2)]}. HF power is almost entirely mediated by the parasympathetic activity to the sinus node directly associated with respiratory activity (Pomeranz et al., 1985). Finally, the LF/HF ratio was calculated as an indicator of sympathetic over parasympathetic balance (Pagani et al., 1986; Malik et al., 1996) even if this interpretation should be viewed with caution (Reyes del Paso et al., 2013; Shaﬀer et al., 2014). Indeed, it The intensity of the stimulation was progressively increased from SOL, GM, and GL response threshold with 2 mA increment until maximal H-reﬂex (HMAX) could be obtained. Since HMAX can be aﬀected by a ceiling eﬀect that could hide potential spinal modulations induced by MI, a submaximal H-reﬂex was recorded November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 5 Neural Responses to Motor Imagery Grosprêtre et al. is followed by a reﬂexive response, called V-wave, which is classically used as an index of the supra-spinal descending neural drive (Grospretre and Martin, 2014). Here, V-wave was used to bring further evidence regarding the descending neural drive modulation during MI in standing condition. For each muscle, all responses were normalized to maximal M-wave evoked in the same condition. Thus, HMAX/MMAX, MatHmax/MMAX, H50/MSUP, MatH50/MMAX, and V/MMAX were considered as dependent variables and compared between the groups. Postural Sway In standing conditions, participants stood on a force plate (Kistler Instrument Corp., Winterthur, Switzerland). The force plate allowed continuous recording of CoP displacements in the mediolateral and antero-posterior axes. Total sway path and amplitudes were determined separately from these CoP variables of the 15 min period of standing in both MI and standing conditions. The area of CoP displacements was also analyzed as the area of the ellipse that includes 90% of the CoP points. The total CoP length and ellipse over the 15 min periods of CTRL and MI were analyzed, as well as over the seven spited parts corresponding to the MI training session (four MI series of 2.5 min and three rest periods of 1.5 min in between). For the latter analysis, CoP length was normalized to the time of each period (in mm·s−1). With arterial blood pressure variability (ABPV) spectral analysis, it has been shown that the LF ABPV region could be due to an endogenous neural oscillator acting on the vasculature and the hypothesis of a resonance in the baroreﬂex loop (Castiglioni et al., 2007). Only LF ABPV (i.e., SAP-LF and DAP- LF) was reported here. Beat-by-beat SAP and IBI values were used to assess cardiac baroreﬂex sensitivity (BRS). After having collected the IBI and SAP data, beat-by-beat series have been interpolated at 5 Hz before carrying out the spectral analyses using a cubic interpolation that acts like a ﬁlter because it smooths the transition between points. Under resting conditions, transfer function analysis of gain, phase, and coherence between spontaneous oscillations in SAP and IBI were calculated in accordance with the work of Zhang et al. (1998), i.e., 0.05– 0.15 Hz for the low-frequency (LF) range. The sequence method is based on the identiﬁcation of at least three consecutive beats (sequence) in which an increase (or decrease) in SAP is followed by an increase (or decrease) in the IBI (Pitzalis et al., 1998; Davies et al., 2001). Only sequences with a minimum correlation coeﬃcient of 0.85 were accepted (Laude et al., 2004). To represent arterial pressure control in the upward and downward directions, mean gain values of positive (BRSSeq+) and negative (BRSSeq−) sequences were also computed separately. Frontiers in Physiology \| www.frontiersin.org Electrophysiological Data To account for background myoelectrical activity during the recording of neuromuscular responses, the root mean square (RMS) value of EMG signals was determined with an integration time of 500 prior to the stimulus artifact. SOL, GM, and GL RMS were normalized by the corresponding MMAX recorded in the same condition (MI/rest and sitting/standing). During the MI training sessions, RMS was calculated taking each of the four series as a whole (4 × 2 min30), and the three rest periods (3 × 1 min30). CTRL conditions were split into seven parts reproducing times of the MI training session. For RMS and CoP data in standing posture, the two-way repeated measures analysis of variance (ANOVA) was performed with factors “time” (the seven time periods) and condition (MI vs. CTRL). Regarding PRE–POST neuromuscular data (M-waves and H-reﬂexes), separate analysis was performed for CTRL and MI sessions. In CTRL session a two-way repeated measures ANOVA was performed with factors “time” (PRE vs. POST) and “posture” (sitting and standing). In MI training session, a three three-way repeated measure ANOVA was performed with the addition of the factor “MI” (REST vs. MI). Peak-to-peak amplitudes of myoelectrical responses at rest and during MI were measured for quantitative analysis. It can be noticed that each H-reﬂex, reﬂecting spinal excitability, is generally associated with a small M-wave (noted MatHmax at rest and MatH50), which was also measured. Variation in this response usually reﬂects a shift onto the recruitment curve of the H-reﬂex. Stability of MatH ensure similar nerve stimulation throughout the experiment (Grosprêtre and Martin, 2012). In active condition, i.e., in presence of myoelectrical activity, MMAX When statistical signiﬁcance was identiﬁed, a Sidak post hoc test was used to further delineate diﬀerences between conditions or time. Possible relationships between MI-induced changes in ANS, cardiometabolic, central nervous system, and postural data were screened through Pearson’s correlations. Statistical analysis was completed using Statistica (version 8.00; StatSoft Inc., Tulsa, OK, United States). The level of statistical signiﬁcance was set at p < 0.05. November 2021 \| Volume 12 \| Article 762452 6 Neural Responses to Motor Imagery Grosprêtre et al. For neurophysiological data, a separate analysis was performed for each muscle. For neurophysiological data, a separate analysis was performed for each muscle. weight) and EE values than sitting posture (p < 0.001). RESULTS The data of the present study can be divided into four main domains between which the eﬀects of MI and posture were compared, namely, cardiometabolic ( ˙VO2, EE, RQ, CO, etc.), autonomous nervous system (HRV, baroreﬂex etc.), central nervous system (EMG activities, H-reﬂexes, V-waves, etc.), and posture (CoP length, area, etc.) data. There was no condition eﬀect regarding ˙VE. Overall, standing posture induced signiﬁcantly higher ˙VE values than sitting posture (p < 0.001). ˙VE values while sitting during a CTRL session were signiﬁcantly lower than standing during CTRL and MI sessions (p < 0.001). There was no diﬀerence in ˙VE between sitting CTRL and sitting MI. Standing during CTRL session induced signiﬁcantly lower ˙VE values than during standing during MI (p < 0.05) and signiﬁcantly higher values compared with sitting during MI (p < 0.001). Standing led to increased ˙VE values when compared with sitting during MI sessions (p < 0.001). General Characteristics The general characteristics of the participants are present in Table 1. The self-evaluated MI quality (5.5 ± 1.1 and 5.6 ± 1.1 on average over the whole training session for sitting and standing, respectively) did not change signiﬁcantly from sitting to standing, as no main eﬀect nor interaction has been found for factors “series of MI” and “posture” (p > 0.05). There was no conditional eﬀect regarding Vt but there was a signiﬁcant interaction. Overall, standing posture induced signiﬁcantly higher Vt values than sitting posture (p < 0.05). Vt values while sitting during CTRL session were not signiﬁcantly diﬀerent than standing during CTRL and MI sessions. Sitting during CTRL session induced signiﬁcantly higher Vt values than sitting during MI session (p < 0.05). Standing during CTRL session induced signiﬁcantly higher values of Vt compared with sitting during MI (p < 0.01) while no diﬀerence was observed with standing during MI session. Standing led to increased Vt values when compared with sitting during MI sessions (p < 0.001). Electrophysiological Data During CTRL session, sitting ˙VO2 (absolute and relative to body weight) and EE values were signiﬁcantly lower than during standing (p < 0.05). There was no diﬀerence in ˙VO2 (absolute and relative to body weight) and EE between sitting CTRL and sitting MI. Standing during CTRL session induced signiﬁcantly lower ˙VO2 (absolute and relative to body weight) and EE values than during standing during MI (p < 0.01) and induced signiﬁcantly higher values compared with sitting during MI (p < 0.05). MI while standing led to higher ˙VO2 (absolute and relative to body weight) and EE values than MI during sitting (p < 0.001). Relative changes (in %) from CTRL to MI conditions in each posture were calculated for each variable. Pearson correlations were performed between the relative changes of ANS, cardiometabolic, and neuromuscular parameters in each posture condition. Respiratory and Metabolic Responses Respiratory and Metabolic Responses Table 2 shows the results regarding respiratory and metabolic responses. As there was no duration eﬀect during CTRL sessions and no sequence eﬀect (i.e., no signiﬁcant diﬀerence between series and rest periods) during MI training sessions, the mean value during each posture for each condition (e.g., mean EE during CTRL in sitting position, mean EE during CTRL in standing position, etc.) is considered for the following analyses. No condition, posture, and interaction eﬀects were observed for RQ and Bf. g y There was no condition eﬀect regarding ˙VO2 (absolute and relative to body weight) and EE. Overall, standing posture induced signiﬁcantly higher ˙VO2 (absolute and relative to body Hemodynamic Responses ± 16.3 119.1 ± 14.5 126.1 ± 23.0 112.6 ± 26.9 0.48; 0.54; 0.05 0.0002; 30.21; 0.70 0.29; 1.23; 0.10 DAP (mmHg 61.6 ± 9.0 60.7 ± 6.6 64.9 ± 15.8 60.1 ± 16.2 0.94; 0.006; 0.0006 0.03; 7.4; 0.27 0.32; 1.10; 0.09 MAP (mmHg) 82.9 ± 10.7 78.7 ± 8.5 84.0 ± 17.9 74.8 ± 19.8 0.35; 0.97; 0.08 0.0002; 27.2; 0.68 0.28; 1.28; 0.10 HR (bpm) 67.2 ± 9.8 78.2 ± 14.3 62.9 ± 9.0 77.3 ± 15.5 0.002; 13.18; 0.42 0.0001; 32.38; 0.75 0.11; 3.0; 0.22 CO (L·min−1) 5.7 ± 1.1a 5.4 ± 0.8a 5.4 ± 0.6a 4.9 ± 1.2 0.0003; 29.15; 0.71 0.0001; 35.42; 0.78 0.03; 6.80; 0.25 SV (ml) 92.9 ± 17.5 73.4 ± 15.8 84.4 ± 9.9 63.2 ± 20.7 0.0002; 30.28; 0.72 0.0001; 34.36; 0.76 0.30; 1.17; 0.10 TPR (mmHg·s/mL) 0.96 ± 0.28 0.92 ± 0.29 0.93 ± 0.24 0.95 ± 0.21 0.81; 0.06; 0.005 0.31; 1.14; 0.09 0.55; 0.38; 0.03 Data are presented as Mean ± SD. a: signiﬁcantly different from standing during MI at p < 0.05. CTRL, control; MI, motor imagery; SAP, systolic arterial blood pressure; DAP, diastolic arterial blood pressure; MAP, mean blood arterial pressure; HR, heart rate; CO, cardiac output; SV, stroke volume; TPR, total peripheral resistance. Degree of freedom = 1. TABLE 2 \| Cardiorespiratory and metabolic responses during control (CTRL) and motor imagery (MI) sessions. Hemodynamic Responses CTRL MI p-value; F-value; eta squared Sitting Standing Sitting Standing Condition Position Interaction VO2 (L·min−1) 0.32 ± 0.04 0.33 ± 0.03a 0.32 ± 0.05b 0.36 ± 0.05aaa,bb,ccc 0.60; 0.29; 0.05 0.0001; 31.11; 0.72 0.02; 7; 0.37 VO2 (mL·min−1·kg−1) 4.3 ± 0.8 4.5 ± 0.7a 4.3 ± 0.8b 4.8 ± 0.9aaa,bb,ccc 0.54; 0.40; 0.003 0.0002; 28.40; 0.70 0.02; 7; 0.37 EE (kcal·min−1) 1.54 ± 0.17 1.62 ± 0.17a 1.52 ± 0.25b 1.73 ± 0.26aaa,bb,ccc 0.56; 0.32; 0.04 0.0001; 28.30; 0.71 0.02; 7.1; 0.37 RQ 0.87 ± 0.05 0.87 ± 0.05 0.92 ± 0.1 0.91 ± 0.1 0.13; 2.61; 0.18 0.79; 0.08; 0.01 0.24; 1.55; 0.11 VE (L·min−1) 10.2 ± 1.9 11.6 ± 2.4aaa 10.0 ± 1.7bbb 12.1 ± 2.3aaa,b,ccc 0.47; 0.56; 0.04 0.00003; 41.62;0.78 0.03; 6.7; 0.24 Vt (L) 0.72 ± 0.19 0.77 ± 0.22 0.63 ± 0.13a,bb 0.80 ± 0.28ccc 0.21; 1.75; 0.13 0.05; 4.36; 0.31 0.02; 7.93; 0.40 Bf (breath·min−1) 15.3 ± 3.4 15.8 ± 3.2 16.9 ± 4.2 16.8 ± 4.6 0.15; 2.37; 0.16 0.59; 0.30; 0.02 0.46; 0.58; 0.05 Mean ± SD. a, aaa: signiﬁcantly different from sitting during control session at p < 0.05 and p < 0.001, respectively. b, bb, bbb: signiﬁcantly different from standing during control session at p < 0.05, p < 0.01, and p < 0.001, respectively. ccc: signiﬁcantly different from sitting during MI at p < 0.001. CTRL, control; MI, motor imagery; VO2, oxygen consumption; EE, energy expenditure; RQ, respiratory quotient; VE, ventilation; Vt, tidal volume; Bf, breathing frequency. Degree of freedom = 1. TABLE 2 \| Cardiorespiratory and metabolic responses during control (CTRL) and motor imagery (MI) sessions. ccc: signiﬁcantly different from sitting during MI at p < 0.001. CTRL, control; MI, motor imagery; VO2, oxygen consumption; EE, energy expenditure; RQ, respiratory quotient; VE, ventilation; Vt, tidal volume; Bf, breathing frequency. Degree of freedom = 1. TABLE 3 \| Hemodynamic responses during CTRL and MI sessions. CTRL MI p-value; F-value; eta squared Sitting Standing Sitting Standing Condition Position Interaction SAP (mmHg) 125. Postural Sway A main eﬀect of condition (p < 0.001) has been found for normalized CoP length, with no eﬀect of time or interaction (Figure 3A). The total CoP length was signiﬁcantly lower (p < 0.05) in standing MI as compared to CTRL. A signiﬁcant main eﬀect of condition, and time × condition interaction has been found for CoP ellipse area (p < 0.05). Over the two conditions, CoP ellipse showed a signiﬁcant increase between the last time period and the ﬁrst two in CTRL condition, while remaining unchanged for the MI condition (Figure 3B). A signiﬁcant main eﬀect of condition (p < 0.05) has been found for CoP antero-posterior amplitude, CTRL being greater than MI, with no time eﬀect or interaction (Figure 3C). No signiﬁcant Autonomic Nervous System: Heart Rate Variability and Cardiac Baroreﬂex Sensitivity Analyses condition, TA myoelectrical activity during the second and third rest period was signiﬁcantly higher as compared to the rest of the condition, and signiﬁcantly higher to the three other conditions (CTRL standing, MI, and CTRL sitting) (Figure 2). Overall, for total RMS activities, only a main eﬀect of posture has been found for each of the four tested muscles, MI and CTRL not being statistically diﬀerent in standing and sitting positions. y y Except for Ln-LF (ms2), coherence-LF, and BRS seq+, all the variables showed a position eﬀect. Indeed, IBI, RMSSD, Ln- HF (ms2), Ln-HF (nu), gain-LF, phase-LF, and BRS seq−were signiﬁcantly higher in the sitting than the standing position (Table 4). On the contrary, Ln-LF (nu), Ln-LF/HF, Ln-SAP-LF, Ln-DAP-LF, and the number of positive and negative sequences were signiﬁcantly higher in the standing than the sitting position (Table 4). IBI was signiﬁcantly higher during MI compared with the CTRL condition and Ln-DAP-LF was signiﬁcantly higher during CTRL compared with MI and there was no other condition or interaction eﬀect. Hemodynamic Responses ± 16.3 119.1 ± 14.5 126.1 ± 23.0 112.6 ± 26.9 0.48; 0.54; 0.05 0.0002; 30.21; 0.70 0.29; 1.23; 0.10 DAP (mmHg 61.6 ± 9.0 60.7 ± 6.6 64.9 ± 15.8 60.1 ± 16.2 0.94; 0.006; 0.0006 0.03; 7.4; 0.27 0.32; 1.10; 0.09 MAP (mmHg) 82.9 ± 10.7 78.7 ± 8.5 84.0 ± 17.9 74.8 ± 19.8 0.35; 0.97; 0.08 0.0002; 27.2; 0.68 0.28; 1.28; 0.10 HR (bpm) 67.2 ± 9.8 78.2 ± 14.3 62.9 ± 9.0 77.3 ± 15.5 0.002; 13.18; 0.42 0.0001; 32.38; 0.75 0.11; 3.0; 0.22 CO (L·min−1) 5.7 ± 1.1a 5.4 ± 0.8a 5.4 ± 0.6a 4.9 ± 1.2 0.0003; 29.15; 0.71 0.0001; 35.42; 0.78 0.03; 6.80; 0.25 SV (ml) 92.9 ± 17.5 73.4 ± 15.8 84.4 ± 9.9 63.2 ± 20.7 0.0002; 30.28; 0.72 0.0001; 34.36; 0.76 0.30; 1.17; 0.10 TPR (mmHg·s/mL) 0.96 ± 0.28 0.92 ± 0.29 0.93 ± 0.24 0.95 ± 0.21 0.81; 0.06; 0.005 0.31; 1.14; 0.09 0.55; 0.38; 0.03 Data are presented as Mean ± SD. a: signiﬁcantly different from standing during MI at p < 0.05. CTRL, control; MI, motor imagery; SAP, systolic arterial blood pressure; DAP, diastolic arterial blood pressure; MAP, mean blood arterial pressure; HR, heart rate; CO, cardiac output; SV, stroke volume; TPR, total peripheral resistance. Degree of freedom = 1. TABLE 3 \| Hemodynamic responses during CTRL and MI sessions. Data are presented as Mean ± SD. a: signiﬁcantly different from standing during MI at p < 0.05. CTRL, control; MI, motor imagery; SAP, systolic arterial blood pressure; DAP, diastolic arterial blood pressure; MAP, mean blood arterial pressure; HR, heart rate; CO, cardiac output; SV, stroke volume; TPR, total peripheral resistance. Degree of freedom = 1. Autonomic Nervous System: Heart Rate Variability and Cardiac Baroreﬂex Sensitivity Analyses Hemodynamic Responses Table 3 presents the results regarding hemodynamic responses. Similar to the respiratory and metabolic parameters, as there was no time eﬀect during CTRL sessions and no sequence eﬀect (i.e., series and rest periods) during MI sessions, the mean value during each posture for each condition is presented. There was no condition and interaction eﬀect regarding Ln-SAP, Ln- DAP, and Ln-MAP. Overall, standing position decreased Ln-SAP (p < 0.001), Ln DAP (p < 0.05), and Ln-MAP (p < 0.001) values compared with sitting position. Overall, CTRL sessions induced signiﬁcantly higher Ln-HR and Ln-SV values than MI sessions (p < 0.01 and p < 0.001, respectively). Overall, the standing position led to increased Ln-HR and decreased Ln-SV values compared with the sitting position (p < 0.001). Ln-CO was signiﬁcantly lower in the standing compared to the sitting position (p < 0.01). Ln-CO was signiﬁcantly decreased during MI session in the standing position compared to other conditions (p < 0.05). There was no posture, condition, and interaction eﬀect regarding Ln-TPR. TABLE 1 \| General characteristics of the participants. Age (y) 26.3 ± 4.4 Body weight (kg) 75 ± 10.1 Height (m) 1.77 ± 0.05 BMI (kg·m−2) 23.9 ± 3 WC (cm) 83.5 ± 8.2 HC (cm) 98.4 ± 7.9 WHR 0.85 ± 0.03 FM (%) 15.9 ± 5.1 FFM (kg) 59.6 ± 6.1 Hydration (%) 61.1 ± 4.3 Mean ± SD. BMI, body mass index; WC, waist circumference; HC, hip circumference; WHR, waist-to-hip ratio; FM, fat mass; FFM, fat-free mass. TABLE 1 \| General characteristics of the participants. November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 7 Neural Responses to Motor Imagery Grosprêtre et al. TABLE 2 \| Cardiorespiratory and metabolic responses during control (CTRL) and motor imagery (MI) sessions. Hemodynamic Responses CTRL MI p-value; F-value; eta squared Sitting Standing Sitting Standing Condition Position Interaction VO2 (L·min−1) 0.32 ± 0.04 0.33 ± 0.03a 0.32 ± 0.05b 0.36 ± 0.05aaa,bb,ccc 0.60; 0.29; 0.05 0.0001; 31.11; 0.72 0.02; 7; 0.37 VO2 (mL·min−1·kg−1) 4.3 ± 0.8 4.5 ± 0.7a 4.3 ± 0.8b 4.8 ± 0.9aaa,bb,ccc 0.54; 0.40; 0.003 0.0002; 28.40; 0.70 0.02; 7; 0.37 EE (kcal·min−1) 1.54 ± 0.17 1.62 ± 0.17a 1.52 ± 0.25b 1.73 ± 0.26aaa,bb,ccc 0.56; 0.32; 0.04 0.0001; 28.30; 0.71 0.02; 7.1; 0.37 RQ 0.87 ± 0.05 0.87 ± 0.05 0.92 ± 0.1 0.91 ± 0.1 0.13; 2.61; 0.18 0.79; 0.08; 0.01 0.24; 1.55; 0.11 VE (L·min−1) 10.2 ± 1.9 11.6 ± 2.4aaa 10.0 ± 1.7bbb 12.1 ± 2.3aaa,b,ccc 0.47; 0.56; 0.04 0.00003; 41.62;0.78 0.03; 6.7; 0.24 Vt (L) 0.72 ± 0.19 0.77 ± 0.22 0.63 ± 0.13a,bb 0.80 ± 0.28ccc 0.21; 1.75; 0.13 0.05; 4.36; 0.31 0.02; 7.93; 0.40 Bf (breath·min−1) 15.3 ± 3.4 15.8 ± 3.2 16.9 ± 4.2 16.8 ± 4.6 0.15; 2.37; 0.16 0.59; 0.30; 0.02 0.46; 0.58; 0.05 Mean ± SD. a, aaa: signiﬁcantly different from sitting during control session at p < 0.05 and p < 0.001, respectively. b, bb, bbb: signiﬁcantly different from standing during control session at p < 0.05, p < 0.01, and p < 0.001, respectively. ccc: signiﬁcantly different from sitting during MI at p < 0.001. CTRL, control; MI, motor imagery; VO2, oxygen consumption; EE, energy expenditure; RQ, respiratory quotient; VE, ventilation; Vt, tidal volume; Bf, breathing frequency. Degree of freedom = 1. TABLE 3 \| Hemodynamic responses during CTRL and MI sessions. CTRL MI p-value; F-value; eta squared Sitting Standing Sitting Standing Condition Position Interaction SAP (mmHg) 125. Mean ± SD. a, aaa: signiﬁcantly different from sitting during control session at p < 0.05 and p < 0.001, respectively. Myoelectrical Activities A signiﬁcant eﬀect of “posture” was found on RMS/MMAX for SOL, GM, and GL (p < 0.001, p < 0.01, and p < 0.01, respectively) values being higher in standing as compared to sitting posture during the whole 15 min (Figure 2). There was no other main eﬀect (time or condition) or interaction along the 15 min. However, a signiﬁcant “time” × “posture” × “condition” interaction has been found for TA RMS. In MI standing November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 8 Neural Responses to Motor Imagery Grosprêtre et al. IGURE 2 \| Myoelectrical recordings of leg muscles in standing and sitting postures. The root mean square (RMS) of electromyographic (EMG) activities were alculated for soleus (SOL), Gastrocnemius Medialis (GM), Gastrocnemius Lateralis (GL), and Tibialis Anterior (TA) in MI conditions (right panels) and in CTRL onditions (left panel). For SOL, GM, and GL, RMS is normalized by the maximal M-wave recorded in corresponding condition at PRE (RMS/MMAX). The RMS of the MG recorded continuously during the 15-min conditions was spitted in seven parts: during the four series of MI (S1, S2, S3, and S4) and during the three rest eriods (R1, R2, and R3). In the CTRL conditions the same cutting was applied. (A) Kinetic of SOL RMS/MMAX. (B) SOL RMS/MMAX value of the entire session. C) Kinetic of GM RMS/MMAX. (D) GM RMS/MMAX value of the entire session. (E) Kinetic of GL RMS/MMAX. (F) GL RMS/MMAX value of the entire session. (G) Kinetic f TA RMS. (H) TA RMS value of the entire session. T: time effect; C: condition effect; P: position effect; I: interaction effect between “time,” “condition,” and posture”; ns: non-signiﬁcant. ###: signiﬁcantly different from sitting at p < 0.001. ∗, ∗∗, ∗∗∗: signiﬁcantly different from sitting CTRL at p < 0.05, p < 0.01, and < 0.001, respectively. ϮϮϮ: signiﬁcantly different from standing CTRL at p < 0.001. ££, £££: signiﬁcantly different from sitting MI at p < 0.01 and p < 0.001, espectively. $$$: signiﬁcantly different from other points of standing MI at p < 0.001. FIGURE 2 \| Myoelectrical recordings of leg muscles in standing and sitting postures. The root mean square (RMS) of electromyographic (EMG) activities were calculated for soleus (SOL), Gastrocnemius Medialis (GM), Gastrocnemius Lateralis (GL), and Tibialis Anterior (TA) in MI conditions (right panels) and in CTRL conditions (left panel). Myoelectrical Activities For SOL, GM, and GL, RMS is normalized by the maximal M-wave recorded in corresponding condition at PRE (RMS/MMAX). The RMS of the EMG recorded continuously during the 15-min conditions was spitted in seven parts: during the four series of MI (S1, S2, S3, and S4) and during the three rest periods (R1, R2, and R3). In the CTRL conditions the same cutting was applied. (A) Kinetic of SOL RMS/MMAX. (B) SOL RMS/MMAX value of the entire session. (C) Kinetic of GM RMS/MMAX. (D) GM RMS/MMAX value of the entire session. (E) Kinetic of GL RMS/MMAX. (F) GL RMS/MMAX value of the entire session. (G) Kinetic of TA RMS. (H) TA RMS value of the entire session. T: time effect; C: condition effect; P: position effect; I: interaction effect between “time,” “condition,” and “posture”; ns: non-signiﬁcant. ###: signiﬁcantly different from sitting at p < 0.001. ∗, ∗∗, ∗∗∗: signiﬁcantly different from sitting CTRL at p < 0.05, p < 0.01, and p < 0.001, respectively. ϮϮϮ: signiﬁcantly different from standing CTRL at p < 0.001. ££, £££: signiﬁcantly different from sitting MI at p < 0.01 and p < 0.001, respectively. $$$: signiﬁcantly different from other points of standing MI at p < 0.001. November 2021 \| Volume 12 \| Article 762452 9 Frontiers in Physiology \| www.frontiersin.org Neural Responses to Motor Imagery Grosprêtre et al. al sway during motor imagery (MI) and control (CTRL) in standing condition. In panels (A–D), data are split according to the different phases of the ion (MI, black squares): S1, S2, S3, and S4 are for the four different successive series of MI (2.5 min), while R1, R2, and R3 represent the three n the series (1.5 min). The same cut has been performed in the CTRL session to provide valuable comparison with MI session. In panel (A), of Pressure (CoP) is normalized by the duration of each period (in mm/sec). In panel (B), CoP area (ellipse) is expressed in mm2 in each phase. mean amplitude of the CoP displacement over the different time periods on the antero-posterior (C) and mediolateral (D) axis. ∗and ∗∗∗: main condition (MI vs. CTRL) at p < 0.05 and p < 0.001, respectively. #: signiﬁcant differences between the indicated time points, at p < 0.05. FIGURE 3 \| Postural sway during motor imagery (MI) and control (CTRL) in standing condition. Myoelectrical Activities In panels (A–D), data are split according to the different phases of the motor imagery session (MI, black squares): S1, S2, S3, and S4 are for the four different successive series of MI (2.5 min), while R1, R2, and R3 represent the three rest periods between the series (1.5 min). The same cut has been performed in the CTRL session to provide valuable comparison with MI session. In panel (A), length of the Center of Pressure (CoP) is normalized by the duration of each period (in mm/sec). In panel (B), CoP area (ellipse) is expressed in mm2 in each phase. In panels (C,D), the mean amplitude of the CoP displacement over the different time periods on the antero-posterior (C) and mediolateral (D) axis. ∗and ∗∗∗: main effect of the factor condition (MI vs. CTRL) at p < 0.05 and p < 0.001, respectively. #: signiﬁcant differences between the indicated time points, at p < 0.05. November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 10 Neural Responses to Motor Imagery Grosprêtre et al. TABLE 4 \| Autonomic nervous system responses during CTRL and MI sessions. Myoelectrical Activities CTRL MI p-value; F-value; eta squared Sitting Standing Sitting Standing Condition Position Interaction Heart rate and arterial blood pressure variability IBI (ms) 884 ± 120 759 ± 124 935 ± 92 775 ± 521 0.05; 4.80; 0.30 0.000003; 72.95; 0.87 0.79; 1.03; 0.07 RMSSD (ms) 53.1 ± 18.1 35.5 ± 23.6 60.6 ± 20.7 37.8 ± 24.3 0.24; 1.51; 0.12 0.02; 8.70; 0.44 0.33; 1.03; 0.09 HF (ms2) 1063.82 ± 765.77 506.05 ± 676.59 1156.93 ± 835.42 625.76 ± 855.51 0.92; 0.01; 0.001 0.02;7.22; 0.40 0.68; 0.18; 0.02 LF (ms2) 2438.56 ± 2323.49 1817.42 ± 1027.40 3254.77 ± 2050.10 2356.50 ± 1494.90 0.50; 0.48; 0.04 0.55; 0.46; 0.06 0.38; 0.84; 0.07 HF (nu) 0.35 ± 0.19 0.22 ± 0.16 0.27 ± 0.15 0.18 ± 0.12 0.26; 1.35; 0.10 0.05; 4.79; 0.31 0.81; 0.09; 0;01 LF (nu) 0.65 ± 0.19 0.78 ± 0.16 0.73 ± 0.15 0.82 ± 0.12 0.27; 1.64; 0.10 0.05; 4.78; 0.30 0.79; 0.08; 0.01 LF/HF 2.92 ± 2.42 5.47 ± 4.84 4.45 ± 3.9 7.14 ± 4.71 0.16; 2.24; 0.17 0.004; 13.61; 0.55 0.97; 0.001; 0.0001 SAP-LF (ms2) 33.71 ± 29.03 73.87 ± 141.53 28.10 ± 19.96 35.88 ± 22.40 0.84; 0.05; 0.004 0.04; 5.65; 0.34 0.74; 0.11; 0.01 DAP-LF (ms2) 8.85 ± 6.26 26.53 ± 53.16 10.03 ± 6.39 14.88 ± 8.51 0.05; 4.90; 0.38 0.001; 17.70; 0.62 0.57; 0.34; 0.57 Cardiac Baroreﬂex: Transfer function analysis Gain-LF (ms × mmHg−1) 8.0 ± 3.8 6.9 ± 4.7 9.2 ± 3.1 5.8 ± 2.3 0.65; 0.22; 0.02 0.0006; 22.75; 0.67 0.54; 0.39; 0.03 Phase-LF (rads) 0.008 ± 0.27 −0.17 ± 0.37 0.17 ± 0.21 −0.16 ± 0.22 0.57; 0.34; 0.03 0.008; 0.56; 0.49 0.11; 2.25; 0.26 Coherence-LF 0.60 ± 0.19 0.55 ± 0.23 0.57 ± 0.11 0.63 ± 0.26 0.74; 0.11; 0.01 0.21; 1.80; 0.14 0.99; 0.0001; 0.00001 Cardiac Baroreﬂex: Sequence Method n seq+ 25.6 ± 24.0 35.1 ± 23.0 28.3 ± 17.7 48.8 ± 26.0 0.27; 1.36; 0.11 0.04; 5.35; 0.33 0.20; 1.87; 0.14 n seq− 29.4 ± 31.1 45.8 ± 33.0 30.4 ± 19.7 59.6 ± 26.0 0.53; 0.42; 0.04 0.004; 13.23; 0.55 0.21; 1.81; 0.14 BRS seq+ (ms × mmHg−1) 48.2 ± 100.3 8.6 ± 4.3 13.3 ± 4.5 15.4 ± 17.6 0.86; 0.03; 0.003 0.09; 3.65; 0.25 0.18; 2.02; 0.16 BRS seq− (ms × mmHg−1) 7.7 ± 5.0 5.2 ± 3.0 8.7 ± 3.2 6.1 ± 2.5 0.49; 0.52; 0.05 0.0001; 35.24; 0.76 0.73; 0.13; 0.01 Data are presented as Mean ± SD. Pre-to-Post Neuromuscular Assessment No signiﬁcant main eﬀect nor interaction have been found for maximal and submaximal M-wave in the diﬀerent postures and conditions (p > 0.05). For all tested muscles, MMAX and MatH/MMAX were neither altered by MI nor by the changes from the sitting to standing position showing, respectively, (1) no changes at the level of the neuromuscular junction and (2) no changes in nerve stimulation conditions. As during the MI sessions, the background level of EMG activity measured before each PRE and POST stimulus artifact did not reveal any alteration by MI neither in sitting nor in standing posture. Myoelectrical Activities CTRL, control; MI, motor imagery; IBI, interbeat interval; RMSSD, square root of the sum of successive differences between adjacent normal R–R intervals squared; Ln, logarithm transformation; HF, high frequency; LF, low frequency; DFA, detrended ﬂuctuation analysis; BRS, baroreﬂex sensitivity; n seq+ and n seq−, number of positive and negative BRS sequences, respectively; BRS seq+ and BRS seq−, mean gain values of positive and negative BRS sequences, respectively. Degree of freedom = 1. TABLE 4 \| Autonomic nervous system responses during CTRL and MI sessions. Data are presented as Mean ± SD. CTRL, control; MI, motor imagery; IBI, interbeat interval; RMSSD, square root of the sum of successive differences between adjacent normal R–R intervals squared; Ln, logarithm transformation; HF, high frequency; LF, low frequency; DFA, detrended ﬂuctuation analysis; BRS, baroreﬂex sensitivity; n seq+ and n seq−, number of positive and negative BRS sequences, respectively; BRS seq+ and BRS seq−, mean gain values of positive and negative BRS sequences, respectively. Degree of freedom = 1. main eﬀect or interaction have been found for mediolateral CoP amplitude (Figure 3D). In standing posture, supraspinal modulations were accounted by the concomitant analysis of V-wave accompanying maximal M-wave. In gastrocnemii, no signiﬁcant main eﬀect or interaction have been found for V/MMAX, being unaltered by MI in PRE as in POST condition. In contrast, soleus V/MMAX exhibited a signiﬁcant gain (p < 0.05) by MI in POST, while unchanged in PRE. Frontiers in Physiology \| www.frontiersin.org Relationships Between the Variables p First, no signiﬁcant correlation was found between relative changes from CTRL to MI of each variable in the sitting condition. Second, in the standing posture, only the relative changes from CTRL to MI of CO and those of neuromuscular parameters were signiﬁcantly correlated. Indeed, a signiﬁcant negative correlation was found between the relative change in SOLEUS H-reﬂex and the relative change in CO (r = −0.63, p < 0.05). Participants with the highest increase in H-reﬂex due to MI were those with the greater decrease in CO. On the contrary, relative changes of CO due to MI were positively correlated with relative changes of SOL V/MSUP in standing posture (r = 0.62, p < 0.05). A signiﬁcant correlation (r = 0.65, p < 0.05) was found between the relative change in CO due to MI and the relative change in CoP area due to MI. In other terms, the greater the decrease of the CoP induced by MI, the greater the CO was decreased by MI. Finally, both the relative changes in SOL H-reﬂexes and V-waves were signiﬁcantly and negatively correlated with the change in CoP length (respectively r = −0.97 and r = −0.706, p < 0.01). Regarding spinal excitability, no main eﬀect nor interaction has been found for maximal rest normalized H-reﬂex (HMAX/MMAX) of SOL, GM, and GL for factor “time” (PRE to POST) and condition (MI to REST). However, HMAX/MMAX were signiﬁcantly lower in standing as compared to sitting condition for SOL and GM, a main eﬀect of posture being noted (p < 0.01). Regarding submaximal H-reﬂex (H50/MMAX), condition × time interaction has been found for each muscle (p < 0.05). In sitting as in standing posture, H50/MMAX was greater during MI than during REST only at POST, while not diﬀerent at PRE (Figure 4). To further this analysis, when comparing the relative changes in H50/MMAX with MI, the gains were higher following MI practice in standing posture as compared to sitting posture (Figure 4). November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 11 Neural Responses to Motor Imagery Grosprêtre et al. al excitabilities of the triceps surae during the motor imagery (MI) and control (CTRL) conditions in the sitting and standing postures. In panels (A–C) onses for soleus, gastrocnemius medialis and gastrocnemius lateralis, respectively. Relationships Between the Variables PRE and POST responses are recorded during rest (white bars) ack bars) in sitting (left panel) and in standing condition (middle panel). In panels (A–C) upper panels depict data of the motor imagery session and ct data of the CTRL session. Spinal excitabilities are measured by the H-reﬂex recorded at 50% of its maximal value (H50). This response is e maximal muscle compound action potential (H50/MMAX) recorded in the same condition (MI or rest). The right panel represent the gain associated in the mental session {determined by the formula [(MI-REST)/REST] × 100} in each condition from PRE (white circles) to POST (black circles). This according to the results obtained in H-reﬂexes in MI and at rest, depicted in the left panels. ∗, ∗∗: signiﬁcant differences at p < 0.05 and p < 0.01, gniﬁcant pre–post differences at p < 0.05 for the relative gains associated to MI. FIGURE 4 \| Spinal excitabilities of the triceps surae during the motor imagery (MI) and control (CTRL) conditions in the sitting and standing postures. In panels (A–C) are depicted responses for soleus, gastrocnemius medialis and gastrocnemius lateralis, respectively. PRE and POST responses are recorded during rest (white bars) and during MI (black bars) in sitting (left panel) and in standing condition (middle panel). In panels (A–C) upper panels depict data of the motor imagery session and lower panels depict data of the CTRL session. Spinal excitabilities are measured by the H-reﬂex recorded at 50% of its maximal value (H50). This response is normalized by the maximal muscle compound action potential (H50/MMAX) recorded in the same condition (MI or rest). The right panel represent the gain associated to motor imagery in the mental session {determined by the formula [(MI-REST)/REST] × 100} in each condition from PRE (white circles) to POST (black circles). This gain is calculated according to the results obtained in H-reﬂexes in MI and at rest, depicted in the left panels. ∗, ∗∗: signiﬁcant differences at p < 0.05 and p < 0.01, respectively. #: signiﬁcant pre–post differences at p < 0.05 for the relative gains associated to MI. November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 12 Neural Responses to Motor Imagery Grosprêtre et al. Particularly, the more MI decrease CoP, the more it increases neuromuscular responses. skating athletes. Voluntary? The novelty of the present study lies in the assessment of the MI eﬀects on diﬀerent physiological systems (ANS, cardiometabolic, and neuromuscular) by varying the posture from a relaxed and low energy consuming, i.e., sitting, to a more demanding, albeit automatic, task, the standing posture. The ANS is involved in voluntary muscle contractions through the activation of sympathetic and parasympathetic systems, but also through the stimulation of mechanoreceptors, chemoreceptors, and baroreceptors (Friedman et al., 1992; Iellamo et al., 1997). It has been shown that muscle sympathetic nerve activity and heart rate were increased proportionally with the elevation of tension during submaximal sustained handgrips (Saito et al., 1986). However, Seals (1993) noted that exceeding a minimal force, the sympathetic outﬂows seemed independent of the level of tension during a sustained isometric contraction, suggesting a decoupling relationship between ANS and voluntary contraction. However, these results were observed with a speciﬁc exercise modality (i.e., isometric), and Katayama and Saito (2019) recently reported that changes in muscle sympathetic nerve activity are a function of exercise modality and thus muscle contraction characteristics and position. The ANS activity is inﬂuenced by multiple external factors including, but not exhaustively, physical movement, body position, ingestion of food, cold exposure but also by emotional and cognitive tasks (Porges, 1995; Collet et al., 2013). Collet et al. (2013) provided a comprehensive overview of the interrelationship between ANS and MI, and they highlighted the role of ANS in anticipating cardiovascular and respiratory responses and in providing the necessary resources to cope with impending energy demands and/or a cognitive task. In this regard, previous studies reported that imaged or observed exercise, without any muscle contractions, led to increased arterial pressure or heart rate (Wang and Morgan, 1992; Oishi et al., 2000; Fusi et al., 2005). For instance, Wang and Morgan (1992) reported that internal and external imagery (resistance- imaged exercise) increased SAP to a similar pattern than the actual exercise while no diﬀerence was observed in the CTRL group. The authors highlighted that anticipation of exercise, due to the activation of areas of the brain during MI, triggered ANS activity, which in turn regulated arterial pressure. Similarly, Fusi et al. (2005) observed changes in ANS activity associated with cardiorespiratory responses during imagined walking. In the present study, apart for some cardiometabolic parameters in the standing condition, which is not usual in the literature, the ANS activity was not signiﬁcantly altered by MI. DISCUSSION The aim of the present study was to assess neuromuscular, ANS, and cardiometabolic changes associated with acute bout of MI practice, speciﬁcally in sitting vs. standing condition. While MI did not reveal any eﬀect on ANS, the standing posture increased the indexes of the sympathetic system activity and decreased those of the parasympathetic system activity. Moreover, MI during standing exceeds greater spinal excitability compared with the sitting posture, which was accompanied with higher ˙VO2, EE, ˙VE, and lower CO. Moreover, MI during standing induced greater spinal excitability changes as compared with the sitting posture, as well as signiﬁcant changes in CoP characteristics as compared to the CTRL condition. Here, the results pointed out, for the ﬁrst time, that standing posture during MI magniﬁed the ventilation, oxygen consumption, and EE responses compared to the CTRL condition. Conversely, no signiﬁcant diﬀerence was observed regarding ANS activity and hemodynamic parameters, except for CO which was decreased during MI session in the standing position compared to other conditions. Interestingly, CO was the only cardiometabolic parameter associated with neuromuscular and postural variables. To our knowledge, no study investigated the relationship between cardiometabolic and neuromuscular changes with MI, and it is thus diﬃcult to interpret the present results. However, these results may suggest that CO is associated with the regulation of balance due to the correlations with H-reﬂex and CoP area and the central command during MI in standing posture. The physiological pre-activation generated by the standing position during MI seems to elicit speciﬁc hemodynamic response related to neuromuscular changes. One explanation could be that MI during standing results in increased balance control that leads to a decreased blood ﬂow. Relationships Between the Variables During MI, which consisted of imaged sprint competition with subjects in the supine position, the authors reported an increase in cardiorespiratory parameters (i.e., HR, Bf) and a reduction of the spinal excitability. Conversely, Bunno et al. (2015) observed a greater spinal excitability and index of the cardiac sympathovagal balance (LF/HF ratio) during imaged thenar muscle contraction at 10 and 50% of maximal voluntary contraction. The diﬀerence in the characteristics of subjects and in the MI and methodology used to assess spinal excitability between these two studies may explain the discrepancies observed in terms of neuromuscular responses. Frontiers in Physiology \| www.frontiersin.org The Effects of Motor Imagery on Balance The Effects of Motor Imagery on Balance In the present study, an overall decrease in postural sway was observed when MI was performed in the standing upright posture as compared to the standing CTRL condition. In this regard, results vary in the literature, with CoP oscillations were either increased or decreased by MI. It should be noted that methodologies vary extremely, as does the interpretation of such an eﬀect. For instance, it has been argued that the eﬀect of MI on postural sway is related to the MI ability of the participants (Lemos et al., 2015). Similarly, it has been argued that the pre-activation induced by maintaining balance while standing could facilitate information processing during MI (Stins et al., 2015). Since in the present study, all participants reported high scores on the MIQ-r questionnaire and there was no diﬀerence in MI ability from sitting to standing, no such relationship could be established. Voluntary? Such a discrepancy with other studies could be partly attributed to methodological speciﬁcities. The approach to assessing ANS, the type of MI session (internal vs. external, duration, modality), and the Speciﬁcally, regarding the eﬀect of posture, in the present study, the postural change from sitting to standing increased the indexes of the sympathetic system activity and decreased those of the parasympathetic system activity and resulted in an increase in HR and a decrease in SAP, DAP, MAP, CO, and SV. These changes in response to postural change are commonly observed in the literature (Frey et al., 1994). Yet, signiﬁcant relationships between neuromuscular and hemodynamic variables impacted by MI could only be found in standing posture, demonstrating that it is particularly prone to MI-induced modulations. As MI shares neural circuits with motor execution, one might expect ANS to be activated during MI. Oishi et al. (1994) investigated the inﬂuence of MI on H-reﬂex and HR in elite speed November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 13 Neural Responses to Motor Imagery Grosprêtre et al. characteristics of the subjects (i.e., age, sex, physical activity level, expertise in MI) likely inﬂuence the neurovegetative responses. In addition, the well-known inter-individual variability and low repeatability with respect to ANS responses could conceal potential diﬀerences. Standardization of experimental conditions is therefore crucial when investigating ANS activity, and we paid particular attention to normalization between sessions (i.e., nutritional status, room temperature and humidity, positions during the protocol). The heterogeneity of ANS responses to MI may suggest speciﬁc responder and non-responder proﬁles to MI highlighting the necessity to promote individual strategy for MI practice. Further studies with larger sample size are needed to conﬁrm this hypothesis, especially because we observed in some subjects unusually high values of positive baroreﬂex sequence in the control condition only. Unexpectedly, high baroreﬂex value has been previously reported and explained by sequence that are not driven by the baroreceptor reﬂex (Laude et al., 2004). However, our speciﬁc study design could not help explain this observation. Finally, it should also be mentioned that both supraspinal and spinal autonomic regulation can contribute to speciﬁc changes in HRV indexes. In that sense, previous studies already showed the interest of methods that allows deciphering the speciﬁc spinal and supraspinal contribution to HRV (Introna et al., 1995). November 2021 \| Volume 12 \| Article 762452 Voluntary? In the present protocol, despite greater spinal and supraspinal excitabilities of the voluntary motor system during MI while standing, the HRV indexes were not modiﬁed, and the results showed the non-signiﬁcant eﬀect of MI on autonomic regulation. Concerning substrate oxidation, no diﬀerence was observed in RQ between sessions. However, it is worth noting that important inter-individual variability exists and may have masked potential diﬀerences. In their study, Wang and Morgan (1992) did not observe any diﬀerence in RQ between internal, external imaged exercise, and CTRL session. Troubat et al. (2009) investigated the inﬂuence of a psychologically stressful situation (i.e., chess game) on substrate oxidation (Troubat et al., 2009). They observed an increase in RQ at the onset of the game and then a decrease during the game. The authors had no clear justiﬁcations to explain these results, and the condition diﬀered from MI, but they pointed out the potential inﬂuence of psychological strain and cognitive task on substrate utilization. In addition, cognitive activity aﬀected energy intake, and knowing the close interrelationship between energy intake and substrate oxidation (i.e., fuel storage and utilization), it could be hypothesized that cognitive eﬀort likely alters fuel metabolism (Chaput and Tremblay, 2007; Chaput et al., 2008; Pérusse-Lachance et al., 2013). Indeed, this eﬀect may be mediated via increased cortisol and glucose instability, which has already been found during mental work (Chaput et al., 2008). In this regard, MI session may induce changes in hormonal concentrations (e.g., cortisol, catecholamines), notably in relation to ANS activity and metabolism and substrate availability (i.e., carbohydrates, lipids) due to the increased cognitive demand and/or stress condition. In conclusion, although the present results on energy metabolism and MI are inconclusive, they suggest possible changes in response to MI, particularly in a standing position, and open new avenues for research on this topic. Overall, the results of the present work question the common belief that MI consistently modiﬁed ANS and cardiovascular activity and highlight the speciﬁc nature of the relationship between ANS activity, cardiometabolic regulation, and MI. Frontiers in Physiology \| www.frontiersin.org Additional Effects of Posture and Motor Imagery on the Voluntary Motor System This was conﬁrmed by the lack of change in maximal and submaximal M-waves, which are markers of changes occurring at lower levels, such as at the neuromuscular junction. In fact, many authors rather targeted spinal pre-synaptic inhibition as the potential main contributor of spinal regulation from sitting to standing, although being less involved when the standing posture was perturbed (Baudry and Duchateau, 2014; Johannsson et al., 2015). y g Although the present study also involved kinesthetic MI, the present results depict opposite trends. The ﬁrst clue to explain this discrepancy is related to the speciﬁcity of the imagined task (isometric plantar ﬂexion). Indeed, Grangeon et al. (2011) also found that MI alters postural sway during MI while standing but, interestingly, the direction of the modulation was a function of the imagined task. CoP length, antero-posterior, and mediolateral oscillations were reduced while imagining a ﬁnger movement, whereas these parameters were enhanced while imagining jumping. In addition to an MI task-dependency of the postural adjustments, it can be argued that CoP oscillations are speciﬁcally exacerbated when the imagined task involves a higher postural control (Boulton and Mitra, 2013; Lemos et al., 2015). The MI task in the present study did appear then to be perceived as balance challenging. Therefore, participants were able to focus their attention on mentally activating their triceps surae rather than maintaining balance. Consequently, in the present study, MI during standing could be comparable to a dual- task paradigm. Therefore, a clear allocation of cognitive resources during MI could lead to a higher automatization of postural control compared to quiet standing (CTRL condition). This eﬀect was even more pronounced when the task was prolonged since CoP ellipse increased during the 15 min of quiet standing (CTRL condition), while staying low throughout the whole MI session. y g g Interestingly, although CoP sway can be aﬀected by MI in the standing posture, most authors, in accordance to the present results, found no additional EMG activity of leg muscles during MI, supporting the lack of supplemental voluntary contraction of the calf muscle (Rodrigues et al., 2010; Lemos et al., 2015; Koláˇrová et al., 2016). However, although EMG activity was not quantitatively altered, diﬀerent motor strategies were observed as, for example, strong relationships were found between variations in CoP and EMG during kinesthetic MI (Lemos et al., 2015). Additional Effects of Posture and Motor Imagery on the Voluntary Motor System imagine more dynamic actions such as cycling or jumping, still in a kinesthetic modality (Grangeon et al., 2011; Stins et al., 2015). Additional Effects of Posture and Motor Imagery on the Voluntary Motor System In general, standing requires a greater degree of neuromuscular control than sitting, especially in the triceps surae, to maintain the upright standing posture. The latter is also characterized by a ﬂuctuation of the CoP position (Winter et al., 1998). This leads to a particular neural control of the leg muscles and especially the triceps surae. Indeed, the maintenance of upright standing, although it requires an overall activation of the calf muscles, leading to a greater EMG activity compared to sitting, is not solely a matter of muscular contraction. Indeed, the central command needs to be adjusted depending on these CoP displacements. However, it is important to note that the control of postural sway is mainly attributed to neural mechanisms located at the spinal level (Koceja et al., 1995; Tokuno et al., 2009; Baudry and Duchateau, 2012; Cattagni et al., 2014; Johannsson et al., 2015). Regarding the latter and in line with the present results, it has long been reported that the H-reﬂex, the most usual tool to assess spinal excitability, was in fact depressed from sitting to standing (Katz et al., 1988; Kawashima et al., 2003). In line with the present results, this downward regulation of spinal excitability can be attributed to many mechanisms, from pre- and post-synaptic inhibitory networks to a regulation of motoneuronal pool excitability itself. Notwithstanding, this speciﬁc neural adjustment was attributed to a descending control of spinal networks due to the change in posture, rather than to an increase in the background muscle activity from sitting to standing. In fact, this lowered H-reﬂex has been associated with a cortico-vestibular inﬂuence rather than the leg muscle activation required to maintain posture (Cattagni et al., 2014). This latter assumption is of importance since we did not observe any supplemental motor output with MI in the standing condition, as evidenced by similar triceps surae RMS between the CTRL and MI condition. This raised the fact that neither a greater motoneuronal output nor post- synaptic mechanisms could be accounted for the observed eﬀects of MI in the standing posture. The Effects of Motor Imagery on Energy Metabolism While MI has become a common practice for health and performance over the past decades, to our knowledge, no study speciﬁcally investigated the potential eﬀect of MI on energy metabolism. In the present study, ˙VO2 (absolute and relative to body weight) and EE were signiﬁcantly increased during the standing sessions compared to basal values (i.e., sitting during the CTRL session) and to a greater extent during MI condition. This mean diﬀerence in EE of 13.5% between the sitting CTRL and the standing MI represented a slight increase of ∼26 kcal over 15 min (and less than 2 kcal between standing CTRL and MI) and is unlikely to be of signiﬁcant importance in terms of general energy balance and weight regulation (Hill et al., 2003). One could agree with Hill et al. (2003), however, that the present results underlined, for the ﬁrst time, possible modiﬁcations of ˙VO2 and EE in response to MI session in the standing position, and 13.5% is substantial in the context of non-exercise activity thermogenesis. From the brain perspective, mechanisms may be similar to those of cognitive training eﬀects with increased glucose consumption during MI associated with higher autonomic responses that consequently increase ˙VO2. Then, a wide variety of imagined tasks and modalities can be found in the literature. For instance, Rodrigues et al. (2010) asked healthy individuals to imagine stepping on their toes while assuming an upright posture, using diﬀerent MI modalities (visual or kinesthetic). They found an increase in body sway when participants were in the kinesthetic modality, as evidenced by a greater CoP area. As for oscillations, they were greater during MI only in the antero-posterior axis. They attributed this eﬀect to an increase in descending command emanating from motor regions during MI, targeting the motor system of plantar ﬂexors. The same behavior was observed during CoP when participants had to November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 14 Neural Responses to Motor Imagery Grosprêtre et al. Frontiers in Physiology \| www.frontiersin.org AUTHOR CONTRIBUTIONS SG, UM, PG, GE, and LI contributed to the conception of the work and analyzed the data. SG, PG, GE, and LI performed the experiments. SG, UM, PG, GE, LM, and LI interpreted the data. All authors contributed to drafting the work and revising it critically. CONCLUSION Motor imagery during standing induced greater spinal excitability compared to the sitting posture and was accompanied with greater oxygen consumption, EE, ventilation, and lower CO. MI during standing induced speciﬁc neuromuscular and cardiometabolic changes compared to sitting, but without the expected eﬀect on the ANS. Investigating deeper in detail, the neuronal networks involved, through H-reﬂex conditioning paradigms, for instance, may also allow researchers to endorse the involvement of pre-synaptic circuitry during MI in a standing posture. ETHICS STATEMENT Finally, the lack of change in EMG activity during the 15 min standing CTRL, with no change in PRE–POST neuromuscular data, argues for a lack of neuromuscular fatigue. This emphasized that 15 min of bipedal standing is insuﬃcient to aﬀect neuromuscular parameters in the population tested (young healthy males). Also, performing MI did not induce additional fatigue in this condition, in accordance with a previously reported lack of neuromuscular fatigue induced by a full session of MI (Rozand et al., 2014), but can still involve slight changes in postural strategies. The studies involving human participants were reviewed and approved by Comité de Protection des Personnes (CPP) 2016- A00511-50. The patients/participants provided their written informed consent to participate in this study. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Practical Implications Motor imagery is now recognized as a simple, safe, eﬃcient, and cost-eﬀective modality for enhancing motor rehabilitation and performance. It has thus emerged as a relevant approach for people with prolonged immobilization and/or inactivity (due to, for instance, to injury, surgery, or physical limitations) to prevent motor and cognitive decline or dysfunctions. The present study points out for the ﬁrst time that the posture during Additional Effects of Posture and Motor Imagery on the Voluntary Motor System These authors then suggested that the small cortical outputs may have modiﬁed motoneuronal excitability to optimize the discharge rate of the motor units. In the present study, a surprising result was observed in TA muscular activity, which increased signiﬁcantly during the resting period of the MI standing session. This last result corroborates the idea that motor strategies could diﬀer between MI and rest in the absence of quantitative change in EMG activity, although it suggests a diﬀerent hypothesis than increased motoneuronal excitability. An increase in TA activity during an exaggerated CoP displacement as compared to normal standing has been previously reported showing that TA contribution to maintaining balance may vary depending on the task (Johannsson et al., 2015). This change can be attributed to diﬀerent agonist-antagonist strategies, with the quiet upright posture mainly controlled by the triceps surae. This behavior of the muscle TA may be the reﬂection of altered network activity at the spinal level, in the circuitry mediating TA-triceps surae relationship, rather than in motoneuron excitability itself, such as pre-synaptic circuitry. To this end, the analysis of neuromuscular parameters that change with MI in standing posture, such as spinal excitability, could provide some interesting clues. Interestingly, it has recently been shown that MI speciﬁcally leads to a partial yet complete removal of pre-synaptic inhibition (Grosprêtre et al., 2019). In fact, pre-synaptic inhibition is mediated by speciﬁc structures, particularly by the primary aﬀerent depolarizations interneurons which regulate the Ia-to- alpha motoneuronal synapse and are the main contributor of an H-reﬂex downward ﬂuctuation (Rudomin and Schmidt, 1999). These interneurons were shown to have a lower activation threshold than the alpha motoneurons of the ventral horn of the spinal cord (Daniele and MacDermott, 2009). Therefore, such interneurons are likely more aﬀected by a subliminally cortical output such as generated during MI. It was previously shown November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 15 Neural Responses to Motor Imagery Grosprêtre et al. MI is of great importance to potentiate its neuromuscular and cardiometabolic eﬀects. that MI can therefore enhance H-reﬂex amplitude in the presence of a pre-activation of such structures, such as during muscle stretch, independently of the initial reﬂex size (Grosprêtre et al., 2016). It can be argued that the potential mechanisms leading to an increased H-reﬂex during muscle lengthening are likely to be involved during standing. Additional Effects of Posture and Motor Imagery on the Voluntary Motor System In the presence of an active motor state due to standing posture requirement, an extra quantity of neurotransmitter in the cortico-motoneuronal synapse due to MI would have led to a greater RMS observed during MI as compared to rest. Therefore, the second hypothesis can be ruled out here, in favor of a pre-synaptic inhibitory network modulation. Additional Effects of Posture and Motor Imagery on the Voluntary Motor System Indeed, the standing posture is regulated by the reﬂexive loop activation generated by the successive stretches of the triceps surae during antero-posterior sways (Cattagni et al., 2014). Therefore, the high initial level of pre-synaptic inhibition during standing as compared to sitting lead more room for MI to enhance the H-reﬂex amplitude. This is accounted in the present study by a globally greater eﬀect of MI on H-reﬂex when participants were standing. Furthermore, it should be highlighted that, in the present study, the MI task was an isometric contraction of the calf muscles, which represent a very simple task to mentally represent. Asking the participant to perform MI of such a simple task allows to mentally focus on the motor command that is not challenging regarding balance and may provide speciﬁc cardiometabolic responses. Therefore, this represents a dual task that can be used to reduce postural sway, contrary to the MI of more challenging tasks and could thus be performed by athletes but also patients with physiological limitations. The present results should be put in perspective regarding more complex mental tasks, such as MI of walking. In addition, whether these ﬁndings are consistent amongst athletes and patients warrant further study. Additionally, when the MI was performed repetitively, such as during a full training session, we observed that the eﬀect of MI on spinal excitability was exacerbated. Yet, H-reﬂex was not enhanced by MI in pre-measurements, in sitting nor in standing conditions, while showing a positive eﬀect in post-measurements. In the sitting and relaxed condition, two potential candidates for such eﬀect of prolonged MI practice has already been raised (Grosprêtre et al., 2019). 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FUNDING SG, UM, and PG are funded by the French National Agency of Research (ANR), Grant Number: ANR-20-CE37-0007. SG, UM, and PG are funded by the French National Agency of Research (ANR), Grant Number: ANR-20-CE37-0007. November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 16 Neural Responses to Motor Imagery Grosprêtre et al. REFERENCES Modulation of spinal excitability by a sub-threshold stimulation of M1 area during muscle lengthening. Neuroscience 263, 60–71. doi: 10.1016/j.neuroscience.2014. 01.013 November 2021 \| Volume 12 \| Article 762452 Frontiers in Physiology \| www.frontiersin.org 17 Neural Responses to Motor Imagery Grosprêtre et al. interpretation, and clinical use. Eur. Heart J. 17, 354–381. doi: 10.1093/ oxfordjournals.eurheartj.a014868 interpretation, and clinical use. Eur. Heart J. 17, 354–381. doi: 10.1093/ oxfordjournals.eurheartj.a014868 Guillot, A., Collet, C., Nguyen, V. A., Malouin, F., Richards, C., and Doyon, J. 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https://openalex.org/W2905779404	https://europepmc.org/articles/pmc6337143?pdf=render	English	null	Evaluation of the Performance of Helmet Prototypes Fabricated from Acrylonitrile Butadiene Styrene Composites Filled with Natural Resource	Materials	2,018	cc-by	7,739	Siti Nikmatin 1,, Bambang Hermawan 1, Irmansyah Irmansyah 1, Mohammad Nur Indro 1, Ahmad Beng Hong Kueh 2,3 and Achmad Syaﬁuddin 4 1 Department of Physics, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, 16680 Bogor, Indonesia; bhermawan1000@gmail.com (B.H.); irmansyah@ipb.ac.id (I.I.); mnindro@gmail.com (M.N.I.) 1 Department of Physics, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, 16680 Bogor, Indonesia; bhermawan1000@gmail.com (B.H.); irmansyah@ipb.ac.id (I.I.); mnindro@gmail.com (M.N.I.) 2 Construction Research Centre (CRC), Institute for Smart Infrastructure and Innovative Construction (ISIIC) Faculty of Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia; kbhahmad@utm.my 2 Construction Research Centre (CRC), Institute for Smart Infrastructure and Innovative Construction (ISIIC), Faculty of Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia; kbhahmad@utm.my 3 Department of Civil Engineering, Faculty of Engineering, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 4 Department of Water and Environmental Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia; udenﬁsika@gmail.com 4 Department of Water and Environmental Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia; udenﬁsika@gmail.com Correspondence: snikmatin@apps.ipb.ac.id * Correspondence: snikmatin@apps.ipb.ac.id * Correspondence: snikmatin@apps.ipb.ac.id Received: 20 November 2018; Accepted: 20 December 2018; Published: 22 December 2018 Abstract: The performance of helmet prototypes fabricated from acrylonitrile butadiene styrene composites ﬁlled with oil palm empty fruit bunch ﬁbers was evaluated. The ﬁbers were produced using a milling procedure, while the composites were fabricated using a single-screw extrusion. The physical characteristics of the produced ﬁbers, which are water content, size, and density, were investigated. In addition, the mechanical properties of the produced helmets, including shock absorption, yield stress, frequency, and head injury criterion (HIC), were examined. The impact strength of the produced helmets increases with the rise of ﬁller content. In addition, the helmets were also able to withstand a considerable pressure such that the transmitted pressure was far under the maximum value acceptable by the human skull. The present work also found that HICs exhibited by the investigated helmet prototypes fulﬁll all the practical guidelines as permitted by the Indonesian government. In terms of novelty, such innovation can be considered the ﬁrst invention in Indonesia since the endorsement of the use of motorcycle helmets. Keywords: oil palm; acrylonitrile butadiene styrene; natural ﬁllers; mechanical properties Evaluation of the Performance of Helmet Prototypes Fabricated from Acrylonitrile Butadiene Styrene Composites Filled with Natural Resource Siti Nikmatin 1,*, Bambang Hermawan 1, Irmansyah Irmansyah 1, Mohammad Nur Indro 1, Ahmad Beng Hong Kueh 2,3 and Achmad Syaﬁuddin 4 materials materials 1. Introduction Helmets have been widely worn as a form of protective headgear, with the primary goal of reducing the risk of injury caused by impact incidents [1–6]. Although the development of helmets is well-established, there is a motivated necessity for fabricating helmet shells that meet certain stringent safety requirements, especially with respect to improving their energy absorption. In particular, the capability of motorcycle safety helmets to absorb the impact energy is one of the critical factors for consideration to reduce severe injury. A well-performing helmet must concurrently protect the human skull and brain, since impact damage induced during a motorcycle accident can cause death. Realizing the life-threatening risk of such an event, the understanding of skull fractures and brain damage inﬂicted during accidents, including their protection measure, have been of utmost priority to fabricators. Materials 2019, 12, 34; doi:10.3390/ma12010034 www.mdpi.com/journal/materials Materials 2019, 12, 34 2 of 12 Several investigations have been carried out to study issues concerning head injury worldwide [7–11]. An investigation using 61 real-world accidents to evaluate the head injury criterion (HIC) and the head impact power (HIP), as well as the injury mechanisms, have been carried out [10]. These studies found that the moderate and severe neurological injuries cannot be distinguished solely on the global head acceleration, instead requiring a criterion computed using the intracranial variables. An experimental veriﬁcation to identify the resonance frequencies of two kinds of freely vibrating human dry skulls and extrapolation of the results to living skulls by taking into account all known and estimated differences in mechanical properties has also been conducted [8]. Alternatively, a computational model employing the ﬁnite element model (FEM) to simulate the head injury was also proposed [7]. From these analyses, it was found that the composite shell systems exhibit lower shear performance, provide additional energy absorbing mechanisms, and result in better helmet crashworthiness behavior [9]. A good strategy to reduce the head injury during an accident is by involving a design with reputable criteria, as permitted by regulations given by a standardization. In order to improve the performance of helmets in terms of their energy absorption, recommendations for future helmets include the employment of materials that are capable of absorbing the impact energy during an accident, while keeping the acceleration transmitted to the head at a safe level. 2.1. Materials Oil palm empty fruit bunches (OPEFB) were collected from the PT Perkebunan Nusantara VIII Cikasungka, Bogor, Indonesia. Recycled and virgin acrylonitrile butadiene styrene (Torray Toyulac Resin 100MPJ40049689 NLG) were purchased from the PT MUB Jaya Cibinong, Bogor, Indonesia. A coupling agent, maleic anhydride, from Merck, Darmstadt, Germany was used in this work. In addition, an additive, antioxidant primer (butylated hydroxytoluene) from Tedia, Mumbai, India, was employed. The carvine 0331 Polyurethane 2K Z-331-039, carvine 0331 Polyurethane H-331-014, and carvine 0331.T-0378 were used for painting in three stages, namely, as the base, hardener, and thinner, respectively. The paints were supplied by the PT Murni Cahaya Pratama, Bogor, Indonesia. In addition, a polystyrene foam having a density of 33 g/cm3, wrapped using fabric with a thickness of 10 mm, and a visor fabricated using the polycarbonate were used to complete the overall structure of the helmet prototypes. 2.2. Short Fibers Production The OPEFBs were initially washed using tap water to remove any impurities, and then immersed in tap water for 72 h. To remove water content, the OPEFBs were dried under the sun for 24 h, followed by oven (Xenaco, Guangzhou, China) drying at a temperature of 100 ◦C for 8 h. The drying process was carried out such that the water content in the OPEFBs was less than 10%. Short ﬁber ﬁller production was carried out using a hammer mill (Model HMV-4W-5.5, PT MUB Jaya Cibinong, Bogor, Indonesia) with a rate of 5000 rpm for 10 min. Hammer milling is a mechanical treatment to minimize particle size with the combined actions of collision, shaking, and milling. 1. Introduction Therefore, it is constantly a great challenge to provide the right composition of helmet materials that have the proper set of mechanical properties. Several studies have reported that wearing helmets reduces fatalities by more than 25% [12,13]. Moreover, it has been proven that non-helmeted motorcyclists are up to 3.4-fold more likely to die compared to helmeted riders in trafﬁc crashes [14]. Recently, the exploration and utilization of natural materials have formed huge engineering and commercial interests [15–19]. This is because the use of natural composite materials provides many beneﬁts, encompassing not only the environment but also economy and social aspects [20]. Along with cost-saving and ecological beneﬁts such as improvement in CO2-balance, the main motivation driving these developments is related to the mechanical property proﬁles of natural materials, which offer reinforcement potential [21]. Furthermore, several studies have proven that the performance of some natural materials in ﬁber form is closely comparable to those of synthetic glass ﬁbers [22]. Combined with the low-density of natural ﬁllers, employing natural ﬁber composites results in lighter structures when compared to mineral-, short glass ﬁber-, long glass ﬁber-, and short carbon ﬁber-reinforced materials [23]. In addition, natural ﬁber composites can also be processed similarly to these different composite classes, such as by employing injection molding and extrusion techniques. In terms of processing, it was well established that natural ﬁber composites offer advantages in regard to equipment wear [23]. Considering the aforementioned concerns, the present work aims to evaluate the performance of helmet prototypes produced from acrylonitrile butadiene styrene composites ﬁlled with oil palm empty fruit bunch ﬁbers. Up to this instance, the composite formed by this type of natural ﬁber has not been examined as a helmet material. The present study offers an investigation into the advantage of natural resources as ﬁllers for advanced engineering applications. The present study reveals that the proposed helmet prototype is in general suitable for applications, as regulated by the Indonesian National Standardization (SNI). It is worth noting that such innovation can be considered the ﬁrst invention in Indonesia since the introduction of the use of motorcycle helmets. The ﬁrst Indonesian mandatory helmet law was enacted in 1984. In 1986, the Department of Transportation instituted a regulation making helmet usage mandatory for all motorcyclists. This development is expected to contribute to reducing head and neck injuries and deaths from motorcycle crashes. 3 of 12 Materials 2019, 12, 34 2.3. Granular Production Composition of composites for the granular production for all helmet samples is listed in Table 1. It is noted that SN1, SN2, and SN3 refer to the helmets with ﬁller content of 15% and recycled acrylonitrile butadiene styrene (ABS), ﬁller content of 15% and virgin ABS, and ﬁller content of 20% and recycled ABS, respectively. The granular composite was produced using the single-screw extruder machine (Model HXSJ-125/125, Kaixin, Nanjing, China). In the preparation, the ﬁller, ABS, coupling agent, and additive were mixed at a speed of 15,000 rpm for 15 min. The total mass of the sample was averagely measured as 50 kg. The mixed samples were processed using the single-screw extruder machine and blended with gradient temperatures of 195, 215, 220, 220, 220, 225, 225, and 225 ◦C. Table 1. Material composition of the presently proposed helmets. Type of Helmet Filler (%) Type of ABS Coupling Agent (%) Additive (%) SN1 15 Recycle 2 1 SN2 15 Virgin 2 1 SN3 20 Recycle 2 1 Table 1. Material composition of the presently proposed helmets. In this process, composites having a granular diameter of 3 mm were obtained. In addition, this process can produce composites having a water content of about 30%. Next, the composites were dried under the sun for 24 h to reduce water content down to 13%, followed by oven drying at a temperature of 80 ◦C for 3 h to have a further water content reduction down to 7%. It is recommended that the water content of composites for helmet production using the molding injection machine is kept to be less than 10%. 4 of 12 Materials 2019, 12, 34 Materials 2019, 12, 34 2.5. Shock Absorption Tes 2.5. Shock Absorption Test 2.5. Shock Absorption Tes 2.5. Shock Absorption Test A shock absorption test was performed using the uniaxial impact machine (CADEX, model 1000_00_MIMA, Cadex Inc., Saint‐Jean‐sur‐Richelieu, QC, Canada). This test was carried out according to the Indonesian National Standardization (SNI 1811‐2007). The test was performed in three repetitions. Two different anvil types were employed for the test, i.e., hemispherical and flat. The hemispherical anvil has a spherical surface radius of 48 mm, constituting one‐half of the surface of a full sphere. The flat anvil has a flat surface of minimum side dimensions of 125 mm and a thickness of 24 mm. Additionally, Flat‐1 and Flat‐2 anvils refer to the anvil dropped from 5 m and 3 m in height, respectively. A shock absorption test was performed using the uniaxial impact machine (CADEX, model 1000_00_MIMA, Cadex Inc., Saint-Jean-sur-Richelieu, QC, Canada). This test was carried out according to the Indonesian National Standardization (SNI 1811-2007). The test was performed in three repetitions. Two different anvil types were employed for the test, i.e., hemispherical and ﬂat. The hemispherical anvil has a spherical surface radius of 48 mm, constituting one-half of the surface of a full sphere. The ﬂat anvil has a ﬂat surface of minimum side dimensions of 125 mm and a thickness of 24 mm. Additionally, Flat-1 and Flat-2 anvils refer to the anvil dropped from 5 m and 3 m in height, respectively. 2.4. Helmet Production Helmet prototypes were then fabricated using an injection molding machine (Model HC-250, Hwa Chin, Tainan, China). In the barrel, the samples were blended with gradient temperatures of 195, 215, 220, 220, 220, 225, 225, and 225 ◦C. In addition, the helmets were painted in three stages, namely, for the base, hardener, and thinner. In addition, the polystyrene foam and visor were installed in all the helmets. In general, the helmets were produced according to the National Indonesia Standardization (SNI). For demonstration, various viewing perspectives for the presently fabricated helmet are shown in Figure 1. Materials 2018, 11, x 4 of 12 Figure 1. Design of the presently proposed helmet (a) front; (b) side and (c) back views. (c) (a) (b) Figure 1. Design of the presently proposed helmet (a) front; (b) side and (c) back views. Figure 1. Design of the presently proposed helmet (a) front; (b) side and (c) back views. Figure 1. Design of the presently proposed helmet (a) front; (b) side and (c) back views. 2.6. Yield Stress Estimatio 2.6. Yield Stress Estimation The yield stress was estimated using the procedure proposed by Mills and Gilchrist [24]. It can be computed by considering the contact geometry between a flat impactor and the spherical outer surface of the foam liner. If the amount of liner crush, x, is less than the radius of curvature, R, of the spherical outer surface, the contacted area, A, can be estimated using A = 2πRx. If the impact is applied to a hemispherical anvil, the equation must be modified as Equation 1: The yield stress was estimated using the procedure proposed by Mills and Gilchrist [24]. It can be computed by considering the contact geometry between a ﬂat impactor and the spherical outer surface of the foam liner. If the amount of liner crush, x, is less than the radius of curvature, R, of the spherical outer surface, the contacted area, A, can be estimated using A = 2πRx. If the impact is applied to a hemispherical anvil, the equation must be modiﬁed as Equation 1: 𝐴ൌ2𝜋𝑥ሺ1 𝑅ଵ ൅1 𝑅ଶ ሻିଵ (1) A = 2πx( 1 R1 + 1 R2 ) −1 (1) (1) (1) where R1 and R2 are the radii of the helmet and anvil, respectively. It has been assumed that the foam yields over an area, A, of radius, a, with a constant yield stress, σ. The force transmitted by the foam can be estimated by F=A𝜎. Therefore, the yield stress can be estimated using the following equation: 𝜎 𝑚𝑎 (2) where R1 and R2 are the radii of the helmet and anvil, respectively. It has been assumed that the foam yields over an area, A, of radius, a, with a constant yield stress, σ. The force transmitted by the foam can be estimated by F = Aσ. Therefore, the yield stress can be estimated using the following equation: where R1 and R2 are the radii of the helmet and anvil, respectively. It has been assumed that the foam yields over an area, A, of radius, a, with a constant yield stress, σ. The force transmitted by the foam can be estimated by F=A𝜎. Therefore, the yield stress can be estimated using the following equation: 𝑚𝑎 (2) where R1 and R2 are the radii of the helmet and anvil, respectively. It has been assumed that the foam yields over an area, A, of radius, a, with a constant yield stress, σ. 2.6. Yield Stress Estimatio 2.6. Yield Stress Estimation The force transmitted by the foam can be estimated by F = Aσ. Therefore, the yield stress can be estimated using the following equation: ൌ𝐴 (2) dummy and a is the acceleration of the helmet and anvil. σ = ma A (2) (2) nvil (2) Frequency Analysis re m is the mass of the helmet and head dummy and a is the acceleration of the helmet and anvil 2.7. Frequency Analysis where m is the mass of the helmet and head dummy and a is the acceleration of the helmet and anvil. 2.7. Frequency Analysis where m is the mass of the helmet and head dummy and a is the acceleration of the helmet and anvil. 5 of 12 Materials 2019, 12, 34 2.7. Frequency Analysis 2.7. Frequency Analysis In this analysis, a mass-spring-mass model developed by Gao and Wampler [25] was adopted. In general, it begins by applying Newton’s second law of motion and Hooke’s law formulas as: ma = −kx (3) (3) By deﬁning a function of the position of mass with respect to time as x(t) = Acos(ωt), Equation (3) b By deﬁning a function of the position of mass with respect to time as x(t) = Acos(ωt), Equation (3) becomes: k k m −ω2 (Acos(ωt)) = 0 (4) (4) It can be obtained from Equation 4 that ω = √ k/m. Since ω = 2πf, the frequency, f, can be estimated using the following equation: f = 1 2π r k∗ m with k∗= k1k2 k1 + k2 (5) (5) where m is the total mass of the helmet and head dummy and k1 and k2 are the stiffness constants of the helmet and head dummy, respectively. where m is the total mass of the helmet and head dummy and k1 and k2 are the stiffness constants of the helmet and head dummy, respectively. 2.8. Head Injury Criterion Analysis A head injury can be deﬁned as any incident that results in trauma to the skull or brain. Among all injury criteria, HIC is the most globally used for measuring the severity of injury in the cases where the human head is engaged as the impacted mass. By measuring the energy required to cause concussive effects, a limit between impact intensities causing fatal and non-fatal injuries can be determined. The analytical expression of HIC is described by the following equation [25]: HIC = ([ 1 t2 −t1 t2 Z t1 ˆa(t)dt] 2.5 (t2 −t1)) max (6) (6) where t1 and t2 are the initial and ﬁnal times (in seconds) of the interval, during which HIC attains a maximum value, and acceleration, ˆa, is measured in gs (standard gravity acceleration). It is useful to note that the measurement in gs means that ˆa is a/g, with a as the head acceleration and g as the acceleration of gravity in any compatible units. Therefore, ˆa is deﬁned as the normalized head acceleration. As an alternative, the overall normalized impact resistance index can be estimated for comparative purposes [26,27]. 3.1. Physical Properties of the Produced Fibers The drying process carried out in this work is useful to ensure that the water content of the oil palm short ﬁbers is less than 10%. They were 58.0%, 17.4%, and 7.2% for before drying, after drying under the sun, and after drying in an oven, respectively. It is noticeable that the lowest water content, which was less than 10%, can be obtained using the presently employed method. Figure 2 shows the morphology of the oil palm short ﬁbers. The length and diameter of the short ﬁbers were 230.1 ± 95.3 µm and 58.5 ± 23.0 µm, respectively. These values were obtained by the measurement of random samples using an optical microscopy. 6 of 12 Materials 2019, 12, 34 Figure 2. Morphology of the currently produced OPEFB. 100 μm Figure 2. Morphology of the currently produced OPEFB. Figure 2. Morphology of the currently produced OPEFB. Figure 2. Morphology of the currently produced OPEFB. Table 2. Densities of the studied filler from various works. Type of Oil Palm Fiber Density (g/cm3) Country Reference Short fibers 0.7 to 1.55 India Sreekala et al. [31] Short fibers 1.03 India Rao and Rao [30] Short fibers 1.4 India Joseph et al. [28] Short fibers 1.03 India Rao and Rao [30] Short fibers 1.15 Malaysia Yusoff et al. [32] Short fibers 1.15 Indonesia Karina et al. [29] Table 2 presents the densities of OPEFBs for different studies [28–32]. It is noticeable that the densities reported by various researchers are not identical because of the variation in the kind of oil palm ﬁber used. In addition, there exists some ﬂuctuation in the irregular sectional areas, causing the length determination difference of OPEFBs [33]. Nevertheless, the current study found that the density of the produced ﬁbers was in an acceptable range with those obtained from the previous works. Various investigations have reported that the densities of OPEFB ﬁbers are in the range of 0.7 to 1.55 g/cm3, less than that of glass ﬁber, which is 2.6 g/cm3. This suggests that using OPEFB ﬁbers as the reinforcement of a composite can reduce its total mass compared to the use of glass ﬁber. Therefore, studies on the exploration of OPEFB ﬁbers as an alternative for glass ﬁber have been popular and of high potential for applications. Short fibers 1.35 Indonesia Present work 2. 3.1. Physical Properties of the Produced Fibers Helmet Prototype Helmets produced from ABS composites filled with OPEFB fibers following the SNI 1811‐20 andardization can be well‐expected to improve in their physical‐mechanical properties, particular terms of good impact energy absorption during traffic accidents. Figure 3 shows the prototype e presently produced helmet. In order to get an attractive appearance, the helmet was also painte elmet assembly was done by adding a layer of expanded polystyrene (EPS), soft lining, and oth cessories required in accordance with SNI 1811‐2007. Table 2. Densities of the studied ﬁller from various works. Type of Oil Palm Fiber Density (g/cm3) Country Reference Short ﬁbers 0.7 to 1.55 India Sreekala et al. [31] Short ﬁbers 1.03 India Rao and Rao [30] Short ﬁbers 1.4 India Joseph et al. [28] Short ﬁbers 1.03 India Rao and Rao [30] Short ﬁbers 1.15 Malaysia Yusoff et al. [32] Short ﬁbers 1.15 Indonesia Karina et al. [29] Short ﬁbers 1.35 Indonesia Present work 1.35 Indonesia Table 2. Densities of the studied ﬁller from various works. 3.2. Helmet Prototype Helmets produced from ABS composites ﬁlled with OPEFB ﬁbers following the SNI 1811-2007 standardization can be well-expected to improve in their physical-mechanical properties, particularly in terms of good impact energy absorption during trafﬁc accidents. Figure 3 shows the prototype of the presently produced helmet. In order to get an attractive appearance, the helmet was also painted. Helmet assembly was done by adding a layer of expanded polystyrene (EPS), soft lining, and other accessories required in accordance with SNI 1811-2007. Materials 2019, 12, 34 Helmet assembly accessories require 7 of 12 d other 7 of 12 d other Figure 3. Presently proposed helmet prototype: (a) above and (b) side views. (a) (b) Figure 3. Presently proposed helmet prototype: (a) above and (b) side views. (a) Figure 3. Presently proposed helmet prototype: (a) above and (b) side views. Figure 3. Presently proposed helmet prototype: (a) above and (b) side views. 3.3. Mechanical Propertie 3.3. Mechanical Properties Composite granules were used to produce the helmet specimens using the molding machine. Table 3 lists the impact characteristics of the helmets. As previously mentioned, all testing was conducted according to the SNI. The test was carried out at three different temperatures. This study found that the impacts of SN1 at a temperature of −20 ◦C were in the range of 81.3 to 121.7 G. In addition, their impacts at a temperature of 50 ◦C were in the range of 113.2 to 137.9 G. Table 3. Impact characteristics of the currently produced helmets. Code Temperature (◦C) Clash Position Anvil Type Impact (G) SN1 −20 Backside Flat-1 121.7 Flat-2 103.6 Topside Hemispherical 81.3 50 Backside Flat-1 137.9 Flat-2 113.2 Topside Hemispherical 127.4 SN2 −20 Backside Flat-1 151.7 Flat-2 133.6 Topside Hemispherical 86.1 50 Backside Flat-1 130.3 Flat-2 122.2 Topside Hemispherical 101.7 SN3 −20 Backside Flat-1 317.1 Flat-2 357.2 Topside Hemispherical 310.2 50 Backside Flat-1 240.5 Flat-2 270.5 Topside Hemispherical 230.1 As a comparison, the impact values for SN2 tested at temperatures of −20 ◦C and 50 ◦C ranged from 86.1 to 151.7 G and 101.7 to 130.3 G, respectively. In addition, the corresponding values for SN3 were 310.2 to 357.2 G and 230.1 to 270.5 G, respectively. It was found that the impact values for SN3 were higher compared to SN1 and SN2. Increase in the impact strength was possibly due to the increase in the ﬁller content for SN3 (20%) compared to SN1 (15%) and SN2 (15%). Findings of this study are similar with those obtained from the previous works. For instance, increasing ﬁller contents (snail shell powder) from 0% to 40% increased the impact strength of the produced composites [34]. Table 3. Impact characteristics of the currently produced helmets. As a comparison, the impact values for SN2 tested at temperatures of −20 ◦C and 50 ◦C ranged from 86.1 to 151.7 G and 101.7 to 130.3 G, respectively. In addition, the corresponding values for SN3 were 310.2 to 357.2 G and 230.1 to 270.5 G, respectively. It was found that the impact values for SN3 were higher compared to SN1 and SN2. Increase in the impact strength was possibly due to the increase in the ﬁller content for SN3 (20%) compared to SN1 (15%) and SN2 (15%). Findings of this study are similar with those obtained from the previous works. Materials 2019, 12, 34 Materials 2019, 12, 34 8 of 12 A similar observation was also obtained when the composite material of low-density polyethylene (LDPE) as the base was mixed with raw kaolin [35]. From the relevant study, it was found that increasing the ﬁller content from 5% to 15% increases their composite strength from 0.3 to 0.5 kJ/m2. An increase in the impact strength can be attributed to the elastic behavior of the added ﬁller contents, which have high toughness and extendibility without a permanent deformation [36]. Moreover, the current study has conﬁrmed that the produced helmets offer good performance according to the SNI in terms of their impact strength performance. 3.4. Yield Stress and Frequency The stress yields for SN1 were found to be in the range of 0.99 to 1.32 MPa and 25.94 to 40.65 MPa when the produced helmets were tested at temperatures of −20 ◦C and 50 ◦C using the ﬂat and hemispherical anvils, respectively. At −20 ◦C, the frequency range was 50.74 to 94.21 Hz, while it was from 53.04 to 81.99 Hz at 50 ◦C. In summary, the yield stress and frequency characteristics of SN2 and SN3 are presented in Table 4 (detailed computation is provided in the supporting document, A1). Table 4. Yield stress and frequency of the tested helmets. Code Temperature (◦C) Clash Position Anvil Type Yield Stress (MPa) Frequency (Hz) SN1 −20 Backside Flat-1 1.17 54.99 Flat-2 0.99 50.74 Topside Hemispherical 25.94 44.95 50 Backside Flat-1 1.32 58.54 Flat-2 1.08 53.04 Topside Hemispherical 40.65 56.26 SN2 −20 Backside Flat-1 1.45 61.4 Flat-2 1.28 57.62 Topside Hemispherical 27.48 46.25 50 Backside Flat-1 1.25 56.9 Flat-2 1.17 55.1 Topside Hemispherical 32.45 50.27 SN3 −20 Backside Flat-1 3.04 88.77 Flat-2 3.42 94.21 Topside Hemispherical 98.99 87.80 50 Backside Flat-1 2.30 77.31 Flat-2 2.59 81.99 Topside Hemispherical 73.43 75.62 3.5. HIC Characteristics Table 4. Yield stress and frequency of the tested helmets. Table 4. Yield stress and frequency of the tested helmets. 3.5. HIC Characteristics 3.3. Mechanical Propertie 3.3. Mechanical Properties For instance, increasing ﬁller contents (snail shell powder) from 0% to 40% increased the impact strength of the produced composites [34]. 3.5. HIC Characteristics Based on ASTM-F1292-04, the time interval for evaluation for impact performance is restricted to a maximum of 15 ms, and HIC < 1000 is known as a critical value for avoiding fatal injuries to the human head. This implies that a very high head acceleration is tolerable for a brief period of time. The probabilities of brain injury at different HIC scores are documented in the ASTM-F1292-04 standardization as listed in Table 5. The values shown emphasize the importance of proper and effective protection. 9 of 12 Materials 2019, 12, 34 Table 5. Head injury criteria. HIC Score Moderate Injury (%) Moderate Injury (%) Critical Injury (%) Fatal (%) 0 0 0 0 0 250 40 20 0 0 500 80 40 2 0 750 95 70 4 0 1000 98 90 8 2 1250 100 95 10 2 1500 100 98 20 4 1750 100 100 45 10 2000 100 100 70 30 2250 100 100 90 70 2500 100 100 95 90 2750 100 100 98 95 3000 100 100 100 100 HIC scores of the presently proposed helmet are presented in Table 6. In addition, HIC curve examples of the prototype helmet with testing temperatures of −20 ◦C and 50 ◦C are shown in Figure 4. HIC scores were found in the range of 804.69 to 828.38 when the helmets were tested at −20 ◦C. At 50 ◦C, the HICs were in the range of 769.63 to 792.75. Based on the HIC listed in the table, it is ratiﬁed that the presently proposed helmet can be categorized as offering good protection, with a 95% chance of minor injury, 70% chance of moderate injury, and 4% chance of critical injury. For a comprehensive overview, HIC characteristics of SN2 and SN3 are also presented in Table 6. Therefore, it is obvious that the proposed helmet has a great conformity with the Indonesian standard, SNI 1811-2007. This is because SNI 1811-2007 has regulated that the permitted HIC is <3000. Table 6. Head injury criteria of the presently proposed helmets. 4. Conclusion 4. Conclusions This study was carried out to evaluate the performance of helmet prototypes produced from acrylonitrile butadiene styrene composites filled with oil palm empty fruit bunch fibers. Th mechanical properties of constituents for the helmet prototype have been characterized an presented. The presently fabricated fiber has a water content of less than 10%, density of 1.35 g/cm fiber length of 230.1 ± 95.3 μm, and fiber diameter of 58.5 ± 23.0 μm. The impact values for SN3 wer the highest compared to SN1 and SN2, suggesting an improved impact strength for a higher fille content. Furthermore, it was also found that all HICs produced by the helmet prototypes were <850 as permitted by the SNI regulation. In closing, the prototype helmets are in practical agreement an conform to the Indonesian standard, SNI 1811‐2007. This study was carried out to evaluate the performance of helmet prototypes produced from acrylonitrile butadiene styrene composites ﬁlled with oil palm empty fruit bunch ﬁbers. The mechanical properties of constituents for the helmet prototype have been characterized and presented. The presently fabricated ﬁber has a water content of less than 10%, density of 1.35 g/cm3, ﬁber length of 230.1 ± 95.3 µm, and ﬁber diameter of 58.5 ± 23.0 µm. The impact values for SN3 were the highest compared to SN1 and SN2, suggesting an improved impact strength for a higher ﬁller content. Furthermore, it was also found that all HICs produced by the helmet prototypes were <850, as permitted by the SNI regulation. In closing, the prototype helmets are in practical agreement and conform to the Indonesian standard, SNI 1811-2007. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1 Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/12/1/34/s1. Author Contributions: S.N. conducted the experiments and contributed to the drafting of the manuscript. I.I B.H., M.N.I., and A.B.H.K. involved in the data analysis. A.S. and A.B.H.K. involved in the interpretation o results and contributed to the critical discussion All authors reviewed and approved the manuscript Author Contributions: S.N. conducted the experiments and contributed to the drafting of the manuscript. I.I., B.H., M.N.I., and A.B.H.K. involved in the data analysis. A.S. and A.B.H.K. involved in the interpretation of results and contributed to the critical discussion. All authors reviewed and approved the manuscript. results and contributed to the critical discussion. All authors reviewed and approved the manuscript. 4. Conclusion 4. Conclusions Funding: The present work was funded by the Indonesian Ministry of Research, Technology and Highe Education under the scheme of Penelitian Terapan Unggulan Perguruan Tinggi (PTUPT). Collaboration an support from the Indonesia Oil Palm Estate Fund (Badan Pengelola Dana Perkebunan Kelapa Sawi Funding: The present work was funded by the Indonesian Ministry of Research, Technology and Higher Education under the scheme of Penelitian Terapan Unggulan Perguruan Tinggi (PTUPT). Collaboration and support from the Indonesia Oil Palm Estate Fund (Badan Pengelola Dana Perkebunan Kelapa Sawit [BPDPKS]), Ministry of Finance, Republic of Indonesia are highly appreciated. pp [BPDPKS]), Ministry of Finance, Republic of Indonesia are highly appreciated. Acknowledgments: The authors thank the Bogor Agricultural University for facilitating the research wo [BPDPKS]), Ministry of Finance, Republic of Indonesia are highly appreciated. Acknowledgments: The authors thank the Bogor Agricultural University for facilitating the research work. Acknowledgments: The authors thank the Bogor Agricultural Conﬂicts of Interest: The authors declare no conﬂict of interest. Acknowledgments: The authors thank the Bogor Agricultural University for facilitating the research work. Conﬂicts of Interest: The authors declare no conﬂict of interest. 3.5. HIC Characteristics Code Temperature (◦C) Clash Position Anvil Type HIC SN1 −20 Backside Flat-1 760 Flat-2 398 Topside Hemispherical 335 50 Backside Flat-1 799 Flat-2 433 Topside Hemispherical 453 SN2 −20 Backside Flat-1 1141 Flat-2 662 Topside Hemispherical 334 50 Backside Flat-1 980 Flat-2 587 Topside Hemispherical 411 SN3 −20 Backside Flat-1 904 Flat-2 858 Topside Hemispherical 694 50 Backside Flat-1 591 Flat-2 595 Topside Hemispherical 603 Table 6. Head injury criteria of the presently proposed helmets. Table 6. Head injury criteria of the presently proposed helmets. 10 of 12 10 of 1 10 of 12 10 of 1 Materials 2019, 12, 34 Materials 2018, 11, x Figure 4. Typical head injury criterion curves in accordance with SN1, with testing conditions of (a) −20 °C and (b) 50 °C. Other data for SN2 and SN3 are provided in the supporting document, A1. 0 5 10 15 20 0 50 100 150 200 Tim e (m s) G ravitational unit (G ) 0 5 10 15 20 0 20 40 60 80 100 Tim e (m s) G ravitational unit (G ) (b) (a) Figure 4. Typical head injury criterion curves in accordance with SN1, with testing conditions of (a) −20 ◦C and (b) 50 ◦C. Other data for SN2 and SN3 are provided in the supporting document, A1. l i 0 5 10 15 20 0 20 40 60 80 100 Tim e (m s) G ravitational unit (G ) (b) 0 5 10 15 20 0 50 100 150 200 Tim e (m s) G ravitational unit (G ) (a) Figure 4. Typical head injury criterion curves in accordance with SN1, with testing conditions of (a −20 °C and (b) 50 °C. Other data for SN2 and SN3 are provided in the supporting document, A1. Figure 4. Typical head injury criterion curves in accordance with SN1, with testing conditions of (a) −20 ◦C and (b) 50 ◦C. Other data for SN2 and SN3 are provided in the supporting document, A1. Conflicts o References References 1 Aiello M ; Galvanetto U ; Iannucci L Numerical simulations of motorcycle helmet impact tests Int J 1. Aiello, M.; Galvanetto, U.; Iannucci, L. Numerical simulations of motorcycle helmet impact tests. Int. J. Crashworthiness 2007, 12, 1–7. [CrossRef] 1. Aiello, M.; Galvanetto, U.; Iannucci, L. Numerical simulations of motorcycle helmet impact tests. Int. Crashworthiness 2007, 12, 1–7. 2 Chang L T ; Chang G L ; Huang J Z ; Huang S C ; Liu D S ; Chang C H Finite element analysis of th 2. Chang, L.T.; Chang, G.L.; Huang, J.Z.; Huang, S.C.; Liu, D.S.; Chang, C.H. Finite element analysis of the effect of motorcycle helmet materials against impact velocity. J. Chin. Inst. 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Abo Sabah, S.H.; Kueh, A.B.H.; Al-Fasih, M.Y. Bio-inspired vs. conventional sandwich beams: A low-velocity repeated impact behavior exploration. Constr. Build. Mater. 2018, 169, 193–204. [CrossRef] 27. Abo Sabah, S.H.; Kueh, A.B.H.; Al-Fasih, M.Y. Comparative low-velocity impact behavior of bio-inspired and conventional sandwich composite beams. Compos. Sci. Technol. 2017, 149, 64–74. [CrossRef] 28. Joseph, S.; Kenny, J.M.; Puglia, D.; Thomas, S.; Joseph, K. Oil palm microcomposites: Processing and mechanical behavior. Polym. Eng. Sci. 2010, 50, 1853–1863. [CrossRef] 29. Karina, M.; Onggo, H.; Abdullah, A.D.; Syampurwadi, A. Effect of oil palm empty fruit bunch ﬁber on the physical and mechanical properties of ﬁber glass reinforced polyester resin. J. Biol. Sci. 2008, 8, 101–106. 30. Rao, K.M.M.; Rao, K.M. Extraction and tensile properties of natural ﬁbers: Vakka, date and bamboo. Compos. Struct. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Conflicts o References 2007, 77, 288–295. [CrossRef] 12 of 12 12 of 12 Materials 2019, 12, 34 31. Sreekala, M.S.; George, J.; Kumaran, M.G.; Thomas, S. The mechanical performance of hybrid phenol-formaldehyde-based composites reinforced with glass and oil palm ﬁbres. Compos. Sci. Technol. 2002, 62, 339–353. [CrossRef] 31. Sreekala, M.S.; George, J.; Kumaran, M.G.; Thomas, S. The mechanical performance of hybrid phenol-formaldehyde-based composites reinforced with glass and oil palm ﬁbres. Compos. Sci. Technol. 2002, 62, 339–353. [CrossRef] 2. Yusoff, M.Z.M.; Salit, M.S.; Ismail, N.; Wirawan, R. Mechanical properties of short random oil palm ﬁ reinforced epoxy composites. Sains. Malays. 2010, 39, 87–92. 33. Virk, A.S.; Hall, W.; Summerscales, J. Failure strain as the key design criterion for fracture of natural ﬁbre composites. Compos. Sci. Technol. 2010, 70, 995–999. [CrossRef] 34. Onuegbu, G.C.; Igwe, I.O. The effects of ﬁller contents and particle sizes on the mechanical and end-use properties of snail shell powder ﬁlled polypropylene. Mater. Sci. Appl. 2011, 2, 811–817. [CrossRef] properties of snail shell powder ﬁlled polypropylene. Mater. Sci. Appl. 2011, 2, 811–817. [CrossRef] 35. Mallik, A.; Barik, A.K.; Pal, B. Comparative studies on physico-mechanical properties of composite materials of low density polyethylene and raw/calcined kaolin. J. Asian Ceram. Soc. 2015, 3, 212–216. [CrossRef] 35. Mallik, A.; Barik, A.K.; Pal, B. Comparative studies on physico-mechanical properties of composite materials of low density polyethylene and raw/calcined kaolin. J. Asian Ceram. Soc. 2015, 3, 212–216. [CrossRef] 36. Ruksakulpiwat, Y.; Sridee, J.; Suppakarn, N.; Sutapun, W. Improvement of impact property of natural ﬁber–polypropylene composite by using natural rubber and EPDM rubber. Compos. Part. B Eng. 2009, 40, 619–622. [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W1559203959	https://publicacionesfac.com/index.php/cienciaypoderaereo/article/download/77/75, https://dialnet.unirioja.es/descarga/articulo/5776212.pdf, https://www.redalyc.org/pdf/6735/673571165004.pdf	es		Reorganización para la seguridad aérea en la Fuerza Aérea Colombiana	Revista Ciencia y Poder Aéreo/Ciencia y poder aereo	2,007	cc-by	704	REORiiANIZACIÓN PARA LA !!iEiiURIDAD AÉREA EN LA FUERZA AÉREA COLOMBIANA E5pecialización en Gerenda de la Seguridad Aérea ~c---. MY. EOWARD _lARAMILLO 5ANCHEZ CT. WILLIAM ALBERTO NIETO LOPEZ DESCRIPCIÓN OBJETIVO General La investigación estuvo encaminada a la reorganización de la seguridad aérea en la Fuerza Aérea Colombiana, tomando como referencia el Plan Estratégico Institucional 2003-2010 en donde se plantea como objetivo, "Fortalecer los programas de prevención e investigación de accidentes aéreos para evitar la perdida de vidas, de valiosos recursos y el deterioro de la imagen institucional". Con el ánimo de fortalecer esté objetivo, se propone una reorganización de la seguridad que busca el mejoramiento continuo, para obtener resultados favorables ya que los integrantes de la organización saben el papel que desempeñan a nivel grupal dentro de la organización. Lo anterior conduce a un mejor clima organizacional y a lograr mayor eficacia y eficiencia en el desarrollo organizativo, alcanzando los resultados esperados. Reestructurar la organización actual de la seguridad aérea para la disminución de accidentes e incidentes aéreos y optimizar así los recursos institucionales. Esta estructura organizacional se rediseñó de forma que cada eslabón de la cadena cumple labores específicas, que sumadas favorecen que cada equipo de trabajo sea responsable de ciertos resultados, eliminando así los obstáculos al desempeño ocasionados por la confusión y la incertidumbre de la asignación de tareas. Esto. proporciona redes de toma de decisiones y de comunicaciones, que reflejan y respaldan los objetivos de la Fuerza Aérea Colombiana, disminuyendo la brecha que existe entre la estructura actual y la propuesta. • Rediseñar el modelo de la estructura organizativa de la Fuerza Aérea que permita la solución y prevención de los problemas actuales de la seguridad aérea. OBJETIVOS E§pecíñca§ • Evaluar la estructura actual de la organización de la Fuerza Aérea Colombia con el fin de conocer el estado actual de la organización. • Identificar los procesos y herramientas necesarias para el sistema de gestión de la calidad en la seguridad aérea. • Comparar modelos organizacionales de la seguridad aérea en otras Fuerzas Aéreas con el fin de diseñar un modelo para el caso de la Fuerza Aérea Colombiana. METDDDLD6ÍA Se realizó una investigación de carácter evaluativo, llevando a cabo un análisis funcional de la estructura de la organización y así tener una visión clara de las posibles fal encias a su interior. A .B !i.T RAC El diseño metodológico contó con una fase documental, en donde se recopilaron los datos existentes alrededor de la seguridad aérea aplicada a la FAC; asimismo, se desarrolló una fase de evaluación del modelo para la época de la organización, con la apl icación de la Matriz DOFA, en el Nivel Central, y en el Nivel Base Aérea. RESULTADOS Con base en la información encontrada, se realizó un diagnostico del Departamento de Seguridad Aérea, en dónde se concluyó que en el nivel central el Departamento carece de personal suficiente para realizar todas las actividades que tiene que cumplir, de igual forma, su estructura es rígida, y no ha sido modificada a pesar de que la Fuerza llevó a cabo en su interior cambios significativos a partir del Plan Estratégico Institucional 2003-2010. En las bases aéreas, los Departamentos de Seguridad Aérea cumplen funciones variadas que desvían la atención de los Jefes en actividades de menor importancia que demandan tiempo para su ejecución. Teóricamente, los cambios en la estructura organizacional se encuentran registrados en el Plan 1nstitucional 2003 2010; sin embargo, la actividad de la Fuer· za Aérea refleja que el diseño estructura debía flexibilizarse, porque no se nan evi tado los accidentes aéreos, y por el con trario siguen ocurriendo. Con base en este análisis, se estructuró e redi seño organizativo de la Seguridad Aé rea en la 1nstitución orientado hacia la pre vención de accidentes e incidentes y a un< gestión efectiva del conocimiento como e mecanismo adecuado. Es por ello que en 1< propuesta, se involucró activamente pan la ejecución y seguimiento de las campa ñas de prevención a los responsables dE la ejecución de los procesos ope_rati~o: (Bases Aéreas) y los responsables tune~o nales de la fuerza (Jefaturas). La labor de gestión de la seguridad así como la gestiór del conocimiento se plantean en cabez: del personal de oficiales y suboficiales d1 seguridad aérea.
https://openalex.org/W4366965084	https://www.genesispub.org/resource/images/articles/pdf247.pdf	English	null	Covid Cancer	Journal of Cancer Therapy and Research	2,022	cc-by	491	Covid Cancer ponding author: Causak TE Paul, BSc E, DULE, 23 Park Ave. Saint John, NB E2J 1R2, Canada. Canada Copyright© 2022 genesis pub by Cusack PTE. CC BY NC-ND 4.0 DEED. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License., This allows others distribute, remix, tweak, and build upon the work, even commercially, as long as they credit the authors for the original creation. Citation: Causak TE Paul. (2022) Covid Cancer. J Can Ther Res. 2(1):1-1. Received: September 20, 2022 \| Published: October 12, 2022 Introduction We have already established that COVID long haulers tend toward Chronic Kidney Disease (CKD) that leads to Anemia (or low Iron) . The loss of Iron may lead to cancer. Note the following balanced chemical equation involving iron and hydrogen peroxide: 2 Fe(OH)3 ➔ Fe2O3 + 3 H2O Note the following balanced chemical equation involving iron and hydrogen peroxide: 2 F (OH)3 ➔F 2O3 3 H2O We have established that cancer may be caused by hydrogen peroxide. This is simply hydroxide (OH-). In the equation, we see that low iron mean a surplus of OH- (hydroxide) that is fuel for a cancer to develop. I expect cancer rates to rise among the long haulers after COVID-19. We are fortunate to see researchers who have developed a new blood test to detect 50 types of cancer. It comes in the nick of time [1-3]. The future is brighter for cancer suffers. I suspect a cure for cancer involves simply Hydrogen to reduce hydroxide to water. I’m hoping someone who does experimental research to take up the torch and see if hydrogen is a cure of this plague that has scourged humanity. Journal of Cancer Therapy and Research Genesis-JCTR-2(1)-18 Volume 2 \| Issue 1 Open Access ISSN: 2583-6552 Covid Cancer Editorial \| Causak TE Paul. J Can Ther Res 2022, 2(1)-18. DOI: https://doi.org/10.52793/JCTR.2022.2(1)-18 1. Paul TE C. (2018) Hydrogen Peroxide and Cancer. Open Acc J Oncol Med 2(2) 2. Cusack PTE. (2020) COVID-19: The Progression. Int J Fam Med Prim Care. 1(3): 1013. 3. Paul TE Cusack. (2020) Battle with the Coronavirus. LOJ Nur Heal Car. 2(5). Editorial \| Causak TE Paul. J Can Ther Res 2022, 2(1)-18. DOI: https://doi.org/10.52793/JCTR.2022.2(1)-18 References Editorial \| Causak TE Paul. J Can Ther Res 2022, 2(1)-18. DOI: https://doi.org/10.52793/JCTR.2022.2(1)-18 References 1. Paul TE C. (2018) Hydrogen Peroxide and Cancer. Open Acc J Oncol Med 2(2) 2. Cusack PTE. (2020) COVID-19: The Progression. Int J Fam Med Prim Care. 1(3): 1013. 3. Paul TE Cusack. (2020) Battle with the Coronavirus. LOJ Nur Heal Car. 2(5). 1. Paul TE C. (2018) Hydrogen Peroxide and Cancer. Open Acc J Oncol Med 2(2) 2. Cusack PTE. (2020) COVID-19: The Progression. Int J Fam Med Prim Care. 1(3): 1013. 3. Paul TE Cusack. (2020) Battle with the Coronavirus. LOJ Nur Heal Car. 2(5). Editorial \| Causak TE Paul. J Can Ther Res 2022, 2(1)-18. DOI: https://doi.org/10.52793/JCTR.2022.2(1)-18 2
https://openalex.org/W3167569823	https://zenodo.org/records/7084786/files/25%2023343.pdf	English	null	Stress classification based on human electromagnetic radiation analysis	Indonesian journal of electrical engineering and computer science	2,021	cc-by	5,811	Indonesian Journal of Electrical Engineering and Computer Science Vol. 22, No. 2, May 2021, pp. 826~834 ISSN: 2502-4752, DOI: 10.11591/ijeecs.v22.i2.pp826-834 Indonesian Journal of Electrical Engineering and Computer Science Vol. 22, No. 2, May 2021, pp. 826~834 ISSN: 2502-4752, DOI: 10.11591/ijeecs.v22.i2.pp826-834 Indonesian Journal of Electrical Engineering and Computer Science Vol. 22, No. 2, May 2021, pp. 826~834 ISSN: 2502-4752, DOI: 10.11591/ijeecs.v22.i2.pp826-834 826  Keywords: Biological response Human electromagnetic Radiation Stress classification This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author: Siti Zura A. Jalil Razak Faculty of Technology and Informatics Universiti Teknologi Malaysia Jalan Sultan Yahya Petra, 54100 Kuala Lumpur, Malaysia Email: sitizura.kl@utm.my Stress classification based on human electromagnetic radiation analysis Tengku ‘Afiah Mardhiah Tengku Zainul Akmal1, Abd Hafiz Qayyum Abd Talib2, Siti Zura A. Jalil3 Siti Armiza Mohd Aris4 1,2,3,4Razak Faculty of Technology and Informatics, Universiti Teknologi Malaysia (UTM), Malaysia 2Cluster of Integrative Physiology and Molecular Medicine (CIPMM), Faculty of Medicine, Universiti Kuala Lumpur- Royal College of Medicine Perak, Malaysia ABSTRACT Stress is a feeling of emotional or physical tension due to events that makes one feel frustrated, angry or nervous. It is a situation that trigger biological response when a person encounters a threat or challenge. This paper discussed stress classification based on human electromagnetic radiation (EMR). EMR frequency are captured at seven major chakra points and being analyzed using multivariate analysis of variance (MANOVA) to identify the significance points for the classification. Locally weighted learning (LWL) algorithm is used to classify the collected data. The results show stress classification using EMR based on third eye and throat chakra points obtained accuracy of more than 60%. Article history: Received Oct 13, 2020 Revised Mar 24, 2021 Accepted Mar 30, 2021 Journal homepage: http://ijeecs.iaescore.com 1. INTRODUCTION Stress is defined as a threatened condition to the human body system stability. Human body reaction towards stress is known as adaptive process which involves physiological, biochemical and cognitive behavioral responses to gain body system stability [1]. Adaptive ability in dealing with stress will affect the risk of disease. Stress is a condition in which a person fails to adapt to the right conditions [2]. Stressful events are inevitable in life and need to overcome obstacles as a result of success. A person has the ability to control what they perceive as stress and how to respond to it. Stress response is the body's proactive step in adapting to a situation to encourage survival or motivate success, and can also be catastrophic when the body's response to stress is inappropriate. For instant, when a person experiences excessive situations or recurrent negative trauma, it can cause excessive stress levels and result in an inappropriate stress response that will prolong cortisol secretion [3], [4]. Stress has been a major concern in the current situations as chronic stress can leads to health issues such as heart disease, depression and anxiety. There are several factors that can trigger stress which includes major life changes, financial problems, and work. Many techniques have been used to assess stress. One of the current stress assessment is a lengthy process where patients need to answer multiple sets of questionnaires to be diagnose with stress [5], [6]. Several attempts also have been proposed on assessment and recognition of stress included using electrophysiosignal analysis Journal homepage: http://ijeecs.iaescore.com 827 Indonesian J Elec Eng & Comp Sci ISSN: 2502-4752  [7], [8]. Therefore, this study proposed an alternative stress pre-assessment using human electromagnetic radiation (EMR) analysis. [7], [8]. Therefore, this study proposed an alternative stress pre-assessment using human electromagnetic radiation (EMR) analysis. Several evidences have shown the existence of energy field in the form of electric, magnetic, optical and acoustic emitted from and contained within the human body theoretically and experimentally [9]. Human energy field is defined as an extremely weak electromagnetic (EM) field but measurable EM that are formed from a collection of electromagnetic waves [10]. EM is produced around the human body due to the movement or rotation of particles. Cells, tissues and organs assemble molecules and each molecular interaction in the human body radiate unique energy spectrum. This spectrum is the EM radiation for each respective molecule [11], [12]. 1. INTRODUCTION p Alternative medicine philosophy such as chakra has practiced healing through energy field. It is mentioned that one’s wellbeing is based on body energy balance and energy centered while any blockage or imbalance will affect the person’s health condition. Chakra is derived from a Sanskrit word meaning ‘wheel’. There are seven main chakras located from the perineum in the lower pelvis to the top of the head [13]. Each of these chakra points are associated with organs. When a person has an unresolved stress, this stress will cause disturbance to the body energy field which can contribute to physical illness. As the focus of this study is about stress, thus the chakra points that can be influenced by stress will be further discussed. There are several chakra points that are related to stress which are crown, third-eye, throat, solar plexus and root. Crown chakra is located at the top central of the skull. It is related to pineal gland which produces melatonin hormones for calming. Decreased melatonin production can cause anxiety or stress. Third eye chakra is located on the forehead between the eyebrows. This chakra externalizes the pineal gland thus treatment related to hormonal imbalance is done through this chakra as it governs lower brains, central nervous system, left eyes, ears and nose. Blockage of this chakra can lead to stress and anxiety [14]. Throat chakra is located in the throat to the base of the neck and collar bones. It is associated with communication and expression abilities. Weaken of this chakra can result to introvert behavior. Neck muscle tension due to stress is treated using acupuncture at this point. While throat is related to communication, solar plexus is associate to emotion which controls fear and anxiety. Thus, stress condition will inevitably influence the solar plexus chakra. It is located under the rib cage in the same area of diaphragm [15]. Root or base chakra is located at the base of the spine which is the chakra point that are closest to the earth. It responsible of anchoring the body on the physical plane and provide channel to express oneself. It is associated with adrenal medulla and cortex which produces adrenaline and cortisone. Blockage of this chakra will cause anxiety as the person is no longer grounded and reduced the gland secretion [14]. 1. INTRODUCTION There were five chakra points that are relevant to stress, however the frequency of human EMR is captured at seven chakra points for this study. Further data analysis based on the frequency of human EMR is performed to confirm the points closely related to stress. q y p p y Previous study based on EMR frequencies analysis on gender classification shows that gender can be distinguished using k-nearest neighbour (kNN) classification method [16]. There are 13 out of 23 points that are significant to differentiate gender. Male and female have different distribution of frequency radiations. Males are observed to have higher range of frequencies on both left and right side of the body compare to females [17]. Several studies on human EMR also demonstrates significant result in classifying body segment on upper body, torso, arm and lower body [18], and on left, right and chakra [19]. In addition, studies shown that EMR can be classified based on the person’s health condition [20], [21]. Significant results have been shown for down-syndrome and non-down syndrome person [20] and for stroke patients and to non-stroke participants, which support the assumption of human conditions can affect human EMR [21]. A recent comparative study shows that kNN classifier gives the lowest accuracy when comparing with J48, Bayes Net and locally weighted learning (LWL) algorithm in predicting breast cancer survival rate despite the high performance of kNN in previous studies. The LWL algorithm produces highest accuracy with 66.2% while kNN only able to predict at 56.1% accuracy [22]. The LWL performance also surpass kNN in identifying defective software modules using imbalanced dataset with 92.23% accuracy when validated with 10-fold cross validation and 91.08% accuracy when tested with percentage split of 66%. The result also has been validated using paired t-test with 99% confidence level [23]. Thus in this study, LWL algorithm will be used to classify the stress. 2. METHODOLOGY The proposed study involved four steps which are data acquisition, statistical analysis for pre- processing, classification and validation. Further details about these steps are explained in the next subsections. tress classification based on human electromagnetic… (Tengku ‘Afiah Mardhiah Tengku Zainul Akmal) ISSN: 2502-4752 828  2.1. Sampling The subjects for this study are students from Universiti Teknologi Malaysia, Kuala Lumpur. There are forty (40) volunteer students consist of twenty-nine (29) males and eleven (11) females are involves in this study. The stress is induced through virtual reality (VR) technology [24], [25]. The EMR frequencies are collected two (2) times, i.e before the stress is induced (before VR session) and after the stress is induced (after VR session). During data collection, the measurement room temperature is set constant at 24ºC and the data collection session is limited to four sessions per day. Session 1 is from 9.00am to 10.00am, session 2 is from 10.30am to 11.30am, session 3 is from 2.00pm to 3.00pm and session 4 is from 3.30pm to 4.30pm. 2.2. EMR data collection The EMR data is collected at seven points using body radiation wave detector which are the crown, third eye, throat, heart, solar plexus, sacral and root chakra points. The points’ label and location of each chakra points are described in Table 1. The EMR data acquisition is performed as shown in Figure 1. The EMR readings are taken five times at each point. The average EMR reading for each chakra points are calculated before further analysis. Table 1. Location of chakra points Chakra Points Label Location Crown CA Top of the head Third Eye CB Forehead between the eyes Throat CC Throat Heart CD Centre of chest just above the heart Solar Plexus CE Upper abdomen in the stomach area Sacral CF Lower abdomen, about two inches below the navel Root CG Base of spine in tailbone area Figure 1. EMR data acquisition Table 1. Location of chakra points Chakra Points Label Location Crown CA Top of the head Third Eye CB Forehead between the eyes Throat CC Throat Heart CD Centre of chest just above the heart Solar Plexus CE Upper abdomen in the stomach area Sacral CF Lower abdomen, about two inches below the navel Root CG Base of spine in tailbone area Figure 1. EMR data acquisition 2.3. Statistical analysis y The collected EMR data is analyzed using multivariate analysis of variance (MANOVA) in SPSS Statistics software version 23 to find significant points for the classification of stress state. MANOVA is a generalization of the general linear model of statistical analysis to situations where there are multiple dependent measures. In this study, the independent measures are VR stress session (before and after the stress is induced) and the dependent measures are EMR readings on chakra points of CA to CG. The analysis results in the F-ratio statistic is calculated for the EMR readings to indicate whether different values of before and after VR stress session have statistically significant effect on the EMR readings on chakra points. MANOVA are based on F-test, the larger F value and the smaller p-value (less than 0.05 for the sig. value). Hence, the F value and P value is evaluated to identify which points are significant in identifying the stress. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 2, May 2021 : 826 - 834 829 ISSN: 2502-4752 Indonesian J Elec Eng & Comp Sci  2.4. Data classification The datasets with significant attributes are classified using waikato environment for knowledge analysis (WEKA) software tool by utilizing the locally weighted learning (LWL) classifier. LWL classifier is one of the lazy learner classifiers. Lazy learners are advantageous when performing prediction using single training sets because only the immediate sections of the instance space are occupied by objects to be classified will be modeled. It can improve prediction accuracy by allowing the system to focus on deriving possible decision for exact points of the instance space for prediction. LWL refers to supervised learning of continuous functions which are in the context of kernel regression [26]. LWL forms lazy model around a point of interest whereby only training data that is local to that point is used during classification. LWL can be observed as approximation method function [27]. It is formulated as: 𝐹(𝑥𝑞) = 𝑤0 + 𝑤1𝑎1(𝑥𝑞) + ⋯+ 𝑤𝑛𝑎𝑛(𝑥𝑞) 𝐹(𝑥𝑞) = 𝑤0 + 𝑤1𝑎1(𝑥𝑞) + ⋯+ 𝑤𝑛𝑎𝑛(𝑥𝑞) (1) (1) Where 𝑎𝑖(𝑥𝑞) is the 𝑖𝑡ℎ attributes of point 𝑥𝑞, 𝑤𝑖 is the coefficient for each 𝑎𝑖(𝑥) and 𝐹(𝑥) is the target function determined by 𝑎𝑖(𝑥) and 𝑤𝑖. The lazy model is used to fit nearby data points by defining the error criterion as expressed in (2): 𝐸(𝑥𝑞) = 1 2 ∑ (𝐹(𝑥) −𝑓(𝑥)) 2𝐾( 𝑑(𝑥𝑞,𝑥) 𝛽 ) 𝑥∈𝐷 (2) (2) Where 𝑥𝑞 is the query point, the data point set containing k-nearest data point. 𝐾 is the kernel function to calculate weight for each data point to the distance. While 𝑑(𝑥𝑞, 𝑥) is the distance between query point 𝑥𝑞 to each data point 𝑥. The favorable approximation for the function output 𝐹(𝑥) can be obtained by determining the 𝑤𝑖. Gradient descent method is used to get the best estimation of 𝑤𝑖 with minimal error criterion (𝑥𝑞) . The training criterion is formulated as: ∆𝑤𝑖= 𝜂∑ 𝐾( 𝑑(𝑥𝑞,𝑥) 𝛽 )(𝐹(𝑥) −𝑓(𝑥))𝑎𝑖(𝑥) 𝑥∈𝐷 (3) (3) Where 𝜂 is the learning rate and 𝛽 is the bandwidth. The new weight is obtained through as (4): Where 𝜂 is the learning rate and 𝛽 is the bandwidth. The new weight is obtained through as (4): (4) 𝑤𝑖= 𝑤𝑖+ ∆𝑤𝑖 2.5. Data validation The classification training set is validated using k-fold cross validation. Cross validation is used to evaluate the accuracy of the classifier by repeating the classification process based on define numbers of k. The initial dataset will be randomly partition into k mutually exclusive or also known as folds, 𝐷1,𝐷2, ∷∶, 𝐷𝑘 , each of approximately equal size. The training and testing is executed for k times. For each iteration i, partition 𝐷𝑖 will be used as test set while the remaining partitions are used to train the model. The accuracy estimate is based on the overall number of correct classifications from k iterations and divided by total number of tuples in the initial data [28]. The classification evaluation are measured by true positive (TP) which refer to positive tuples that were correctly labeled by the classifier, true negative (TN) which is the negative tuples that were correctly labeled by the classifier, false positive (FP) are the negative tuples that were incorrectly classified and false negative (FN) are the positive tuples that were incorrecly labeled by the classifier. 3. RESULTS Table 2 shows the result of MANOVA analysis for the overall data. The results demonstrate that throat chakra is the most significant to differentiate stress state as the sig. or p-value is less than 0.05, then followed by third eye and solar plexus chakra. The overall classification results by each chakra points are tabulated in Table 3. The third eye chakra has the highest accuracy with 65% correctly classified and 90% TP Rate for classification of before VR session and 40% TP Rate for after VR session. The second highest correctly classified accuracy is the throat chakra with 53.80% and followed by root chakra with 52.50%. The overall classification results by combination of multiple chakra points are displayed in Table 4. Combination of chakra points are based on significant points found in the statistical analysis. The combination of third eye (CB) and throat chakra (CC) produce a highest accuracy of classification up to 66.25% and 90% of TP rate for before VR session. The second highest correctly classified accuracy is tress classification based on human electromagnetic… (Tengku ‘Afiah Mardhiah Tengku Zainul Akmal) ISSN: 2502-4752  830 followed by combination of crown (CA), third eye (CB), throat (CC), sacral (CF) and root (CG) with 65% accuracy. Table 2. MANOVA analysis for overall data Chakra Points F Sig. (p-value) Crown CA 1.839 0.179 Third Eye CB 3.883 0.052 Throat CC 6.881 0.011 Heart CD 2.007 0.161 Solar Plexus CE 3.586 0.062 Sacral CF 1.684 0.198 Root CG 1.414 0.238 Table 2. MANOVA analysis for overall data Chakra Points F Sig. (p-value) Crown CA 1.839 0.179 Third Eye CB 3.883 0.052 Throat CC 6.881 0.011 Heart CD 2.007 0.161 Solar Plexus CE 3.586 0.062 Sacral CF 1.684 0.198 Root CG 1.414 0.238 Table 3. Classification of chakra points Chakra Points Correctly Classified Incorrectly Classified TP Rate (After VR session) TP Rate (Before VR session) Crown CA 50.00 50.00 0.300 0.700 Third Eye CB 65.00 35.00 0.400 0.900 Throat CC 53.80 46.20 0.200 0.900 Heart CD 42.50 58.50 0.200 0.600 Solar Plexus CE 43.80 56.20 0.300 0.400 Sacral CF 46.30 54.70 0.400 0.600 Root CG 52.50 48.50 0.500 0.500 Table 3. Classification of chakra points Table 3. Classification of chakra points Table 4. 3. RESULTS Classification of combination chakra points CA CB CC CD CE CF CG Correctly Classified Incorrectly Classified TP Rate (After VR session) TP Rate (Before VR session)        63.75 36.25 0.400 0.875    -    63.75 36.25 0.400 0.875    - -   65.00 35.00 0.400 0.900    - - -  62.25 37.75 0.375 0.850 -   - - -  62.50 37.50 0.375 0.875 -   - - - - 66.25 33.75 0.425 0.900 3.1. EMR stress data The result of MANOVA analysis for EMR stress subject’s shows no significant difference between before and after VR stress session. The chakra points that are close to significant are solar plexus, throat, heart, third eye and sacral as shown in Table 5. The classification result for stress subject’s is shown in Table 6. The results indicate throat chakra has the highest classification accuracy of 63.64% with 100% TP rate for before VR stress session. The next highest accuracy is sacral chakra with 59.09% and solar plexus chakra with 54.55% accuracy. y In Table 7, the combination of throat (CC), solar plexus (CE) and sacral (CF), and combination of throat (CC) and sacral chakra (CF) produces a highest accuracy of correctly classified up to 59.09%. The combination of throat and sacral chakra yields a highest TP rate up to 90.9% for classification before VR session. Meanwhile, combination of throat, solar plexus and sacral chakra produces slightly lower TP rate at 81.8%. This finding is consistent with previous studies showing the relation of several chakra points to stress [19]. Table 5. MANOVA analysis for stress subjects Chakra Points F Sig. (p-value) Crown CA 0.565 0.461 Third Eye CB 1.931 0.180 Throat CC 2.475 0.131 Heart CD 2.048 0.168 Solar Plexus CE 4.147 0.055 Sacral CF 1.655 0.213 Root CG 0.64 0.433 Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 2, May 2021 : 826 - 834 Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 2, May 2021 : 826 - 834 831 Indonesian J Elec Eng & Comp Sci ISSN: 2502-4752  Table 6. Stress subjects classification by chakra points Chakra Points Correctly Classified Incorrectly Classified TP Rate (After VR session) TP Rate (Before VR session) Crown CA 40.91 59.09 0.273 0.545 Third Eye CB 45.45 54.55 0.545 0.364 Throat CC 63.64 36.36 0.273 1.00 Heart CD 50.00 50.00 0.273 0.727 Solar Plexus CE 54.55 45.45 0.364 0.727 Sacral CF 59.09 40.91 0.364 0.818 Root CG 50.00 50.00 0.636 0.364 Table 7. 3.2. EMR result based on subjects’ feedback EMR result based on subjects’ feedback Based on the subjects’ feedback on stress after the VR stress session experiment, majority of the subjects shows doesn’t feel stress. Figure 2 illustrates the finding. There is only 19% of the male subjects and 43% of female subjects were experienced stress after VR stress session, giving a total of 11 subjects out of 40 subjects. From the findings, it demonstrates gender differences on stress experiences in which female more susceptible of developing stress when exposed to physiological stress as compared to male. This finding is in line to previous studies [6], [8]. Studies also shows that females have slower rate of adapting to virtual reality environment compared to the males, indicating that VR or any type of stressor will give higher impact in females with compared to males [29], [30]. Figure 2. Post VR experience feedback on stress Figure 2. Post VR experience feedback on stress 3.1. EMR stress data Stress subjects classification by combination of chakra points CA CB CC CD CE CF CG Correctly Classified Incorrectly Classified TP Rate (After VR session) TP Rate (Before VR session)        36.36 63.64 0.273 0.455 -       40.91 59.09 0.273 0.545 - -      40.91 59.09 0.273 0.545 - -  -    50.00 50.00 0.273 0.727 - -  -   - 59.09 40.91 0.364 0.818 - -  - -  - 59.09 40.91 0.273 0.909 REFERENCES [11] Z Movaffaghi and M Farsi "Biofield threrapies : Biophysical basis and biological regulations? " Complementary [10] J. L. Oschman, “Science and The Human Energy Field,” Reiki News Magazine, vol. 1, no. 3, pp. 27-44, 2002 [11] Z. Movaffaghi and M. Farsi, "Biofield threrapies : Biophysical basis and biological regulations?," Complem h l l l 15 1 35 3 2009 d i 10 1016/j 2008 0 001 [10] J. L. Oschman, Science and The Human Energy Field, Reiki News Magazine, vol. 1, no. 3, pp. 27-44, 2 [11] Z. Movaffaghi and M. 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Gilmore et al., "Gender differences in subjective stress and neuroendocrine response to a stress task among individuals with opioid dependence: A pilot study," Addictive Behaviors, vol. 92, pp. 148-154, 2019, doi: 10.1016/j.addbeh.2018.12.022. j [14] S. Shienfield, “The Art of Chakra Balancing”, Andrews McMeel Publishing, 2005. g [15] J. R. Cross, N. Ellis, and J. Amaro, “Acupuncture and the Chakra Energy System: Treating the Cause of Disease”, North Atlantic Books, 2012. [16] S. Z. A. Jalil, M. N. Taib, H. A. Idris, and M. M. Yunus, "Gender Classification Based on Human Radiation Wave Analysis," in UkSim 13th International Conference on Computer Modelling and Simulation, 2011, pp. 59-63, doi: 10.1109/UKSIM.2011.21. [17] S. Z. A. Jalil, M. N. Taib, H. Abdullah, and M. M. Yunus, "Frequency Radiation Characteristic Around the Human Body," International Journal of Simulation: Systems, Science and Technology, vol. 12, no. 1, pp. 35-39, 2011, doi: 10.5013/IJSSST.a.12.01.05. [18] S. Z. A. Jalil, S. A. M. Aris, N. A. Bani, H. 4. CONCLUSION Stress has been a major concern in the current situations as chronic stress can leads to health issues such as heart disease, depression and anxiety. This paper discussed stress classification based on human electromagnetic radiation (EMR) analysis. There are twenty-nine males and eleven female’s student involved in this study. The human EMR data are analyse using statistical analysis of MANOVA and classified using LWL algorithm. Based on the overall EMR dataset, the finding shows that throat is the most significant point and followed by third eye. The result shows that the classification accuracy of this combinations is more than 60% accuracy. This finding in line with the previous studies indicating the relation of several chakra points to tress classification based on human electromagnetic… (Tengku ‘Afiah Mardhiah Tengku Zainul Akmal)  832 ISSN: 2502-4752 stress. However, since the number of subjects are unbalanced with the majority of the subjects are males, and based on the subjects’ feedback on stress after the VR stress session, most of male subject’s did not feel stressed while using VR. This factor may contribute to the percentage accuracy obtained of correctly classify. Although this study is capable of classifying stress using human EMR, further investigation with more and balance number of subject will be performed to distinguish and classify the human EMR on stress. In addition, this study will be associate with a well-established bio feedback instruments on stress identification such as electroencephalogram (EEG) for future research. stress. However, since the number of subjects are unbalanced with the majority of the subjects are males, and based on the subjects’ feedback on stress after the VR stress session, most of male subject’s did not feel stressed while using VR. This factor may contribute to the percentage accuracy obtained of correctly classify. Although this study is capable of classifying stress using human EMR, further investigation with more and balance number of subject will be performed to distinguish and classify the human EMR on stress. In addition, this study will be associate with a well-established bio feedback instruments on stress identification such as electroencephalogram (EEG) for future research. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Universiti Teknologi Malaysia for supporting the research. This work is supported in part by Ministry of Higher Education under Fundamental Research Grant Scheme (FRGS/1/2018/ICT04/UTM/03/3). REFERENCES [1] M. Stults-Kolehmainen and R. Sinha, "The Effects of Stress on Physical Activity and Exercise," Sports medicine (Auckland, N.Z.), vol. 44, no. 1, pp. 81-121, 2013, doi: 10.1007/s40279-013-0090-5. [1] M. Stults-Kolehmainen and R. 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Keren, "Stress Inducing Demands in Virtual Environments," Proceedings of the Human Factors and Ergonomics Society Annual Meeting, vol. 62, no. 1, pp. 2066-2070, 2018, doi: 10.1177/1541931218621466. [25] J. Santl, S. Youssef, A. Plab, S. Wüst, B. Kudielka, and A. Mühlberger, "Gender Differences in Stress Responses during a Virtual Reality Trier Social Stress Test," International Journal of Virtual Reality, vol. 19, no. 2, pp. 2-15, 2019, doi: 10.20870/IJVR.2019.19.2.2912. , [26] J.-A. Ting, S. Vijayakumar, and S. Schaal, “Locally Weighted Regression for Control,” C. Sammut & G. I. Webb (Eds.), Encyclopedia of Machine Learning. Boston, MA: Springer US, vol. REFERENCES 11, pp. 613-624, 2010, doi: 10.1007/978-1-4899-7502-7_493-1. [27] C.-C. Wei, “Comparing lazy and eager learning models for water level forecasting in river-reservoir basins of inundation regions,” Environmental Modelling & Software, vol. 63, pp. 137-155, 2015, doi: 10.1016/j.envsoft.2014.09.026. j [28] J. Han, M. Kamber, and J. Pei, “Classification: Basic Concepts,” J. Han, M. Kamber, & J. Pei (Eds.), Data Mining (Third Edition), Boston: Morgan Kaufmann, pp. 327-391, 2012. [29] M. Wei, J. Luo, H. Luo, and R. Song, "The effect of gender on vection perception and postural responses induced by immersive virtual rotation drum," in 8th International IEEE/EMBS Conference on Neural Engineering (NER), 2017, pp. 473-476, doi: 10.1109/NER.2017.8008392. [30] P.-A. Fransson et al., "Postural instability in an immersive Virtual Reality adapts with repetition and includes directional and gender specific effects," Scientific Reports, vol. 9, no. 1, pp. 1-10, 2019, doi: 10.1038/s41598-019- 39104-6. tress classification based on human electromagnetic… (Tengku ‘Afiah Mardhiah Tengku Zainul Akmal) BIOGRAPHIES OF AUTHORS Tengku ‘Afiah Mardhiah Tengku Zainul Akmal was born in Malaysia, on 28 May, 1990. She received Bachelor of Aircraft Eng. Tech.(Hons) Mechanical from Universiti Kuala Lumpur (UniKL) and MSc in Systems Engineering from Universiti Teknologi Malaysia (UTM) in 2009 and 2019 respectively. She has been working on image classification and bio-signals analysis during her Masters’ studies. She is currently a data scientist in NXP Semiconductors and her present work are related with image processing, pattern recognition and anomalous signals detection in various semiconductor testing areas. Abd Hafiz Qayyum Abd Talib was born in Malaysia, on 11 September, 1985. He received Bachelor Degree in Bachelor of Aircraft Engineering Technology (Hons.) Mechanical from Universiti Kuala Lumpur Malaysian Institute of Aviation Technology (UniKL MIAT) and Master of Medical Science (Physiology) from Universiti Kuala Lumpur Royal Collage of Medicine Perak (UniKL RCMP). He is currently pursuing his PhD degree level in Electrical and Electronic Engineering (Biomedical Engineering) at Universiti Teknologi Malaysia (UTM) Kuala Lumpur. He involved many research work related with Electrical Power, Biomedical Engineering, Integrative Physiology and Molecular Medicine including Electrophysiology, Human Gaits, Artificial Intelligence (AI) and Internet of Things (IoT). Siti Zura A. Jalil was born in Malaysia, on 31 August, 1975. She received a B. Eng. (Hons) degree and M. Eng. degree in Electrical Engineering from Universiti Technologi Malaysia in 1998 and 2001, respectively, and the PhD Degree in Biomedical Engineering in 2014 from Universiti Teknologi MARA, Malaysia. She is a senior lecturer at Razak Faculty of Technology and Informatics, Universiti Teknologi Malaysia, Kuala Lumpur, Malaysia. She has led research in advanced signal processing and data analytics with applications in biomedical engineering particularly in human electromagnetic radiation and bio-signal analysis. She is also interested in the field of biomechanic engineering of gait studies. She is member to IEEE Malaysia chapters of Signal Processing Society, and Engineering in Medicine and Biology Society.  834 ISSN: 2502-4752 Siti Armiza Mohd Aris was born in Malaysia, on 11 September, 1975. She received the B.Eng degree in Electrical Engineering (Microelectronics) from Universiti Teknologi Malaysia in 1998, and the M.Eng. as well as Ph.D degrees in Electrical Engineering from Universiti Teknologi Malaysia in 2001 and Universiti Teknologi MARA in 2016 respectively. She started as a tutor in 1998 and now has become a senior lecturer at Universiti Teknologi Malaysia, Kuala Lumpur. BIOGRAPHIES OF AUTHORS In 2012, she joined the UTM Razak School of Engineering and Advanced Technology as a Lecturer and Researcher, a school that offers undergraduate and postgraduate students from various disciplines. Her current research interests include EEG signal processing, bio-signal processing, psycho-physiological interactive tools, and bio-signal monitoring tools. She is a member of IEEE Malaysia Section, IEEE EMBS Malaysia Chapter and IEEE Signal Processing Society Malaysia Chapter. In 2016, her research paper has been recognised by the IEEE WIE and awarded as the best research paper for her outstanding work. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 2, May 2021 : 826 - 834
https://openalex.org/W2903648430	http://nrl.northumbria.ac.uk/id/eprint/36989/8/Adams%20et%20al%20-%20Feasibility%20of%20trial%20procedures%20for%20a%20randomised%20controlled%20trial%20of%20a%20community%20based%20group%20exercise%20intervention%20for%20falls%20prevention%20for%20visually%20impaired%20older%20people%20OA.pdf	English	null	Feasibility of trial procedures for a randomised controlled trial of a community based group exercise intervention for falls prevention for visually impaired older people: the VIOLET study	BMC geriatrics	2,018	cc-by	12,424	p // g / / / This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/36989/ This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/36989/ This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/36989/ Northumbria University has developed Northumbria Research Link (NRL) to enable users to access the University’s research output. 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To read and/or cite from the published version of the research, please visit the publisher’s website (a subscription may be required.) Northumbria Research Link Citation: Adams, Nicola, Skelton, Dawn A., Howel, Denise, Bailey, Cathy, Lampitt, Rosy, Fouweather, Tony, Gray, Joanne, Coe, Dorothy, Wilkinson, Jennifer, Gawler, Sheena, De Jong, Lex D., Waterman, Heather, Deary, Vincent, Clarke, Michael and Parry, Steve (2018) Feasibility of Trial Procedures for a Randomised Controlled Trial of a Community Based Group Exercise Intervention for Falls Prevention in Visually Impaired Older People: The VIOLET Study. BMC Geriatrics, 18. ISSN 1471-2318 Published by: BioMed Central RESEARCH ARTICLE Open Access Feasibility of trial procedures for a randomised controlled trial of a community based group exercise intervention for falls prevention for visually impaired older people: the VIOLET study Nicola Adams1* , Dawn A. Skelton2, Denise Howel3, Cathy Bailey1, Rosy Lampitt4, Tony Fouweather3, Joanne Gray1, Dorothy Coe1, Jennifer Wilkinson4, Sheena Gawler1, Lex D. de Jong5, Heather Waterman6, Vincent Deary1, Michael Clarke7 and Steve W Parry8 Adams et al. BMC Geriatrics (2018) 18:307 https://doi.org/10.1186/s12877-018-0998-6 Adams et al. BMC Geriatrics (2018) 18:307 https://doi.org/10.1186/s12877-018-0998-6 Open Access Feasibility of trial procedures for a randomised controlled trial of a community based group exercise intervention for falls prevention for visually impaired older people: the VIOLET study Nicola Adams1* , Dawn A. Skelton2, Denise Howel3, Cathy Bailey1, Rosy Lampitt4, Tony Fouweather3, Joanne Gray1, Dorothy Coe1, Jennifer Wilkinson4, Sheena Gawler1, Lex D. de Jong5, Heather Waterman6, Vincent Deary1, Michael Clarke7 and Steve W Parry8 Abstract 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. Tyne NE7 7XA, UK Full list of author information is available at the end of the artic © 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. 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: Visually impaired older people (VIOP) have a higher risk of falling than their sighted peers, and are likely to avoid physical activity. The aim was to adapt the existing Falls Management Exercise (FaME) programme for VIOP, delivered in the community, and to investigate the feasibility of conducting a definitive randomised controlled trial (RCT) of this adapted intervention. Methods: Two-centre randomised mixed methods pilot trial and economic evaluation of the adapted group-based FaME programme for VIOP versus usual care. A one hour exercise programme ran weekly over 12 weeks at the study sites (Newcastle and Glasgow), delivered by third sector (voluntary and community) organisations. Participants were advised to exercise at home for an additional two hours over the week. Those randomised to the usual activities group received no intervention. Outcome measures were completed at baseline, 12 and 24 weeks. The potential primary outcome was the Short Form Falls Efficacy Scale – International (SFES-I). Participants’ adherence was assessed by reviewing attendance records and self-reported compliance to the home exercises. Adherence with the course content (fidelity) by instructors was assessed by a researcher. Adverse events were collected in a weekly phone call. Results: Eighteen participants, drawn from community-living VIOP were screened; 68 met the inclusio participants were randomised with 33 allocated to the intervention and 31 to the usual activities arm. 94% of participants provided data at the 12 week visit and 92% at 24 weeks. Adherence was high. The intervention was found to be safe with 76% attending nine or more classes. Median time for home exercise was 50 min per week. There was little or no evidence that fear of falling, balance and falls risk, physical activity, emotional, attitudinal or quality of life outcomes differed between trial arms at follow-up. (Continued on next page) * Correspondence: nicola.adams@northumbria.ac.uk 1Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne NE7 7XA, UK Full list of author information is available at the end of the article © 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. Background Falls in older people are common [1, 2] and are associated with considerable morbidity and mortality [3], with ap- proximately 10% of falls resulting in fractures [4]. Costs ac- count for 0.85–1.5% Western economies’ total health-care expenditure [5]. In the UK, in 2015, falls in the over 65 s were estimated to cost £4.6 million a day [6] and thus rep- resent a high cost to health and social care budgets [7, 8]. The incidence and prevalence of older people with sig- nificant sight loss is increasing [9]. Older people with visual impairment have a 1.7 times higher risk of falling than the general population, requiring more hospital and nursing home admissions. More contact with their gen- eral practitioner (GP) than sighted peers is also reported. [10]. Eight per cent of falls-related hospital admissions are likely to occur in people who are visually impaired [8], which accounts for approximately 21% of the total cost of treating falls. Visual impairment thus acts as an independent risk factor for falls [11–13] with falls risk factors including poor visual acuity and contrast sensi- tivity, decreased depth perception and reduced visual field [14–18], in addition to more general factors such as muscle weakness and balance. q Older people with visual impairment are therefore at higher risk of falls and fear of falling and its associated adverse psychosocial effects. Evidence of the effective- ness of exercise programmes and adherence to them in such individuals is lacking, though there is some evi- dence to suggest that group based exercise may be of more benefit than individual programmes. In addition, there is an issue with case ascertainment in those with falls and visual impairment, with only half of falls clinics in the UK assessing routinely for visual impairment [34]. The current study used a known effective community-based exercise intervention, routinely used in falls services in the UK [35]. The aims of the study were to conduct a feasibility study to inform the design and conduct of a future definitive multicentre rando- mised controlled trial (RCT) and economic evaluation of an adapted group-based exercise programme to prevent falls and reduce fear of falling among VIOP. Specific aims of the feasibility study were to assess recruitment and retention to the study, willingness to be randomised and to test the trial methodology, including the identifi- cation of candidate outcome measures and rehearse the methodology for cost effectiveness analysis. Background Participants’ adherence to the exercise programme was also exam- ined. The acceptability of the intervention was examined qualitatively, and will be reported in a separate paper. Older people with visual impairment are therefore at higher risk of falls and fear of falling and its associated adverse psychosocial effects. Evidence of the effective- ness of exercise programmes and adherence to them in such individuals is lacking, though there is some evi- dence to suggest that group based exercise may be of more benefit than individual programmes. In addition, there is an issue with case ascertainment in those with falls and visual impairment, with only half of falls clinics in the UK assessing routinely for visual impairment [34]. Fear of falling (FoF), an umbrella term for the psycho- social consequences of falls, is common and a significant predictor of a future fall alongside a cycle of restricting daily activity and mobility with loss of confidence, re- duced social participation, increased frailty and reduced quality of life [19–22]. A vision charity found that older people are highly likely to avoid activity because of their visual impairment [23]. Anxiety and depression are also common in those with visual impairment with concomi- tant reduced activity [3]. A Cochrane review found that exercise can reduce fear of falling in the short term, with insufficient evidence for longer term efficacy [24, 25]. A high risk of bias was noted in most included trials. There are also limited health eco- nomic data about fear of falling interventions [26, 27]. Multifactorial falls intervention programmes are effective in reducing falls among older people [28–31]. A Cochrane systematic review reported that home safety assessment and multi-component group and home-based exercise programmes reduce the rate of falls and the risk of falling in community dwelling older people [30]. For example, a (Continued from previous page) (Continued from previous page) Conclusions: The intervention, FaME, was implemented successfully for VIOP and all progression criteria for a main trial were met. The lack of difference between groups on fear of falling was unsurprising given it was a pilot study but there may have been other contributory factors including suboptimal exercise dose and apparent low risk of falls in participants. These issues need addressing for a future trial. Trial registration: Current Controlled Trials ISRCTN ID: 16949845 Registered: 21 May 2015. Keywords: Falls management, Exercise, Visual impairment, Older people, Feasibility clinical trial New Zealand based randomised controlled trial (RCT), [32], showed no benefit from a multicomponent home-based exercise programme in visually impaired par- ticipants, though those with stricter adherence to the exer- cise programme had fewer falls. Adherence to the home based exercise programme in VIOP was poor with only 18% of VIOP completing all home exercise sessions over a year period. In a subsequent 3-armed UK based feasibility trial (VIP2 UK), all participants who completed the trial reported partially or completely adhering to home safety recommendations, but evidence for adherence to home exercise was equivocal [33]. New Zealand based randomised controlled trial (RCT), [32], showed no benefit from a multicomponent home-based exercise programme in visually impaired par- ticipants, though those with stricter adherence to the exer- cise programme had fewer falls. Adherence to the home based exercise programme in VIOP was poor with only 18% of VIOP completing all home exercise sessions over a year period. In a subsequent 3-armed UK based feasibility trial (VIP2 UK), all participants who completed the trial reported partially or completely adhering to home safety recommendations, but evidence for adherence to home exercise was equivocal [33]. Adams et al. BMC Geriatrics (2018) 18:307 Page 2 of 15 Randomisation Randomisation has been previously described [36], was stratified by centre and was administered cen- trally via Newcastle Clinical Trials Unit using a se- cure web based system using a blocked allocation system to allocate participants to the two groups. Par- ticipants were informed of their allocated treatment group following randomisation. Study intervention h l l d l The logistical delivery of the group-based Falls Manage- ment Exercise (FaME) programme was adapted for VIOP by stakeholders and is reported in a separate paper. The exercise component and progression content of FaME remained as the original. The exercise programme (the intervention) consisted of one hour weekly sessions over 12 weeks and these were held in community venues with a maximum capacity of ten participants per group. Two ex- ercise groups were held at each site, with a third in New- castle added to maximise recruitment. Participants were offered taxi transport and also brought a companion or support if they wished. Study design The study adheres to CONSORT guidelines for the de- sign and reporting of clinical trials. Page 3 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 The Ophthalmology Department in Newcastle, New- castle Society for Blind People (NSBP), and Visibility in Glasgow identified potential participants and passed on the expressions of interest to the research team. VIOP who expressed an interest in participating in the study received further detailed information (in English only). This study adapted an existing effective group-based health promotion intervention (FaME) for VIOP. Be- cause of the lack of relevant information for a full RCT on this topic, a randomised mixed methods feasibility study was designed to inform the design and conduct of a future definitive multi-centre RCT on an adapted ver- sion of a community-based exercise intervention for falls management, the FaME programme. Following initial low referrals from the Newcastle Low Vision Clinic, and with appropriate ethical approval, Eye Clinic Liaison Officers (ECLOs) were empowered to identify and approach potential participants. With per- mission, expressions of interest were forwarded to the research team who assessed for eligibility in the same way as the participants identified through the low vision clinic and NSBP. The design was a two-centre (Newcastle and Glasgow) randomised pilot trial and economic evaluation of an adapted exercise programme for older visually impaired people versus no intervention with embedded qualitative evaluation. Interviews were conducted to explore accept- ability and applicability of the intervention, the research methods and the outcome measures. The results of these interviews are being reported in a separate paper. Participants gave permission for the study team to contact their General Practitioner (GP), via letter, to ensure medical fitness of the participant to take part in the study, and once eligibility confirmed, the par- ticipant was randomised. Visually impaired community-dwelling older people were recruited from the 2 study sites and were rando- mised into one of two groups. Group One was a 12-week exercise programme (one-hour session per week), and Group Two was usual activities. Inclusion and exclusion criteria The inclusion and exclusion criteria have been published in a previous protocol paper [36]. In brief these were: aged 60 years and over, community dwelling and attend- ing a low vision clinic and/or were members of organisa- tions for the visually impaired. Exclusion criteria were acute or uncontrolled medical conditions and inability to comprehend simple movement instructions. Ethical approval and consent to participate Favourable ethical opinion from the Newcastle and North Tyneside Research Ethics Committee and R&D approval was obtained prior to commencement of the intervention. Glasgow Caledonian University was ap- proved as a non-NHS site with local ethics approval. Information sheets were provided to all eligible partici- pants and written informed consent obtained prior to any study procedures. Signed or verbal consent was sought and if participants were unable to sign, consent was sought by a third party who signed the witness sec- tion of the consent form. pp y All participants completed a health screening tool, normally administered by the exercise instructors prior to delivery of the FaME programme. The exercises con- sisted of balance specific, individually-tailored and tar- geted training for dynamic balance, strength, endurance, flexibility, gait and functional skills, training to improve ‘righting’ or ‘correcting’ skills to avoid a fall and backward-chaining i.e. retraining of the ability to get down to and up from the floor. Functional floor exer- cises and adapted Tai Chi exercises were also carried out with progressively more challenging content over the 12 weeks. Resistance bands and mats were used [37]. Identification, screening, and recruitment Previous studies have identified that recruitment and ad- herence in frail older people can be difficult, although data for recruitment and retention rates in VIOP was relatively unknown. Within the VIP2UK study [33], only 10% of those initially screened and 51% of eligible partic- ipants agreed to take part. Recruitment in the current study was from both National Health Service (NHS) and non NHS sources, in order to maximise recruitment as this has been found to be difficult in previous trials. Page 4 of 15 Page 4 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 Participants were also advised to exercise at home for up to two hours per week. The exercises were to be performed if possible daily, on the days the participant was not attend- ing the exercise class. All home programmes contained ‘prompts’ that linked exercises to daily tasks e.g. performing heel raises whilst waiting for the kettle to boil, in order to improve adherence. Exercises were provided in a large text booklet, DVD or audio format. Exercises were designed to be completed in 10 to 20 min blocks, becoming more chal- lenging and graduating into longer periods. Thus the inter- vention comprised up to 36 h over the 12 week period (with full adherence), concordant with current evidence [38]. co-morbidities, current medication, socio-economic in- formation and the main study outcome measures were collected at the baseline visit. The selected outcome measures were standardised assessment instruments that have been used in falls research. These were assessed at baseline, 12 weeks and 24 weeks (Table 1, Fig. 1). Fear of falling (FoF) was selected as the primary outcome variable. The Short Falls Efficacy Scale – International (SFES-I), captures the participants’ concerns about doing every- day activities without falling [39]. Secondary outcome measures included: Activity avoidance [40]; Timed Up & Go test (TUG), [41] Falls Risk (FRAT) [42]; Physical Activity (Phone-FITT) [43]; Loneliness (Six-Item Scale for Overall, Emotional, and Social Loneliness) [44]; Hospital Anxiety and Depres- sion Scale (14 item) [45]; Work and Social Adjustment Scale (WSAS) [46]; Health related quality of life (EQ-5D-5 L) [47] and quality of life (ICECAP-O) [48]. Currently performed physical activities/exercise were also assessed using a short bespoke self-report inven- tory. Identification, screening, and recruitment Because of their visual impairment, assistance was often required to fill in the questionnaires, either by the researcher reading out the questions and filling out the forms, or by providing the questionnaires in a format accessible to the participant. Where no more than 20% of questions were missing or uninterpretable on specific scales, the score was calculated by using the mean or median value (as appropriate) of the respondent-specific completed responses on the rest of the scale to replace the missing items [49]. Control group Those participants who were randomised to the usual activities group received no intervention and continued with their usual activities. They were offered an equiva- lent exercise programme after the 24-week follow-up data collection at both sites. Outcomes The progression criteria to judge the feasibility of pro- gressing to a full trial were that following the six-month follow-up data collection [36]: 1) ≥50% of eligible participants recruited into the feasibility study; 2) ≥70% of the participants in the intervention arm completed nine to twelve sessions in the exercise programme. g 3) ≥70% of participants had data collected on main outcomes at six-month follow-up; Each participant completed a falls diary each week, with assistance from a Researcher, during a weekly tele- phone call. Details of any adverse events and near misses were also recorded at this time and are described in the safety analysis. When a fall did occur, the researcher completed the falls resource/expenses form on behalf of the participant retrospectively during the weekly tele- phone at 12 and 24 weeks follow up. 4) < 10% of serious adverse events deemed due to the intervention. Sample size As this was a feasibility study, a formal power calculation was not appropriate. We aimed to obtain a minimum of 30 responses in each trial arm at 6 month follow-up to estimate critical parameters to the necessary degree of precision [50]. To provide feasibility data, we aimed to recruit a total of 80 community-living VIOP to allow for loss to follow-up. Cost effectiveness analysis was rehearsed from an NHS and personal social services perspective using the EQ-5D-5 L and ICECAP-O as outcome measures and via a health economic self-report service receipt inven- tory. Costs of the intervention were micro-costed using the Violet feasibility study records. For each trial partici- pant, all components of treatment costs stratified by cat- egory of resource use were computed by multiplying units of resource use by their unit costs. These were then summed over all resource use categories to obtain a total cost for each participant. This was then used to generate the average cost per patient in each arm of the trial. All unit costs were expressed in GBP (£) and Economic evaluation Economic evaluation Outcome measures Following consent, a researcher assessed all participants, either on site or in their own home, depending on the participants’ preferences. Information on demographics, Table 1 Trial Procedures Intervention Control Weeks 1–12 Daily completion of falls diary 1 h weekly exercise session Advice to carry out up to two hours of additional home exercise per week Weekly telephone call to/from researcher to record any adverse events Completion of falls resource/expenses form with the researcher (if required). Daily completion of falls diary Weekly telephone call to/from researcher to record any adverse events. Completion of falls resource/expenses form with the researcher (if required). Week 12 ((+/−2 weeks) Following information collected: Co-morbidities, current medication, any changes in socioeconomic information, and incidental costs (intervention group only) and the outcome measures were completed. Information collected as per intervention group Weeks 12–24 Daily completion of falls diary Weekly telephone call to/from researcher to record any adverse events. Completion of falls resource/expenses forms with researcher if required Information collected as per intervention group. Week 24 (+/−2 weeks) Assessed on all measures Assessed on all measures Table 1 Trial Procedures Intervention Control Weeks 1–12 Daily completion of falls diary 1 h weekly exercise session Advice to carry out up to two hours of additional home exercise per week Weekly telephone call to/from researcher to record any adverse events Completion of falls resource/expenses form with the researcher (if required). Daily completion of falls diary Weekly telephone call to/from researcher to record any adverse events. Completion of falls resource/expenses form with the researcher (if required). Week 12 ((+/−2 weeks) Following information collected: Co-morbidities, current medication, any changes in socioeconomic information, and incidental costs (intervention group only) and the outcome measures were completed. Information collected as per intervention group Weeks 12–24 Daily completion of falls diary Weekly telephone call to/from researcher to record any adverse events. Completion of falls resource/expenses forms with researcher if required Information collected as per intervention group. Week 24 (+/−2 weeks) Assessed on all measures Assessed on all measures Assessed on all measures Adams et al. BMC Geriatrics (2018) 18:307 Page 5 of 15 Fig. 1 Schematic Representation of the Randomised Controlled Trial Fig. 1 Schematic Representation of the Randomised Controlled Trial Identification of potential participants In Newcastle, a dedicated member of NSBP tele- phoned members aged over 60 years to gain permis- sion to forward their contact details to the research team. It is not known precisely how many potential participants were approached by NSBP, but anecdotal information suggests that over two hundred calls were made. From these, thirty-five expressions of interest were passed to the researchers. The primary source of potential participants at the Glasgow site was Visibil- ity. Anecdotally, over one hundred direct contacts were made by Visibility staff, and the research team received forty-eight expressions of interest. The process of identifying potential participants was time consuming and incumbent upon the resources of the third sector organisations. Safety All adverse events judged as having a reasonable suspected causal relationship to a study procedure (i.e. definitely, probably or possibly related) were considered to be related adverse events. The opinion of a physician was sought if re- quired. Severity of all AEs was graded on a three-point scale of intensity (mild, moderate, and severe). Expected adverse events included: Fall/trip/slip and its consequences: cuts and abrasions, soft tissue injury, fracture; muscular/joint pain associated with the above or with increased physical activity; minor illness not requiring GP intervention (cold, flu etc.); minor illness requiring GP intervention (chest in- fection, urinary tract infection etc.) Identification of participants from the RVI Low Vi- sion Clinic in Newcastle initially proved problematic: very few of the staff referred potential participants to the researchers, only seven were initially identified. After the involvement of ECLOs, fifteen further po- tential participants were identified, and their expres- sions of interest were forwarded to the research team. No screening or recruitment took place from the Low Vision Clinic in Glasgow as it was closed over the period of recruitment. All participants who had expressed an interest in the pilot trial were contacted by the research team and assessed for eligibility in a consistent manner. Any serious adverse events were recorded until a par- ticipant reached their 24-week follow-up visit and in- cluded injurious falls, serious falls and/or hospitalisation due to falls. Fidelity of the intervention Instructors submitted basic lesson plans for the 12-week programme prior to the start of the intervention. Their adherence with the course content (fidelity) was assessed by a researcher (SG) attending 20% sample of exercise sessions. A standardised checklist was used, similar to that used in a previously published trial [35], and these sessions were videotaped for quality assurance purposes. Adherence Adherence to the group exercise programme (register) and home exercise programme (self-report) was assessed. Participants were classed as adherent to group exercise if they attended nine out of 12 of the group sessions [36]. Page 6 of 15 Page 6 of 15 Page 6 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 valued at 2015–16 prices. The mean cost of the inter- vention across the two sites was estimated. Utilities were estimated and reported for each trial arm using means, standard deviations, the median and the range. Participant flow Participant flow is illustrated in the CONSORT diagram in Fig. 2. Results Identification of potential participants Statistical analysis After assessment for eligibility, 68 people were asked if they would be willing to be randomised and 66 agreed to do so (97%). This consent rate was much higher than the target of 50%, but will be biased upwards as a num- ber implicitly declined to enter trial at an earlier stage when they failed to send back an expression of interest. Since this was a feasibility trial, the main analyses were descriptive, in order to inform the design, choice of primary outcome, sample size and approach to analysis for a future definitive study. The main out- comes were feasibility outcomes; the numbers of eli- gible participants seen over the recruitment period, and the resulting rates of recruitment, compliance with randomisation, and data completion were pre- sented. Data completeness of the instruments and any potential bias in the completion of follow-up data to inform the choice of instruments in a future trial was ascertained. The majority of the outcome data is pre- sented in simple descriptive tables, presenting per- centages, means and standard deviations or 5-number summary (as appropriate), for each arm of the study. y p Recruitment to the trial took place between June and No- vember 2015. Recruitment was closed when 64 participants had been randomised, which was below the target of 80. After randomisation, 33 VIOP were allocated to the intervention arm, and 31 to usual activities. Of the 33 VIOP allocated to the intervention arm, 3 did not attend any classes: 2 of those who didn’t attend any classes nevertheless provided study data. During the study, 1 person was lost to follow-up and 4 people in the intervention arm withdrew completely from the study. The remaining 59 subjects provided data that was included in the statistical analysis: this was only slightly below the target of follow-up data on 60 participants. There was potential for clustering effects, particu- larly class-based clustering in the intervention group and this study aimed to investigate and estimate the size of any such effects. Page 7 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Fig. 2 CONSORT Diagram of the VIOLET Study Fig. 2 CONSORT Diagram of the VIOLET Study Baseline participant characteristics respectively and, in Newcastle, class sizes were six, five and four respectively. Demographic and baseline characteristics at randomisa- tion were compared across treatment groups (Tables 2 and 3). The distributions of demographic variables were similar across the trial arms: the only noticeable differ- ence was that there were more who lived alone in the usual activities arm. Four withdrew completely from the study and attended 0, 2, 3 and 11 classes prior to withdrawing. One participant died while in the trial, after attending 6 exercise classes. One participant withdrew from the intervention after 3 classes but continued to provide trial data. Two additional participants randomised to the intervention arm did not attend any classes as their GP consent was not received in time, but con- tinued to provide follow-up. Despite this, 76% attended 9 or more classes, which was one of the feasibility criteria for a future trial. Delivery of intervention and intervention adherence A staggered start was used to facilitate the running of the first set of classes in both Newcastle and Glasgow. This enabled those for whom the GP confirmation of eli- gibility had not been returned promptly to start at any time within the first three weeks and continue to complete the twelve sessions. In Newcastle, a third set of classes was provided for those who had been recruited later, or whose eligibility checks took a long time to complete. In Glasgow, the class sizes were six and nine Table 4 summarises the number of sessions attended by participants randomised to the intervention arm on the basis of class registers, and how often they exercised at home (self-report) with its frequency and duration. It can be seen that on average they spent 50 min per week, Adams et al. BMC Geriatrics (2018) 18:307 Page 8 of 15 Page 8 of 15 Table 2 Baseline demographic characteristics, by treatment arm Variable Intervention Arm (n = 33) Usual activities Arm (n = 31) Gender Male 14 (42%) 11 (35%) Female 19 (58%) 20 (65%) Age (years) Median (IQR) 80 (75, 87) 78 (68, 83) Mean (SD) 79.3 (8.7) 76.5 (9.7) Range 61–95 62–95 Participants’ first language: English 32 (97%) 28 (90%) Other 1 (3%) 3 (10%) Ethnicity White 33 (100%) 29 (94%) Asian or Asian British 0 (0%) 2 (6%) Marital status: Married/living as married 16 (48%) 8 (26%) Living with other family members 0 (0%) 2 (6%) Living alone 11 (33%) 16 (52%) Widowed 6 (18%) 5 (16%) Employment status Full time employment 0 (0%) 1 (3%) Retired 29 (88%) 28 (90%) Other 4 (12%) 2 (6%) Table 2 Baseline demographic characteristics, by treatment arm Variable Intervention Arm (n = 33) Usual activities Arm (n = 31) one was lost to follow up from the study at this point). In two cases, assessments were completed outside the two-week limit, due to other commitments or extenuat- ing circumstances. All those participants in the intervention arm remaining in the trial at each time point completed each of the questionnaires. There were two occasions when participants in the usual activity arm only par- tially completed a questionnaire (but this was still usable using missing data rules), and two occasions on which whole questionnaires were not completed. The elements of the phone-FITT questionnaire were completed for all those remaining in the trial at each time point. Questionnaire data throughout the trial Questionnaire data throughout the trial Table 5 summarises numeric outcome measures by trial arm and data collection point, for the SFES-I, WSAS, FRAT and phone-FITT. It can be seen that, based on the SFES-I score, the majority of participants had low or mod- erate concern over falling at baseline. The change from baseline in SFES-I at 12 and 24 weeks was minimal in both arms. There was a very wide range of WSAS impact scores at baseline, but the median changes over time were small. For FRAT, the scores were generally low at baseline and showed little change over time. Using the Phone-FITT summary, it can be seen that typical physical activity levels rose slightly over the follow-up period in the intervention arm, and less so in the control arm, though no formal com- parison was made. These illustrate that concern over falling (the proposed primary outcome for a definitive trial), and assessment of falls risk was low in the participants re- cruited, so there was little progress to be made by any intervention. though there was a large variation in the amount of time spent exercising. This was much less than the 2 h per week that they were encouraged to so. Data completeness Sixty out of 64 (94%) provided data at 12-week visit (four had withdrawn completely from the study at this point). Fifty nine out of 64 (92%) participants completed the 24-week visit (four had withdrawn completely and Delivery of intervention and intervention adherence This suggests that all the chosen scales were suitable for use in a future trial, though the need to have researchers help participants, proved time consuming. Safety analysis l f A total of 180 Adverse Events (AEs) were reported; these were categorised as 16 Serious Adverse Events (SAEs) and 164 AEs. The majority of AEs reported were due to minor illness and unrelated to the intervention. Table 3 Baseline numbers of self-reported co-morbidities, by treatment arm Comorbidities Intervention Arm (N = 33) Usual activities Arm (N = 31) Total (N = 64) Any comorbidities reported Yes 25 (76%) 20 (65%) 45 (70%) No 8 (24%) 11 (35%) 19 (30%) Numbers of comorbidities per participant Min 0 0 0 LQ 1 0 0 Median 2 2 2 UQ 5 6 5 Max 9 13 13 Table 3 Baseline numbers of self-reported co-morbidities, by treatment arm There were 81 reported AEs in the intervention arm and 83 in the usual activities arm. No AEs or SAEs oc- curred during the exercise classes. There were 9 SAEs in the intervention arm and 7 SAEs in the usual activities arm. Of the 9 SAEs in the intervention arm, 4 were frac- tures due to falls. There were no fractures due to falls in the usual activities arm. However, of the 4 participants who sustained fractures due to falls in the intervention arm, 2 of these participants did not actually begin the intervention and had a self- reported history of repeated previous falls. With regard to the other two participants Page 9 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Table 4 Summary statistics for the exercise classes attended and home exercise min LQ Median UQ max Number classes attended 0 9 10 12 12 Weeks when exercised at home during interventiona 0 5 9 11 14 Average weekly exercise frequencyb 0 1.8 3.4 4.6 6.7 Average exercise duration per week (min)b 0 17.3 50.4 75.7 122 a ‘during intervention’ is any calls made in the period between 1st exercise class and last class + 7 days b is the average frequency or duration for all weeks when participant reported exercising at home in the valid timeframe (note that these include zeros for weeks where no exercise at home was done) Table 7 shows the EQ-5D-5 L utilities and ICECAP-O capability scores for both trial arms at baseline and each of the two follow up periods. Safety analysis l f Re- sults show that for both treatment groups at baseline, EQ-5D scores were poor, indicating ‘a state worse than death’ with the health state being worse in the intervention group. in the intervention arm who sustained fractures: one had taken part in 7 exercise classes when they tripped and fell in their own home, whilst the other had taken part in 10 exercise classes when they tripped and fell outside. There appears to be no evidence of a link be- tween taking part in the intervention and being at greater risk of a fracture due to a fall. One intervention participant died during the study, but the death was un- related to the intervention. This highlights that these VIOP had multiple medical conditions and syndromes of ageing that affected their participation. Compared to baseline the utility, scores at both follow up periods were improved for both groups but still showing average health related quality of life scores being poor. The range of utility scores were much larger in the usual activ- ities arm compared to the intervention arm with more ob- servations showing positive health related quality of life. The capability score in the intervention arm was slightly higher compared to the usual activities arm at baseline. At 12 weeks, capability was slightly higher with both groups having on average 80% capability. Economic evaluation Data regarding the intervention itself and the associated costs were fully recorded. Though 31 falls were reported (in 20 participants), only six participants utilised health and social care interventions post-fall, with none of these being quantifiable in terms of costs, due to missing data regarding the type of intervention utilised. At 24 weeks, capability was maintained at an average of 80% capability in the intervention arm, with a slight reduction in the usual activities arm (78% capability). Table 8 identifies the main issues raised and implica- tions for a future trial. The average total cost of the intervention per patient across both sites was £310 and is shown in Table 6. Delivery of the intervention A key component of the VIOLET study was the success- ful delivery of the adapted FaME programme in which instructors adapted their delivery style to the needs of participants. Overall, recruiting from third-sector (voluntary and community)organisations was successful, though organ- isational feedback suggested that the conversion rate from initial contact to expression of interest was poor. Also, one of these organisations was able to provide a dedicated member of staff for recruitment, but the other was not. Thus, identification of potential participants was more burdensome to the voluntary and community organisations than expected. In future multi-site studies, a recruitment strategy should be discussed and agreed across recruiting organisations with provision of add- itional resource and support. Further stakeholder in- volvement may improve recruitment and should be considered for a future definitive study. For each cohort, quality assurance checks were per- formed, ensuring fidelity of the programmes at both sites. We recommend that for future studies, participat- ing instructors should have an opportunity to attend two workshops, to share and discuss findings regarding suc- cessful delivery of the intervention. Within the exercise classes, a wide range of partici- pant ability led to some participants reporting not be- ing physically challenged nor understanding the relevance of specific exercises. There is a need for falls prevention programmes to emphasise facilitating independence as well as other positive benefits, such as socialising and receiving useful health education, in addition to an exercise component. However, it is possible that the dose and duration of exercise classes were insufficient for many of these participants to no- tice tangible benefits, as many exhibited low FoF and low to moderate falls risk scores. It proved difficult to recruit from the NHS Low Vision clinic. Since this was a regional centre, many patients lived too far away from the exercise classes provided. Economic evaluation Table 5 Numeric outcome measures by trial arm and data collection point (weeks 0, 12 & 24) Week Intervention arm Usual activities arm n min LQ Med UQ max n min LQ Med UQ max SFES-I (concerns over falling) Scores 7–28: Higher scores= > more concern 0 32 7 8 9 10 15 31 7 7 9 11 23 12 29 7 8 9 11 14 30 7 7 8 10 20 24 28 7 7 8 10 21 31 7 7 8 11 17 Impact of visual impairment (WSAS) Scoring (0–40): Higher scores = > more impact 0 32 0 5.6 11.9 18.1 27.5 30 0 5.0 12.5 23.8 38.8 12 29 0 10 15 20 35 31 0 3.8 11.3 17.5 37.5 24 28 0 12.5 20 25.6 40 31 0 5 15 26.3 33.8 Falls risk assessment tool (FRAT) Scoring (0–5): Higher scores = > more risk 0 33 0 0 1 3 5 31 0 1 1 3 4 12 29 0 1 1 3 4 31 0 1 2 3 4 24 28 0 1 1 3 4 31 0 1 2 3 4 Phone-FITT Total Frequency and Duration (FD) 0 33 0 34 49 89.5 296 31 1 16 42.5 66 222 12 29 0 41 55 96 209 31 0 25 47 67 377 24 28 9 30.8 52.1 100.5 410 31 0 18 43 66 1506 Functional test (TUG) Time in seconds to complete the test 0 33 8.8 9.8 13.3 16.8 35.6 31 7.4 10.7 13.3 20 120 12 28 8.9 11 13.5 18.4 30.2 31 6 11.1 17 19.2 975 24 28 8.2 10.3 13.8 16.3 28.5 31 6.6 10.4 15.1 18.2 100 Table 5 Numeric outcome measures by trial arm and data collection point (weeks 0, 12 & 24) Week Intervention arm Usu Table 5 Numeric outcome measures by trial arm and data collection point (weeks 0, 12 & 24) Page 10 of 15 Page 10 of 15 Page 10 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 Discussion staff to identify potential participants and pass on ex- pressions of interest. This recruitment strategy met the progression criteria with near to 50% conversion rate, though low overall numbers. Only two of those origin- ally found eligible for the study declined to take part. The study has shown that it is feasible to conduct a RCT of a modified FaME exercise intervention in visu- ally impaired older people. Recruitment of eligible par- ticipants to the randomised feasibility study exceeded the pre-planned progression criteria, as did the adher- ence of participants to the intervention and data collec- tion from the participants. In addition, as planned, less than 10% of serious adverse events were due to the intervention itself. Given the difficulties experienced in recruiting via third-sector organisations and an NHS low vision clinic, in a future study it may be advisable to use the primary care setting with the dual purpose of GP-led assessment of health exclusions and recruit- ment. Previous research suggests that a ‘personal’ in- vite by a health care professional increases uptake of community exercise classes [51]. Improving recruitment Variation in the way participants were identified across the two study sites led to some discrepancies in the re- cording of eligibility for the study. We are aware that over 300 direct contacts (phone or letter) were made to potential participants, which converted to 105 expres- sions of interest. An accurate count of contacts would be required for future studies. Delivery of the intervention On the other hand, the NHS ECLOs dedicated a member of Table 6 Costing of intervention Resource use Cost (£) Unit Source Costs of Consumables Yoga Mats and exercise bands 420 Total cost VIOLET study files DVDs and CDs 65 Total cost VIOLET study files Costs of Staff Time Newcastle PSI (12 sessions × 3 cycles) 66.66 Per hour HealthWorks Glasgow PSI (12 sessions X 2 cycles) 61.50 Per hour LLT Glasgow PSI set up time (0.5 h per session for 24 sessions) 30.75 Per 30 mins LLT Glasgow PSI travel and parking costs (per session) 7.24 Per session LLT Costs of Room Hire and Refreshments Newcastle (3 × 12 week cycles) 1925 Total Cost HealthWorks Glasgow (2 × 12 week cycles) 1476 Total Cost Visibility KEY: PSI postural stability instructor, LLT Later Life Training Adams et al. BMC Geriatrics (2018) 18:307 Page 11 of 15 Table 7 EQ-5D-5 L utility scores and ICECAP-O for health-related quality of life by trial arm Intervention Arm Usual Activities Arm N Mean (SD) Median (IQR) Range N Mean (SD) Median (IQR) Range EQ-5D-5 L Utility Scores Baseline 28 −0.23 (0.25) −0.25 (0.39) 0.83 31 −0.15 (0.24) −0.19 (0.18) 1.15 12 weeks 28 −0.2 (0.22) −0.2 (0.29) 0.78 31 −0.12 (0.27) −0.15 (0.35) 0.95 24 weeks 28 −0.21 (0.27) −0.26 (0.47) 0.9 31 −0.06 (0.28) −0.09 (0.43) 1.14 ICECAP-O Capability Scores Baseline 27 0.79 (0.14) 0.84 (0.22) 0.46 31 0.77 (0.12) 0.77 (0.2) 0.43 12 weeks 27 0.8 (0.11) 0.82 (0.13) 0.46 31 0.8 (0.13) 0.83 (0.19) 0.48 24 weeks 27 0.8 (0.14) 0.83 (0.14) 0.67 31 0.78 (0.15) 0.82 (0.24) 0.54 Table 7 EQ-5D-5 L utility scores and ICECAP-O for health-related quality of life by trial arm 1 h 50 min in total when the weekly group exercise inter- vention was included. Home exercising was hard to sus- tain, particularly once the 12 weekly group classes were completed: this concords with other research [32, 33, 35]. The VIP and VIP2UK studies [32, 33] and ProAct65+ 1 h 50 min in total when the weekly group exercise inter- vention was included. Home exercising was hard to sus- tain, particularly once the 12 weekly group classes were completed: this concords with other research [32, 33, 35]. Delivery of the intervention The VIP and VIP2UK studies [32, 33] and ProAct65+ Guidelines recommend at least 36 h of exercise per falls prevention exercise programme, over the 12 weeks of par- ticipation, equating to a total of three hours per week [2, 29, 38]. However, VIOLET study participants spent an average of 50 min on home exercise, which only provided Table 8 Summary of issues raised and implications for a future definitive trial Issues Findings Implications for future trial Identifying potential participants a) Via third sector organisations Not clear how many had been contacted Very time consuming Impractical to use research staff b) Via low vision clinic Only few expressed interest when Eye Clinic Liaison Officers involved Would need to support third sector staff more Debatable whether Eye Clinic is worthwhile route First contact may be better via GP Did eligible participants consent? 66/68 (97%) of those eligible consented to enter trial: exceeded target of 50% Estimate biased upwards since some declined before being screened Unreliable estimate from this pilot trial Met recruitment target? Randomised 64 participants to trial: below target of 80 Recruitment was more difficult than anticipated: need to improve procedures Compliance with intervention 25/33 (76%) completed at least 9/12 group sessions: exceeded target of 70% Compliance with exercise class regime is possible Home exercise duration per week Median duration 50 min: encouraged to exercise for 120 min Need to find ways of encouraging participants to exercise at home for longer Intervention FaME intervention successfully adapted for VIOP Need exercises appropriate to ability of individual, providing sufficient challenge Retention throughout study 59 participants retained to end of study: narrowly missed target of 60 Retention was better than anticipated Outcome assessments completed 60/64 (94%) provided data at 12 week visit 59/64 (92%) provided data at 24 week visit: exceeded target of 70% Very few items of missing data on any scale Once recruited, retention and data collection was very good. No problem with completion if researchers can help, but this is time-consuming Suitability of candidate outcome measures Suitable, howeverelements of standard assessments were occasionally inappropriate for VIOP Other outcome assessments may additionally be included. Safety issues? Delivery of the intervention 16 serious adverse events, but none deemed due to intervention: less than target of < 10% No safety issues Sample size calculation for definitive trial Calculation very imprecise because of lack of estimates of parameters for SFES-1 Better estimates necessary Data collection for health economic analysis Data often missing from resources form Data collection form needs to be more structured Summary of issues raised and implications for a future definitive trial Findings Implications for future trial Page 12 of 15 Page 12 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 [35], reported poor adherence to the home exercise com- ponent, although the studies suggested that stricter adher- ence was associated with fewer falls. Access to home exercise information could be enhanced technologically by providing audio material that can be easily paused, revisited and generally modified to individual preference, such as DAISY (Digital Accessible Information System), screen reader, voice synthesiser, MP3, talking book and Braille. Continued development of strategies to increase adherence to home exercise, or an alternative may be to offer more group sessions in a week, is recommended, ra- ther than reliance upon home exercise. Classes may also provide a platform for an informal sharing and exchanging of broad health information. Because reduction in falls risk does require sustained engagement with exercise to maintain strength and balance as we age, irrespective of VIOP, it is important that future studies attempt to in- corporate strategies to encourage behaviour change be- yond the intervention. Many falls interventions attempt to do this by encouraging self management and self-efficacy with engagement in home exercise, but as engagement with home exercise in VIOP appears so challenging, other strategies may need to be adopted. and thus open to individual interpretation. This was also the case when assessing the participant’s ability to walk in- doors and outdoors with or without aid. Mixing together able and less able participants, impacts on the challenge and potential effectiveness of the programme. Stratifica- tion by functional ability, as well as falls risk, is recom- mended for a definitive trial. There were a number of potential participants who ‘self-reported’ an uncontrolled medical condition as rea- son for exclusion, however the degree of concordance with a GP was not assessed. Methodological issues The completion rates of the outcome measures were very high. Ninety-four percent of trial participants pro- vided data at 12 weeks and 92% at 24 weeks, although as researchers often were needed to aid completion, the time allocated to this task requires consideration. In general, these participants took longer to perform the TUG time/functional ability assessment than that re- ported for healthy community dwelling subjects 65–84 years [52]. Additional outcome measures, such as frailty, may be included in future, in order to indicate whether exercise is maintaining the level of resilience, even if it does not lead to an improvement. This may be more ap- propriate and realistic for an older population with many co-morbidities. Further, a longer follow-up period of 12 to 18 months would also be recommended in future studies to explore effects of discontinuation and longer term effects (30). Delivery of the intervention If recruitment were to be carried out in primary care in a future study, the assess- ment would be carried out by a GP, rather than by a par- ticipant or a researcher, who may not be medically trained, although our method replicates current practice in falls services. Although the progression criteria to judge the feasi- bility of progressing to a full trial were all met, suggest- ing that this intervention could be taken to a full study, recommendations from the research team and partici- pants suggested that most VIOP in the pilot trial could have been integrated into a mainstream class. Training (CPD) of instructors to accommodate a range of VIOP in their mainstream sessions is minimal and easy to fa- cilitate through online training so would be affordable and have sustainable reach. Only those with multiple co-morbidities (such as extreme deafness or extreme frailty) required significantly more supervision. Main- stream classes can be much larger, so if VIOPs were to join, extra supervisors may be necessary. Economic evaluation The current feasibility study has shown that whilst it is possible to collect most of the data necessary for a full cost-effectiveness analysis of the exercise interven- tion compared with usual care (cost of intervention per se, utility values and capability values), there were some practical issues in accessing information regard- ing participant self-reporting of resource use post intervention. It is unclear whether this was due to their visual impairment. Participant inputs on use of health and social care services and broader service use were often missing, despite telephone calls by re- searchers. The data collection instrument for this was the participant self- reported Falls Resources / Ex- penses Form (including informal care givers time). This was based on a series of open ended questions as it was initially thought by study team that carers filling out the form would find these types of ques- tions easier to respond to. However, from this data capture form, it was unclear whether an absence of recorded data signified missing or whether partici- pants had not actually received any formal care. Fur- thermore, detail of the type of care was also lacking, Within the VIOLET feasibility study there was no as- sessment of visual impairment. This was largely a prag- matic decision; however, this is recommended for future studies as it would allow an assessment of whether a VIOP might be able to join a mainstream class, or whether they might require one to one intervention, or more intensive supervision. The inclusion criteria did not allow screening out for ‘deafness’. People who are profoundly deaf and have a VI are difficult to accommodate in a group setting. The assessment of whether potential participants were ‘physically able to take part in a group exercise class’ cat- egory was, on the whole, carried out when face to face, Page 13 of 15 Page 13 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 Although the progression criteria were met, a future definitive trial should consider the development of strategies to increase physical activity and structured exercise at home, in order to reach recommended levels of activity. Stratification of those with low, moderate and high falls risk should also be explored and that should include an assessment of visual im- pairment and functional ability as those with better functional vision may be able to be integrated into mainstream programmes. Abbreviations AE Ad E AE: Adverse Event; DAISY: Digital Accessible Information System; ECLO: Eye Clinic Liaison Officer; FaME: Falls Management Exercise; FoF: Fear of Falling; FRAT: Falls Risk Assessment Tool; GCP: Good Clinical Practice; GP: General Practitioner; HADS: Hospital Anxiety and Depression Scale; ICC: Intra-class correlation coefficient; ICE-CAP-O: ICEpop Capability Measure for Older People; NHS: National Health Service; NIHR: National Institute of Health Research; NRES: National Research Ethics Service; NSBP: Newcastle Society for Blind People; NuTH: The Newcastle upon Tyne Hospitals NHS Foundation Trust; PAG: Project Advisory Group; PHR: Public Health Research; PI: Principal Investigator; PIS: Patient Information Sheet; PSI: Postural Stability Instructor; QoL: Quality of Life; R&D: Research and Development; RCT: Randomised Controlled Trial; REC: Research Ethics Committee; SAE: Serious Adverse Event; SFES- I: Short Falls Efficacy Scale - International; TMG: Trial Management Group; TOC: Trial Oversight Committee; TUG: Timed Up and Go; VI: Visual Impairment; VIOLET: Visually Impaired Older Adults Exercise Programme for Falls Prevention; VIOP: Visually Impaired Older Person; WSAS: Work and Social Adjustment Scale Economic evaluation A recruitment strategy should be discussed and agreed across recruiting or- ganisations which should also could include primary care practices and involve the multidisciplinary team. rendering any estimation of costs of the use of health and personal social care resources difficult. The use of a more structured previously piloted data collection tool may have mitigated against some of these issues though it is well documented that reliance on pa- tients/carers as a data collection method is limited by biases in recall, nonresponse, and evasiveness [53]. Missing data is a common problem for economic evaluations that run alongside clinical trials [54]. g The HRQoL scores showed that participants in both arms of the trial at all time points were poor. How- ever, capability scores were towards the high end of being capable. The ICECAP measures potentially offer a broader assessment of quality of life and well-being, in comparison to measures routinely used in eco- nomic evaluation, such as the EQ-5D-3 L [55]. This broader assessment may allow measurement of the full effects of an intervention or treatment. Previous research has indicated that the ICECAP-O (for older people) and EQ-5D-3 L measures provide complemen- tary information and are not substitutes [56]. The same may be true for the EQ-5D-5 L however, this is currently not addressed in the literature. Limitations The main limitations were that the sample recruited typically exhibited low to moderate falls risk and rela- tively low fear of falling. The intervention may have benefited higher risk fallers and these should be tar- geted for recruitment in future studies. Recruitment proved difficult and we did not recruit the originally planned sample of 80 participants, though retention was better than anticipated. Availability of data and materials It was possible to adapt successfully an existing, widely used exercise intervention for falls prevention (FaME) for people with visual impairment. Adherence to the intervention was high with very low attrition rates. It was to be expected that there would be no difference between the two groups in the main out- come measure, fear of falling, but a further two rea- sons may have contributed to the finding: there was possible a sub-optimal dose of exercise and the ma- jority of participants were found to be of low risk. The dataset is held at an institutional repository. To access the dataset, please contact the lead author (NA), who was Chief Investigator and representative for the Sponsor of the study. The full protocol has been previously published [36]. Acknowledgements We wish to thank foremost the participants in this study. We would also like to thank the members of the VIOLET’s study Trial Oversight Committee (TOC), Chaired by Dr. Chie Wei Fan. We would like to thank members of the study’s Advisory Group for their invaluable input and also, the two organisations, Visibility and Newcastle Society for Blind People (NSBP) for their support in shaping the project and in recruitment. Finally, we thank the study’s Postural Stability Instructors, Vivienne Thompson (Health Works, Newcastle) and Allison Sampson (Later Life Training, Visibility, and Glasgow) for the delivery of our intervention. It is possible that there was potential bias by having the weekly telephone call, which may have acted like an intervention in itself. There was no assessment of visual impairment and we only recruited English speaking participants. These findings limit the gener- alisability of the findings to a wider, more ethnically diverse population. There was a lack of appropriate estimates upon which to calculate sample size for a definitive trial. Funding h This project was funded by the Public Health Research Programme of the National Institute for Health Research, (NIHR) United Kingdom, Health Promotion Interventions for People with Impairments Programme (project Number: 12/81/07). The funding body did not participate in the conception, design, analysis or conduct of the study. This was carried out independently by the authors. Competing interests Dawn Skelton (DS) is a Director of Later Life Training Ltd., a company that runs as a not for profit organisation, delivering FaME (PSI) training to health and fitness professionals across the UK. 16. Rubenstein L. Falls in older people: epidemiology, risk factors and strategies for prevention. Age Ageing 2006;35(S2):ii37–ii41. 17. Dhital A, Pey T, Stanford MR. Visual loss and falls: a review. Eye. 2010;24:1437–46. 17. Dhital A, Pey T, Stanford MR. Visual loss and falls: a rev 18. Brundle C, Waterman H, Ballinger C, Olleveant N, Skelton D, Stanford P, et al. The causes of falls: views of older people with sight impairment. Health Expect. 2015;42:2021–31. Received: 20 March 2018 Accepted: 27 November 2018 23. Campbell S. Deteriorating vision, falls and older people: the links. Glasgow: Visibility; 2005. 24. Kendrick D, Kumar A, Carpenter H, Zijlstra G, Skelton D, Cook J, et al. Exercise for reducing the fear of falling in older people living in the community. Cochrane Database Syst Rev. 2014;28(11):CD009848. https://doi.org/10.1002/14651858.CD009848.pub2. Consent for publication 15. Lord S, Smith S, Menant J. Vision and Falls in older people: risk factors and intervention strategies. Clinical Geriatric Medicine. 2010;26:569–81. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 19. Scheffer A, Schuurmans M, van Dijk N, van der Hooft T, SE. dR. fear of falling: measurment strategy, prevalence, risk factors and onsequences amoung older persons. Age Ageing 2008;37:19–24. Authors’ contributions k ll Prevalence of visual imparment in people aged 75 years older in Britian: results from the MRC trial of assessment and management of older people in the community. British Journal Opthalmology. 2002; 86:795–800. 10. Steinman B, Nguyen A, Pynoos J, Leland N. Falls-prevention interventions for persons who are blind or visually impaired. Research and Practice in Visual Impairment Blindness. 2011;4:83–91. 11. Lamoureux E, Gadgil S, Pesudovs K, Keeffe J, Fenwick E, Dirani M, et al. The relationship between visual function, duration and main causes of vision loss and falls in older people with low vision. Graefe's Archive of Clinical Experimental Opthalmology. 2010;248:527–33. Authors’ contributions k ll NA took overall responsibility for the study and the writing of the manuscript. DS contributed to the design of the study, led on the production of the adaptation of the FaME exercise intervention manual and contributed to the drafting of the manuscript. CB contributed to its design and the writing of the manuscript. DH and TF, contributed to the design of the study, conduct of the trial, managed the analysis and Page 14 of 15 Page 14 of 15 Page 14 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 interpretation of the quantitative and statistical results and co-drafted relevant sections of the manuscript. RL and JW to the design of the study, managed the conduct of the trial. DC managed the Newcastle site data and LD de Jong managed the Glasgow site. SG managed quality assurance and the production of the FaME exercise intervention manual. JG conducted the health-economic analysis. VD contributed to the design of the study. MC, was PI for the study NHS site, NHS low vision clinic recruitment and clinical overseeing of the study’s serious adverse events. HW and SP, provided expertise and guidance throughout on all aspects of design. All authors contributed to drafting the manuscript. All authors read and approved the final manuscript. 8. Boyce T. Falls—costs, numbers and links with visual impairment RNIB, evidence and service impact. London: RNIB; 2011. 8. Boyce T. Falls—costs, numbers and links with visual impairment RNIB, evidence and service impact. London: RNIB; 2011. interpretation of the quantitative and statistical results and co-drafted relevant sections of the manuscript. RL and JW to the design of the study, managed the conduct of the trial. DC managed the Newcastle site data and LD de Jong managed the Glasgow site. SG managed quality assurance and the production of the FaME exercise intervention manual. JG conducted the health-economic analysis. VD contributed to the design of the study. MC, was PI for the study NHS site, NHS low vision clinic recruitment and clinical overseeing of the study’s serious adverse events. HW and SP, provided expertise and guidance throughout on all aspects of design. All authors contributed to drafting the manuscript. All authors read and approved the final manuscript. 9. Evans J, Fletcher A, Wormald R, Ng E, Stirling S, Smeeth L, et al. Author details 1 20. Yardley L, Smith H. A prospective study of the relationship between feared consequences of falling and avoidance of activity in community-living older people. Gerontologist. 2002;42:17–23. 1Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne NE7 7XA, UK. 2Institute of Applied Health Research, School of Health & Life Sciences, Glasgow Caledonian University, Glasgow, UK. 3Institute of Health and Society, Baddiley-Clark Building, Newcastle University, Newcastle upon Tyne, UK. 4Newcastle Clinical Trials Unit, Newcastle University, 1-4 Claremont Terrace, Newcastle upon Tyne, UK. 5School of Physiotherapy and Exercise Science, Curtin University, Bentley, Western Australia. 6Healthcare Sciences, Cardiff University, Cardiff, UK. 7Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. 8Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. 21. Zijlstra GA, van Haastregt J, van Eiijk J, van Rossum E, Stalenhoef P, Kempen G. Prevalence and correlates of fear of falling, and assoicated avoidance of activity in the general population of community-living older people. Age Ageing. 2007;36:304–9. 22. Grue E, Finne-Soveri H, Stolee P, Poss J, Wergeland Sorbye L, Noro A, Hirdes J, Hylen Ranhoff A. Recent visual decline-a health hazard with consequences for social life: a study of home care clients in 12 countries. . Current Gerontology and Geriatric Research 2010., Article ID 503817, 9 pages. https://doi.org/10.1155/2010/503817 Received: 20 March 2018 Accepted: 27 November 2018 Ethics approval and consent to participate Favourable ethical opinion from the Newcastle and North Tyneside Research Ethics Committee and R&D approval was obtained prior to commencement of the intervention. Glasgow Caledonian University was approved as a non-NHS site with local ethics approval. Information sheets were provided to all eligible subjects and written informed consent obtained prior to any study procedures. Favourable ethical opinion from the Newcastle and North Tyneside Research Ethics Committee and R&D approval was obtained prior to commencement of the intervention. Glasgow Caledonian University was approved as a non-NHS site with local ethics approval. 12. de Boer M, Pluijm S, Lips P, Moll A, Volker-Dieben H, Deeg D, et al. Different aspects of visual impairment as risk factors for falls and fractures in older men and women. J Bone Miner Res. 2004;19:1539–47. 13. Klein B, Moss S, Klein R, Lee K, Cruickshanks K. Associations of visual function with physical outcomes and limitations 5 years in an older opoulation. The Beaver Dam eye study Opthalmology. 2003;110:644–50. Information sheets were provided to all eligible subjects and written informed consent obtained prior to any study procedures. 14. Lamoreux E, Chong E, Wang J, Saw S, Aung T, Mitchell P, et al. Visual impairment, causes of vision loss, and falls: the Singapore Malay eye study. Investig Ophthalmol Vis Sci. 2008;49:528–33. 7. Tian Y, Thompson J, Buck D. Sonola L. London: Exploring the system-wide costs of falls in older people in Torbay; 2013. References 1. Painter J, Elliott S, Hudson S. Falls in community-dwelling adults aged 50 years and older: prevalence and contributing facotrs. J Allied Health. 2009; 38:201–7. 1. Painter J, Elliott S, Hudson S. Falls in community-dwelling adults aged 50 years and older: prevalence and contributing facotrs. J Allied Health. 2009; 38:201–7. 25. Kumar A, Delbaere K, Zijstra G, Carpenter H, Illifie S, Masud T, Skelton D, Morris R, Kendrick D. Exercise for reducing fear of falling in older people living in the community: Cochrane systematic review and meta-analysis. Age Ageing. 2016;45(3):345–52. 2. Panel on Prevention of Falls in Older Persons. American Geriatrics Society, British geriatrics society. Summary of the updated American Geriatircs society/British geriatrics society clinical practice guideline for prevention of falls in older persons. Journal of American Geriatric Society. 2011;59:148–57. 26. Hadjistavropoulos T, Delbaere K, Fitzgerald T. Reconceptualizing the role of fear of falling and balance confidence in fall risk. Journal of Aging Health. 2011;23:3–23. 3. Parry S, Bamford C, Deary V, Finch T, Gray J, MacDonald C, et al. Cognitive- behavioural therapy-based intervention to reduce fear of falling in older people: therapy development and randomised controlled trial - the strategies for increasing Independence, confidence and energy (STRIDE) study. Health Technol Assess. 2016:56. 27. Parry S, Finch T, Deary V. How should we manage fear of falling in older adults living in the community? Br Med J. 2013;346:f2933. 28. Cameron ID, Gillespie LD, Robertson MC, Murray GR, Hill KD, Cumming RG, Ngaire K. Interventions for preventing falls in older people in care facilities and hospitals. Cochrane database of systematic reviews. 2012;12:CD005465– 1–CD005465–63. 4. American Geriatrics Society. British geriatrics society, American Academy of Orthopadic surgeons panel on Falls prevention. Guideline for the prevention of Falls in older persons. Journal of American Geriatric Society. 2001;49:664–72. 29. NICE 21 Falls Guideline. Falls: assessment and prevention of falls in older people. In: National Institute of clinical evidence; 2010. 5. Heinrich S, Rapp K, Rissman U, Becker C, Konig H. Cost of falls in old age: a systematic review. Osteoporos Int. 2010;21:891–902. 30. Gillespie LD, Robertson M, Gillespie WJ, Sherrington C, Gates S, Clemson LM, Lamb SE. Interventions for preventing falls in older people living in the community. Cochrane Database of Systematic Reviews 2012, Issue 9. Art. No.: CD007146. DOI: https://doi.org/10.1002/14651858.CD007146.pub3. 6. Age UK. Falls in the over 65s cost £4.6 million a day. In: Age UK, editor. 2015. 7. 5. Heinrich S, Rapp K, Rissman U, Becker C, Konig H. Cost of falls in old age: a systematic review. Osteoporos Int. 2010;21:891–902. 6. Age UK. Falls in the over 65s cost £4.6 million a day. In: Age UK, editor. 2015. References Tian Y, Thompson J, Buck D. Sonola L. London: Exploring the system-wide costs of falls in older people in Torbay; 2013. 7. Tian Y, Thompson J, Buck D. Sonola L. London: Exploring the system-wide costs of falls in older people in Torbay; 2013. Page 15 of 15 Adams et al. BMC Geriatrics (2018) 18:307 Adams et al. BMC Geriatrics (2018) 18:307 31. Lamb S, Gates S, Fisher J, et al. Scoping exercise on fallers’ clinics, report. London: National Co-ordinating Centre for NHS Service Delivery and Organisation R & D, 2007. 32. Campbell AJ, Robertson MC, Grow SJL, Kerse NM, Sanderson GF, Jacobs RJ, Sharp DM, Hale LA. Randomised controlled trial of prevention of falls in people aged > or =75 with severe visual impairment: the VIP trial. BMJ. 2005;331:817–23. https://doi.org/10.1136/bmj.38601.447731.55. 33. Waterman H, Ballinger C, Brundle C, et al. A feasibility study to prevent falls in older people who are sight impaired: the VIP2UK randomised controlled trial. Trials. 2016;17:464. https://doi.org/10.1186/s13063-016-1565-0. 34. Royal College of Physicians. Older people’s experience of therapeutic exercise as part of a falls prevention service. London: R Coll P, 2012. https:// www.laterlifetraining.co.uk/wp-content/uploads/ 2012/03/patient-and- public-involvement-report-march-2012.pdf. 35. Iliffe S, Kendrick D, Morris R, Masud T, Gage H, Skelton D, Dinan S, Bowling A, Griffin M, Haworth D, Swanwick G, Carpenter H, Kumar A, Stevens Z, Gawler S, Barlow C, Cook J, Belcher C. Multicentre cluster randomised trial comparing a community group exercise programme and homebased exercise with usual care for people aged 65 years and over in primary care. Health technol Assess. 2014;18(49):1–105. 36. Skelton DA, Bailey C, Howel D, et al. Visually impaired OLder people's exercise programme for falls prevenTion (VIOLET): a feasibility study protocol. BMJ Open. 2016;6:e011996. https://doi.org/10.1136/bmjopen-2016-011996. 37. Skelton DA, Dinan SM. Exercise for falls management: rationale for an exercise programme to reduce postural instability. Physiotherapy: Theory and Practice. 1999;15:105–20. 38. Power V, Clifford A. Charateristics of optimum falls prevention exercise programmes for community-dwelling older adults using the FITT principle. Eur Rev Aging Phys Act. 2013;10(2):95–106. 39. Kempen GI, Yardley L, van Haastregt JC, et al. The short FES-I: ashortened version of the falls efficacy scale-international to assess fear of falling. Age Ageing. 2008;37:45–50. 40. Zijlstra GA, van Haastregt JC, van Eijk JT, et al. Prevalence and correlates of fear of falling, and associated avoidance of activity in the general population of community-living older people. Age Ageing. 2007;36:304–9. 41. Podsiadlo D, Richardson S. 55. Keely T, Coast J, Nicholls N, Foster S, Jowett S, Al-Janabi H. An analysis of the complementarity of ICECAP-A and EQ-5D-3L in an adult population of patients with knee pain. Health Qual Life Outcomes. 2016;14:36. References The timed ‘up & go’: a test of basic functional mobility for frail elderly persons. J Am Geriatr Soc. 1991;39:142–8. 42. Nandy S, Parsons S, Cryer C, et al. on behalf of the Falls Prevention Pilot Steering Group. Development and preliminary examination of the predictive validity of the Falls risk assessment tool (FRAT) for use in primary care. J Public Health. 2004;26:138–43. 43. Gill DP, Jones GR, Zou GY, et al. The phone-FITT: a brief physical activity interview for older adults. J Aging Phys Act. 2008;16:292–315. 44. De Jong Gierveld J, Van Tilburg T. A 6-item scale for overall, emotional, and social loneliness: confirmatory test on survey data. Res Ageing 2006;28:582–598. 45. Zigmund AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983;67:361–70. 46. Mundt JC, Marks LM, Shear MK, et al. The work and social adjustment scale: a simple measure of impairment in functioning. Br J Psychiatry. 2002;180:461–4. 47. Herdman M, Gudex C, Lloyd A, et al. Development and preliminary testing of the new five-level version of EQ-5D (EQ-5D-5L). Qual Life Res. 2011;20:1727–36. 47. Herdman M, Gudex C, Lloyd A, et al. Development and preliminary testing of the new five-level version of EQ-5D (EQ-5D-5L). Qual Life Res. 2011;20:1727–36. 48. Davis JC, Liu-Ambrose T, Richardson CG, et al. A comparison of the ICECAP- O with EQ-5D in a falls prevention clinical setting: are they complements or substitutes? Qual Life Res. 2013;22:969–77. 49. Peyre H, Leplege A, Coste J. Missing data methods for dealing with missing items in quality of life questionnaires. Qual Life Res. 2011;20:287–300. 50. Lancaster G. Design and analysis if pilot studies: recommendations for good practice. J Eval Clin Pract. 2004;10(2):307–12. 51. Bunn F, Barnet-Page E, McInnes E, Dickenson A, Horton K. A systematic review of older people's perceptions of facilitators and barriers to participation in falls-prevention interventions. Ageing Soc. 2008;4:449–72. 52. Medley A, Thompson M. The effect of assistive devices on the performance of community dwelling elderly on the timed up and go test. Issues Aging 1997;20(3):7.44.53. 53. Bowling A. Mode of questionnaire administration can have serious effects on data quality. J Public Health 2005;27:281–91. 53. Bowling A. Mode of questionnaire administration can have serious effects on data quality. J Public Health 2005;27:281–91. 54. Petrou S, Gray A. Economic evaluation alongside randomised controlled trials: design, conduct, analysis, and reporting. Br Med J. 2011;342:d1548. 54. Petrou S, Gray A. 56. Davis J, Liu-Ambrose T, Richardson C, Bryan S. A comparison of the ICECAP- O with EQ-5D in falls prevention clinical setting: are they complements or substitutes? Qual Life Res. 2012;22(5):969–77. 55. Keely T, Coast J, Nicholls N, Foster S, Jowett S, Al-Janabi H. An analysis of the complementarity of ICECAP-A and EQ-5D-3L in an adult population of patients with knee pain. Health Qual Life Outcomes. 2016;14:36. 56. Davis J, Liu-Ambrose T, Richardson C, Bryan S. A comparison of the ICECAP- O with EQ-5D in falls prevention clinical setting: are they complements or substitutes? Qual Life Res. 2012;22(5):969–77. References Economic evaluation alongside randomised controlled trials: design, conduct, analysis, and reporting. Br Med J. 2011;342:d1548.
https://openalex.org/W4288072132	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/9A252DC8B168076189EAABB54899F76B/S1073110522000547a.pdf/div-class-title-restricting-unhealthy-food-and-beverage-advertising-in-brazil-challenges-and-opportunities-div.pdf	English	null	Restricting Unhealthy Food and Beverage Advertising in Brazil: Challenges and Opportunities	The journal of law, medicine & ethics/The Journal of law, medicine & ethics	2,022	cc-by	5,454	Isabel Barbosa, LL.M., LL.B., is an Adjunct Professor at Georgetown University and a Senior Associate at the O’Neill Institute for National and Global Health Law. Fábio Leite, Ph.D., M.A., LL.B., is a Professor of Constitutional Law at the Pontifícia Universidade Católica do Rio de Janeiro and the Coordinator of PLEB – Pesquisa sobre Liberdade de Ex- pressão no Brasil. Carla Britto Pereira, LL.M., M.A., LL.B., is a Researcher at PLEB – Pesquisa sobre Liberdade de Ex- pressão no Brasil. Restricting Unhealthy Food and Beverage Advertising in Brazil: Challenges and Opportunities Isabel Barbosa1, Fábio Leite2,3, and Carla Britto3 1: GEORGETOWN UNIVERSITY, WASHINGTON, DC, USA, 2: PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, RIO DE JANEIRO, BRAZIL, 3: PLEB – PESQUISA SOBRE LIBERDADE DE EXPRESSÃO NO BRASIL, RIO DE JANEIRO, BRAZIL Keywords: Advertising Restrictions, Unhealthy Food and Beverages, Dispute Resolution, Com- mercial Speech, Freedom of Speech Keywords: Advertising Restrictions, Unhealthy Food and Beverages, Dispute Resolution, Com- mercial Speech, Freedom of Speech Abstract: In Brazil, the normative landscape around advertising is complex, not the least because of limitations inherent to dispute reso- lution mechanisms. Focusing on unhealthy food and beverages, this case study identifies some challenges and opportunities around advertising restrictions, including in relation to freedom of speech. O O ver the years, the advertising of unhealthy food and beverages has become the object of legal debate in Brazil. On the one hand, civil society has developed tools to identify ads deemed to be misleading or abusive, including the Observatory of Food Advertisement, a platform that allows for such ads to be reported by the public and analyzed by legal teams, eventually leading to proceedings filed before administrative or judicial bodies.1 Their premise is that the food and beverage industry frequently engages in advertising that infringes on existing laws and regula- tions, and — most importantly — that it must be held accountable by third parties. On the other hand, the food and beverage industry has participated in cam- paigns of responsible advertising, such as Take respon- sible advertising seriously,2 a series of posts in social media aimed at corporations launched in partnership with, among others, advertisers and trademark asso- ciations.3 They rely on the premise that companies are capable of holding themselves to high standards. 1: GEORGETOWN UNIVERSITY, WASHINGTON, DC, USA, 2: PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, RIO DE JANEIRO, BRAZIL, 3: PLEB – PESQUISA SOBRE LIBERDADE DE EXPRESSÃO NO BRASIL, RIO DE JANEIRO, BRAZIL These opposing narratives are best exemplified by the issue of advertising to children, including but not limited to food and beverages. Civil society has repeat- edly stated that advertising to children is necessarily abusive and therefore illegal, pointing to decisions issued by judges of the Superior Tribunal of Justice (hereinafter STJ) — the highest court for federal law interpretation in Brazil.4 By contrast, the Advertis- ers Association has openly questioned this position, recently issuing guidelines on Responsible Market- ing: Safeguards and Limits of Advertising to Chil- Isabel Barbosa, LL.M., LL.B., is an Adjunct Professor at Georgetown University and a Senior Associate at the O’Neill Institute for National and Global Health Law. Restricting Unhealthy Food and Beverage Advertising in Brazil: Challenges and Opportunities Isabel Barbosa1, Fábio Leite2,3, and Carla Britto3 1: GEORGETOWN UNIVERSITY, WASHINGTON, DC, USA, 2: PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, RIO DE JANEIRO, BRAZIL, 3: PLEB – PESQUISA SOBRE LIBERDADE DE EXPRESSÃO NO BRASIL, RIO DE JANEIRO, BRAZIL Fábio Leite, Ph.D., M.A., LL.B., is a Professor of Constitutional Law at the Pontifícia Universidade Católica do Rio de Janeiro and the Coordinator of PLEB – Pesquisa sobre Liberdade de Ex- pressão no Brasil. Carla Britto Pereira, LL.M., M.A., LL.B., is a Researcher at PLEB – Pesquisa sobre Liberdade de Ex- pressão no Brasil. 291 commercial speech and commercial determinants of health • summer 2022 ercial speech and commercial determinants of health • summer 2022 The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © The Author(s), 2022. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: https://doi.org/10.1017/jme.2022.54 https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press 0.1017/jme.2022.54 Published online by Cambridge University Press SYMPOSIUM dren, where they argue that advertising to children is not necessarily abusive and that the specific situa- tions in which children get taken advantage of should be assessed on a case-by-case basis.5 Setting aside this normative discussion, advertisement to children, including of unhealthy food and beverages, is still common in Brazil.6 Constitution and the Consumer Protection Code. We then analyze existing dispute resolution mechanisms, exploring specifically the shortcomings of self-regula- tion by the National Council of Advertisement Self- Regulation (hereinafter CONAR) and landmark court decisions. Finally, we discuss one recent state law, with potential to increase the protection of public health in primary schools, which was recently questioned in court on the grounds of free commercial speech. From the legal standpoint, the dynamics of unhealthy food and beverage advertising in Brazil is both a product of, and results in, structural challenges This case study seeks to identify and analyze some challenges and opportunities relevant to the debate around advertising restrictions in Brazil, focusing specifically on the advertising of unhealthy food and beverages. We start with a brief overview of the legal framework on free speech and advertisement, with an emphasis on legislation, as opposed to all normative acts, exploring only select provisions of the Brazilian Constitution and the Consumer Protection Code. We then analyze existing dispute resolution mechanisms, exploring specifically the shortcomings of self-regulation by the National Council of Advertisement Self-Regulation (hereinafter CONAR) and landmark court decisions. Finally, we discuss one recent state law, with potential to increase the protection of public health in primary schools, which was recently questioned in court on the grounds of free commercial speech. worth noting. First, identifying, reporting and eventu- ally litigating unlawful ads is a lengthy and cumber- some process that demands considerable resources. By the time this process concludes, even if corpora- tions are ordered to take down the ad in question and/ or to pay a fine, airing it may still have been profitable. journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press ercial speech and commercial determinants of health • summer 2022 Second, the cases that make it to court can lead to rel- evant — even landmark — decisions, but these do not always carry the same weight as precedents would in common law countries.7 In this sense, every ad iden- tified, reported and challenged before a court of law does not necessarily prevent other potentially unlaw- ful ads, which might then need to undergo the same process, leading to a whack-a-mole situation that can prove extremely costly for society. Brief Overview of the Normative Landscape The adoption of the Constitution of 1988, which re- established democracy following the military dicta- torship in Brazil, and the enactment of the Consumer Protection Code in 1990, set important limits to advertising. The Constitution establishes freedom of expression as a fundamental right, protecting the free expression of thoughts (article 5, IV) and of intellectual, artistic, scientific and communications activities regardless of censorship or licensing (article 5, IX). Regarding advertisement, the Constitution lays the foundation for its restriction by establishing that “it is within the competence of federal law to: (...) establish legal means which afford persons and families the possibil- ity of defending themselves against (…) the advertis- ing of products, practices, and services which may be harmful to health or to the environment” (article 220, § 3, II). More specifically, it states that “the advertising of tobacco, alcoholic beverages, pesticides, medicines and therapies shall be subject to legal restrictions (…) and shall contain, whenever necessary, a warning con- This case study seeks to identify and analyze some challenges and opportunities relevant to the debate around advertising restrictions in Brazil, focusing specifically on the advertising of unhealthy food and beverages. We start with a brief overview of the legal framework on free speech and advertisement, with an emphasis on legislation, as opposed to all normative acts, exploring only select provisions of the Brazilian 292 Barbosa, Leite, and Britto cerning the damages which may be caused by their use” (article 220, §4).8 ment when prompted, necessarily after the circulation of the advertising piece in question. If the advertis- ing piece is considered not to have violated the rules of the Brazilian Self-Regulation Advertising Code, the Council of Ethics can dismiss the complaint. ercial speech and commercial determinants of health • summer 2022 On the procedural side, CONAR’s governance rules clearly favor advertisers over consumers, consumer associations and consumer protection entities.20 For instance, despite governance rules setting aside space in the Council of Ethics for civil society representa- tives, the selection of such representatives is done by CONAR’s Superior Council, which is in turn made up by representatives of CONAR’s founding members, who are all connected to the advertising industry.21 Finally, the volume of cases considered by CONAR is arguably small in a country of continental proportions with massive investment in advertising:22 according to data found on CONAR’s website, the average is 236 cases per year.23 y The Consumer Protection Code also contains important provisions related to advertising, especially the prohibition of “all misleading or abusive adver- tisement” (article 37).11 In order for advertisement to be considered misleading, the information conveyed needs to be fully or partially false or withhold essential information, inducing consumers to make a mistake.12 In turn, abusive advertising is essentially anti-ethical advertising that preys upon consumers’ vulnerability and goes against basic social values, harming society as a whole.13 The general ban on misleading and abu- sive advertising has been particularly relevant in the context of unhealthy food and beverages due to the fact that, unlike tobacco and alcohol, there is no spe- cific federal law restricting their advertising.14 How- ever, as institutional communications by the STJ itself show, the limits of advertising in relation to consumer protection are not always clear, leading to constant disputes that need to be resolved.15 On the substantive side, CONAR openly frames its mission in defense of free commercial speech; includ- ing the “promot[ion of] free speech in advertising and [the] defen[se of] the constitutional prerogatives of… advertising.”24 This sets the tone of their reason- ing in decision-making. ercial speech and commercial determinants of health • summer 2022 If the advertising piece is instead considered to have violated those rules, the Council of Ethics can recom- mend the suspension or modification of the advertis- ing piece, or issue a mere warning to the advertisers behind it. Notably, it cannot issue fines to the adver- tisers, although in any case fines could fall short of disincentivizing companies from airing ads that are expected to be highly profitable.19 , § Law No. 9.294 in 1996 fleshed out the constitutional provision on advertising. In particular, it defined the time period when the advertisement of alcoholic bev- erages and tobacco could be aired in radio and televi- sion, as well as set rules on the creation of an adver- tising piece, including, for example, restrictions on associating cigarettes and alcoholic beverages with sports and suggesting that tobacco products have calming or stimulating properties.9 In 2011, this law underwent an important reform that prohibited “the commercial advertising of cigarettes, cigarillos, cigars, pipes or any other smoking product, derived from tobacco or not.”10 To date, however, no specific fed- eral law has yet implemented article 220, §3, II of the Constitution, and thereby given “persons and families the possibility of defending themselves against (…) the advertising of products, practices and services which may be harmful to health or to the environment.” Self-regulation by CONAR presents considerable problems of both procedural and substantive nature. ercial speech and commercial determinants of health • summer 2022 For example, in 2011 CONAR dismissed a complaint by Alana, a non-governmental organization, about a McDonald’s ad exhibited dur- ing the trailer of animated film “Rio.”25 In the decision, the rapporteur of the case portrayed Alana as a witch who hates children, stating that “[w]hen the witch Alana comes into scene, children live on bread and water… [N]o more cheeseburgers, fries, milkshake or soda.” They went on to frame the complaint as part of a broader strategy of demonizing advertising to ideo- logically control children.26 In response, Alana said it no longer recognized CONAR as a serious entity to safeguard ethics in advertising due to the open mock- ery displayed in this decision.27 https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press Considering Emerging Opportunities for the Limitation of Commercial Speech i Attempts to adopt specific federal laws regulating the advertisement of unhealthy food and beverages have not yet been successful in Brazil, despite the introduc- tion of several bills in Congress that moved past the general prohibition of misleading and abusive adver- tising.34 In general, these initiatives tend to face strong resistance from both the advertising industry and the food and beverage industry, who have argued in the past that such limitation violates freedom of commer- cial speech among other arguments.35 At the national level, two decisions issued by judges in the STJ are especially relevant. In March 2016, the 2nd Chamber upheld the conviction of a food com- pany for the “Time for Shrek” marketing campaign, which targeted children by using the well-known animated character. Upon the purchase of five cook- ies of the brand, plus a payment of R$ 5.00, the con- sumer could receive a Shrek watch. The judges in the STJ found that the marketing campaign was abusive because it aimed, directly or indirectly, at children, in addition to other arguments.29 In April 2017, the 2nd Chamber of the STJ considered a similar advertising campaign, known as “Sadia Mascots.” Upon the pur- chase of five products of the same brand, plus a pay- ment of R$ 3.00, the consumer could receive a collect- ible stuffed animal, the company mascot. Though the product here — frozen food — was meant for an adult audience, the judges concluded that the kind of prize indicated that the marketing campaign targeted chil- dren and was therefore abusive and unlawful.30 In March 2021, the Supreme Federal Tribunal (hereinafter STF) — the highest constitutional court in Brazil — upheld State Law No. 13.582 of 2016, as amended by State Law No. 14.045 of 2018, which prohibits commercial communication to children in primary schools in the state of Bahia.36 State Law No. 14.045 significantly altered the content of State Law No. 13.582. Before the reform, Law No. 13.582 prohibited the advertisement of unhealthy food and beverages aimed at children on radio and television at certain times, as well as in schools at all times. Since the reform, Law No. Considering Emerging Opportunities for the Limitation of Commercial Speech i 13.582 prohibits not only the advertisement of unhealthy food and beverages, but all commercial communication to children, though this prohibition is now limited to primary schools.37 These are landmark decisions for consumer protec- tion, but ultimately their legal effects are limited to the concrete cases considered, meaning that despite their persuasive power they may or may not be followed by the other judges adjudicating over similar cases across the country.31 The food and beverage industry has already taken advantage of this characteristic of the legal system in Brazil. For example, when the National Agency of Health Surveillance issued Resolution 24 in 2010 — a binding normative act of lower rank than legislation — restricting the advertisement of unhealthy food and beverages, multiple associations from the private sector questioned it in court, filing diffuse actions across different jurisdictions that led to conflicting decisions. The issue is yet to be definitively resolved by high courts.32 The constitutionality of this law was questioned in the STF by the Association of Radio and Television Networks, based in part on the alleged violation of the freedom of commercial speech. In the ruling, the judges indicated that advertising “instrumentalizes free enterprise under commercial speech,”38 and unan- imously held that (i) commercial speech is included in freedom of speech; (ii) freedom of speech is not absolute and can therefore be subject to restrictions; and (iii) restrictions must be proportionate. Though the decision did not lay out a detailed roadmap for the assessment of proportionality, it did list some factors that had shaped their analysis, namely the scope of the ban (commercial communication to children, primary schools). However, the judges did not analyze the nature of commercial speech in detail; for example, whether the protection granted to commercial speech rises to the same level of non-commercial speech.39 Taking advertising to children as an example again, the fact that the industry continues to push the legal narrative that it is not necessarily abusive33 — irre- spective of these recent rulings by judges in the STJ — signals that they are likely to continue such practices. The situation is ever more complex in the context of unhealthy food and beverages, in the absence of spe- cific federal laws limiting advertising. In the end, the broader the legislation, such as general bans on mis- leading and abusive advertising, the greater the lee- The STF also touched upon other relevant points. Confronting Limitations in Dispute Resolution The prevailing model of dispute resolution has long centered on CONAR,16 charged with applying the Bra- zilian Self-Regulation Advertising Code.17 This model was adopted in the mid-1970s in a context of mistrust of government agencies — particularly in relation to limitations to free speech — during the military dicta- torship (1964-1985).18 The judiciary can also exert control over advertis- ing to the extent that it potentially violates relevant laws and regulations, including the Consumer Protec- tion Code. For example, at the state level, the court of appeals of São Paulo ruled that McDonald’s engaged To this day, the self-regulatory model is in force and provides that complaints against misleading or abu- sive advertising must be assessed by CONAR’s Council of Ethics. The Council of Ethics conducts this assess- 293 commercial speech and commercial determinants of health • summer 2022 The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) SYMPOSIUM way for decision-makers across the judiciary to ascer- tain their sometimes-conflicting views. in abusive and therefore unlawful advertising to chil- dren by holding Ronald McDonald concerts — which they framed as educational — in nurseries and pri- mary schools, both public and private. The lawsuit had been filed by the Public Defender’s Office in São Paulo, following reports to government entities.28 Considering Emerging Opportunities for the Limitation of Commercial Speech i journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press Conclusion 7. L. G. Marinoni, “Aproximação Crítica entre as Jurisdições de Civil Law e de Common Law,” Revista de Processo no. 172 (2009): 171-232; R. Cramer, Precedentes Judiciais: Teoria e Dinâmica (Rio de Janeiro: Forense, 2016): 24-235. In Brazil, the normative landscape around advertis- ing is complex, not so much due to the lack of appli- cable norms, but rather to the limitations inherent to dispute resolution mechanisms. The Constitution and existing legislation, not least the Consumer Protec- tion Code, already impose restrictions on advertising. In particular, misleading and abusive unhealthy food and beverage advertising is prohibited. However, in the judiciary, there are structural difficulties related to the limited effects of some high-court decisions that interpret these general provisions. In this sense, more specific laws like the one in the state of Bahia can be strategic in moving forward with protecting public health — not least because it confronts the argument of free commercial speech in relation to other fun- damental rights in the context of advertising restric- tions, possibly setting an example for other states in the country. 8. Brasil, Constituição da República Federativa do Brasil, October, 1988, available at <http://www.planalto.gov.br/ ccivil_03/constituicao/constituicao.htm> (last visited May 2, 2022). The relationship between freedom of speech, com- mercial speech and advertisement will be addressed at a later moment. 9. Brasil, Lei 9.294, July, 1996, available at <http://www.plan- alto.gov.br/ccivil_03/leis/l9294.htm> (last visited May 2, 2022). 10. Brasil, Lei 12.546, December, 2011, available at <http://www. planalto.gov.br/ccivil_03/_ato2011-2014/2011/lei/l12546. htm> (last visited May 2, 2022). Importantly, though there has been no specific federal law, other normative acts have restricted advertising. For example, Resolution 163/2014 by the National Council of the Rights of Children and Adoles- cents (CONANDA) and Resolution 24/2010 by the National Agency of Health Surveillance (ANVISA).i 11. Article 37 of the Consumer Protection Code defines these con- cepts as follows (free translation): §1 It is considered mislead- ing any form of information or communication with advertis- ing nature, entirely or partially false, or in any other way, even by omission, capable of misleading the consumer regarding the nature, characteristics, quality, quantity, properties, ori- gin, price and any other data about products and services. Considering Emerging Opportunities for the Limitation of Commercial Speech i Importantly, it clarified that the advertising restric- tions listed in the Constitution under article 220, §4º are not exhaustive, but rather constitute a list of examples. In other words, by enumerating tobacco, 294 https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press Barbosa, Leite, and Britto alcoholic beverages, pesticides, medicines and thera- pies, the Constitution merely illustrated one possible pathway to the restriction of the freedom of com- mercial speech, in order to promote or protect other fundamental rights, including health — framed not only as an individual’s right, but also as the State’s duty, and one of utmost priority. In particular, the STF stressed the role of government in leading the con- trol of unhealthy food and beverage advertising, rely- ing on recommendations issued by the World Health Organization.40 This indicates that health consider- ations can be paramount in the analysis of advertising restrictions. Note Carvalho Leite and Britto Pereira report grants from Global Center for Legal Innovation on Food Environments at the O’Neill Insti- tute for National and Global Health Law, during the conduct of the study. Isabel Barbosa reports grants from Bloomberg Philanthro- pies, during the conduct of the study. References 1. Instituto Brasileiro de Defesa do Consumidor, Observatório de Publicidade de Alimentos, available at <https://idec.org.br/ publicidadedealimentos> (last visited May 1, 2022). 1. Instituto Brasileiro de Defesa do Consumidor, Observatório de Publicidade de Alimentos, available at <https://idec.org.br/ publicidadedealimentos> (last visited May 1, 2022). p y 2. Original in Portuguese: “Com publicidade responsável não se brinca.” 3. Associação Brasileira da Indústria de Alimentos, “Associações se unem e lançam campanha “Publicidade responsável: No digital também não se brinca,” available at <https://www.abia. org.br/noticias/associacoes-se-unem-e-lancam-campanha- publicidade-responsavel-no-digital-tambem-nao-se-brinca> (last visited May 1, 2022). Overall, this decision paves the way for other states to pass legislation restricting commercial communica- tion in primary schools, given that it weakens proba- ble legal arguments grounded on the freedom of com- mercial speech, provided that the measures restricting advertising — including that of unhealthy food and beverages — are proportionate. Most importantly, even if at the state level, laws like the one in Bahia are relevant because they move the needle from more gen- eral to more specific regulation of advertising, leaving less (though far from non-existent) room for interpre- tation in a context of potentially widespread litigation, as described above. y 4. Publicidade Infantil Não, “Manifesto Publicidade Infantil Já É Proibida,” available at <https://publicidadeinfantilnao.org. br/secao/manifesto/#:~:text=Especificamente%20nas%20 rela%C3%A7%C3%B5es%20de%20consumo,de%20direcio- nar%20publicidade%20a%20crian%C3%A7as> (last visited May 1, 2022). y 5. Associação Brasileira de Anunciantes; Magalhães e Dias Advocacia, “Marketing Responsável: Garantias e Limites da Publicidade Infantil. A ABA em Prol da Publicidade Respon- sável”, 2020, p. 8-9, available at <https://www.aba.com.br/ wp-content/uploads/2019/05/guia-aba-mkt-infantil-edit- 2603-digital.pdf> (last visited May 1, 2022). 5. Associação Brasileira de Anunciantes; Magalhães e Dias Advocacia, “Marketing Responsável: Garantias e Limites da Publicidade Infantil. A ABA em Prol da Publicidade Respon- sável”, 2020, p. 8-9, available at <https://www.aba.com.br/ wp-content/uploads/2019/05/guia-aba-mkt-infantil-edit- 2603-digital.pdf> (last visited May 1, 2022). g p y 6. Observatório de Publicidade de Alimentos, “Casos Documen- tados: Publicidade Infantil,” available at <https://publicidad- edealimentos.org.br/casos-documentados/publicidade-infan- til/> (last visited May 1, 2022). https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press Conclusion § 2° It is abusive, among others, discriminatory advertising of any nature, advertising which incites violence, exploits fear or superstition, takes advantage of the child’s lack of judg- ment and experience, disrespects environmental values, or is capable of inducing consumers to behave in a manner that is harmful or dangerous to their health or safety. Acknowledgements Acknowledgements The authors are grateful to the guest editors and to Mateus Piva Adami for comments on earlier drafts, as well as to Natasha Mar- tín Lauletta for her research assistance. 295 commercial speech and commercial determinants of health • summer 2022 The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press SYMPOSIUM 12. C. L. Marques, “Artigo 37,” in C. L. Marques et al, Comentários ao Código de Defesa do Consumidor (São Paulo: Revista dos Tribunais, 2013): 860-880, at 862-863. 26. CONAR, Representação no 085/2011, June 16, 2011, avail- able at <https://criancaeconsumo.org.br/wp-content/ uploads/2014/07/ArcosDouradosRio_Decisao_2014.pdf> (last visited May 5, 2022). , , 13. C. L. Marques, “Artigo 37,” in C. L. Marques et al, Comentários ao Código de Defesa do Consumidor (São Paulo: Revista dos Tribunais, 2013): 860-880, at 864. y 27. Migalhas, “Conar arquiva representação contra comercial do Mc Donald´s no trailer de ‘Rio’”, July 6, 2011, available at <https://www.migalhas.com.br/quentes/136882/conar-arqui- va-representacao-contra-comercial-do-mc-donald-s-no-trailer- -de--rio-> (last visited May 5, 2022). 14. The exception being breastmilk substitutes. Brasil, Lei 11.265, January, 2006, available at <http://www.planalto.gov.br/ ccivil_03/_ato2004-2006/2006/lei/l11265.htm> (last visited may 8, 2022). y 28. Relator Desembargador Guilherme G. Strenger, Câmara Espe- cial, Tribunal de Justiça de São Paulo, Embargos de Declara- ção no 1127739-71.2016.8.26.0100, February 8, 2021. y 15. Superior Tribunal de Justiça, “Os limites da publicidade diante dos direitos do consumidor,” August 15, 2021, available at <https://www.stj.jus.br/sites/portalp/Paginas/Comunicacao/ Noticias/15082021-Os-limites-da-publicidade-diante-dos-di- reitos-do-consumidor.aspx> (last visited May 2, 2022). 29. Relator Ministro Humberto Martins, Segunda Turma, Superior Tribunal de Justiça, Brasil, “Recurso Especial 1.558.086/SP”, March 10, 2016, available at: <https://scon.stj.jus.br/SCON/ GetInteiroTeorDoAcordao?num_registro=201500615780&dt_ publicacao=15/04/2016> (last visited May 2, 2022). 16. A. Pasqualotto, “Autorregulamentação da publicidade: um estudo de modelos europeus e norte-americano,” Revista de Direito do Consumidor 112, a. 26 (São Paulo, July-August, 2017): 115-148, at 135-138. p / / y 30. Relator Ministro Herman Benjamin, Segunda Turma, Supe- rior Tribunal de Justiça, Brasil, “Recurso Especial 1.613.561/ SP,” April 25, 2017, available at <https://scon.stj.jus.br/SCON/ GetInteiroTeorDoAcordao?num_registro=201600171682&dt_ publicacao=01/09/2020> (last visited May 2, 2022). 17. Conclusion On advertising to children: Associação Brasileira de Anuncian- tes and Magalhães e Dias Advocacia, “Marketing Responsável: Garantias e Limites da Publicidade Infantil. A ABA em Prol da Publicidade Responsável,” 2020: 8-9 and 16-18, available at <https://www.aba.com.br/wp-content/uploads/2019/05/guia- aba-mkt-infantil-edit-2603-digital.pdf> (last visited May 1, 2022). Also, on the partnership between the advertising indus- try and the food and beverage industry: Associação Brasileira da Indústria de Alimentos, “Associações se unem e lançam campanha ‘Publicidade responsável: No digital também não se brinca,’” October 4, 2021, available at <https://www.abia. org.br/noticias/associacoes-se-unem-e-lancam-campanha- publicidade-responsavel-no-digital-tambem-nao-se-brinca> (last visited May 3, 2022). In the case of RDC 24/2014, ABIA filed a lawsuit against ANVISA’s normative act: Sexta Turma, Tribunal Regional Federal 1ª Região, Brasil, 0042882- 45.2010.4.01.3400/DF, Desembargador Federal Jirair Aram Meguerian, February 22, 2013, available at <https://proces- sual.trf1.jus.br/consultaProcessual/processo.php> (last visited May 3, 2022). 35. On advertising to children: Associação Brasileira de Anuncian- tes and Magalhães e Dias Advocacia, “Marketing Responsável: Garantias e Limites da Publicidade Infantil. A ABA em Prol da Publicidade Responsável,” 2020: 8-9 and 16-18, available at <https://www.aba.com.br/wp-content/uploads/2019/05/guia- aba-mkt-infantil-edit-2603-digital.pdf> (last visited May 1, 2022). Also, on the partnership between the advertising indus- try and the food and beverage industry: Associação Brasileira da Indústria de Alimentos, “Associações se unem e lançam campanha ‘Publicidade responsável: No digital também não se brinca,’” October 4, 2021, available at <https://www.abia. org.br/noticias/associacoes-se-unem-e-lancam-campanha- publicidade-responsavel-no-digital-tambem-nao-se-brinca> (last visited May 3, 2022). In the case of RDC 24/2014, ABIA filed a lawsuit against ANVISA’s normative act: Sexta Turma, Tribunal Regional Federal 1ª Região, Brasil, 0042882- 45.2010.4.01.3400/DF, Desembargador Federal Jirair Aram Meguerian, February 22, 2013, available at <https://proces- sual.trf1.jus.br/consultaProcessual/processo.php> (last visited May 3, 2022). 23. A. Pasqualotto, “Autorregulamentação da publicidade: um estudo de modelos europeus e norte-americano,” Revista de Direito do Consumidor 112, a. 26 (São Paulo, July-August, 2017): 115-148, at 143. In the section of CONAR’s website called “Decisões” there is information on how many new pro- cedures were opened per year. There were 286 new cases in 2021; 276 in 2020; 302 in 2019; 324 in 2018; 300 in 2017; 308 in 2016; 241 in 2015; 308 in 2014. 24. Free translation from CONAR’s website in the section called “Missão.” Original in Portuguese: “CONAR é uma organização não-governamental que visa promover a liberdade de expres- são publicitária e defender as prerrogativas constitucionais da propaganda comercial”, available at <http://www.conar.org. br/> (last visited May 2, 2022). 25. Conclusion CONAR, Código Brasileiro de Autorregulamentação Publici- tária, São Paulo, 2021-2022, available at <http://www.conar. org.br/pdf/codigo-conar-2021_6pv.pdf> (last visited May 2, 2022). 31. Brasil, Código de Processo Civil – Lei 13.105, article 506, March, 2015, available at <http://www.planalto.gov.br/ ccivil_03/_ato2015-2018/2015/lei/l13105.htm> (last visited May 2, 2022). 18. G. M. P. Bezerra, Antecedentes do Conar: contexto histórico e político da organização corporativa do setor publicitário brasileiro, paper presented at Congresso Brasileiro de Ciên- cias da Comunicação, Rio de Janeiro, Brasil, September 4-7, 2015, available at <https://repositorio.ufc.br/bitstream/ riufc/30246/1/2015_eve_gmpbezerra.pdf> (last visited May 2, 2022). Also CONAR, “História: contra a censura na publi- cidade,” available at <CONAR-Conselho Nacional de Autorre- gulamentação Publicitária> (last visited May 3, 2022). É 32. IDEC, “Unhealthy Food Marketing: Barriers and Regulation Perspectives in Brazil,” Cadernos IDEC (São Paulo, 2014): 31-37, at 33-35, available at <https://idec.org.br/publicacao/ publicidade-de-alimentos-nao-saudaveis-os-entraves-e-per- spectivas-de-regulacao-no-brasil> (last visited May 3, 2022). p g y 33. Associação Brasileira de Anunciantes and Magalhães e Dias Advocacia, “Marketing Responsável: Garantias e Limites da Publicidade Infantil. A ABA em Prol da Publicidade Res- ponsável,” 2020: 8-9, available at <https://www.aba.com. br/wp-content/uploads/2019/05/guia-aba-mkt-infantil-edit- -2603-digital.pdf> (last visited May 1, 2022). Again, though there has been no specific federal law, other normative acts have restricted advertising. For example, resolution 163/2014 by the National Council of the Rights of Children and Adoles- cents (CONANDA) and resolution 24/2010 by the National Agency of Health Surveillance (ANVISA). 19. CONAR, Regimento Interno do Conselho de Ética, arti- cle 27, available at <https://fagali.com/wp-content/ uploads/2017/10/CONAR-RICE-Regimento-Interno-do-Con- selho-de-%C3%89tica.pdf> (last visited May 2, 2022) and CONAR, Código de Ética, article 50, available at <http:// www.conar.org.br/pdf/codigo-conar-2021_6pv.pdf> (last visi- ted May 2, 2022). y , ) 20. CONAR, Estatuto Social, article 8, available at <http://conar. org.br/pdf/estatuto-social-conar.pdf> (last visited May 2, 2022) ) 21. CONAR, Estatuto Social, article 30, available at <http:// conar.org.br/pdf/estatuto-social-conar.pdf> (last visited May 2, 2022). g y 34. IDEC, “Unhealthy Food Marketing: Barriers and Regulation Perspectives in Brazil,” Cadernos IDEC (São Paulo, 2014): 54-54, available at <https://idec.org.br/publicacao/publici- dade-de-alimentos-nao-saudaveis-os-entraves-e-perspectivas- de-regulacao-no-brasil> (last visited May 3, 2022). 22. According to Deloitte’s research, in 2020, the amount that Brazil spent on advertising was R$ 49 billions of reais. Deloi- tte and Conselho Executivo de Normas-Padrão, “O Valor da Publicidade no Brasil: o impacto do setor nos negócios, na economia e na sociedade,” 2021, available at <https://static. poder360.com.br/2021/09/relatorio-deloitte-publicidade-bra- sil-2020.pdf> (last visited May 2, 2022). g y , 35. https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) 37. Instituto Brasileiro de Defesa do Consumidor, “STF define que lei que proíbe publicidade em escolas na BA é constitucional,” April 15, 2021, available at <https://idec.org.br/noticia/stf- -define-que-lei-que-proibe-publicidade-em-escolas-na-ba- -e-constitucional> (last visited May 3, 2022). ( y , ) 38. Relator Ministro Edson Fachin, Supremo Tribunal Federal, Ação Direta de Insconstitucionalidade 5631, March 25, 2021, p. 21, available at <https://redir.stf.jus.br/paginadorpub/pagi- nador.jsp?docTP=TP&docID=755977807> (last visited May 1, 2022). ) 39. Relator Ministro Edson Fachin, Supremo Tribunal Federal, Ação Direta de Insconstitucionalidade 5631, March 25, 2021, y , ) 40. Relator Ministro Edson Fachin, Supremo Tribunal Federal, Ação Direta de Insconstitucionalidade 5631, March 25, 2021, p. 21 e 49, available at <https://redir.stf.jus.br/paginadorpub/ paginador.jsp?docTP=TP&docID=755977807> (last visited May 1, 2022). p. 21-22, available at <https://redir.stf.jus.br/paginadorpub/ paginador.jsp?docTP=TP&docID=755977807> (last visited May 1, 2022). Conclusion Migalhas, “Conar arquiva representação contra comercial do Mc Donald´s no trailer de ‘Rio,’” July 6, 2011, available at <https://www.migalhas.com.br/quentes/136882/conar-arqui- va-representacao-contra-comercial-do-mc-donald-s-no-trai- ler-de--rio-> (last visited May 5, 2022). y 36. Relator Ministro Edson Fachin, Supremo Tribunal Federal, Ação Direta de Insconstitucionalidade 5631, March 25, 2021, available at <https://redir.stf.jus.br/paginadorpub/paginador. jsp?docTP=TP&docID=755977807> (last visited May 1, 2022). 296 The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) Barbosa, Leite, and Britto 297 https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press commercial speech and commercial determinants of health • summer 2022 The Journal of Law, Medicine & Ethics, 50 (2022): 291-297. © 2022 The Author(s) https://doi.org/10.1017/jme.2022.54 Published online by Cambridge University Press
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https://openalex.org/W4393985734	https://jcs.greenpublisher.id/index.php/jcs/article/download/362/355	Indonesian	null	Upaya Kepolisian Dalam Penanggulangan Kasus Investasi Bodong (Studi Kasus Wilayah Hukum Kepolisian Resor Pohuwato)	Journal of Comprehensive Science	2,023	cc-by-sa	5,343	1 Diana Tambunan, Ida Hendarsih. “Waspada Investasi Ilegal di Indonesia”. Jurnal Ekonomi dan Manajemen Universitas Bina Sarana Informatika.Vol. 20.No. 1 Maret. 2022. Hal. 110 Keywords: Police Efforts, Handling Fraudulent Investment Cases Keywords: Police Efforts, Handling Fraudulent Investment Cases Keywords: Police Efforts, Handling Fraudulent Investment Cases p-ISSN: 2962-4738 e-ISSN: 2962-4584 Vol. 2 No. 5 Mei 2023 UPAYA KEPOLISIAN DALAM PENANGGULANGAN KASUS INVESTASI BODONG (STUDI KASUS WILAYAH HUKUM KEPOLISIAN RESOR POHUWATO) Ista Ismail, Fence M Wantu, Avelia Rahmah Y Mantali Fakultas Hukum Universitas Negeri Gorontalo, Indonesia Email: gusasianastasia@gmail.com Abstrak Penelitian ini di latar belakangi dari sebuah kasus investasi bodong yang terjadi di Kabupaten Pohuwato, berdasarkan hasil wawancara dengan wilayah hukum kepolisian Polres Pohuwato ada 2 upaya yang di lakukan yaitu upaya represif dan upaya preventif. Penelitian ini bertujuan untuk mengetahui upaya yang di lakukan oleh kepolisian dalam penanggulangan kasus investasi bodong di wilayah hukum Kepolisian Resor Pohuwato dan untuk mengetahui kendala kepolisian dalam penangulangan kasus investasi bodong. Kata Kunci: upaya kepolisian, penanggulangan kasus investasi bodong Abstract This research is in the background of a fraudulent investment case that occurred in Pohuwato Regency, based on the results of interviews with the jurisdiction of the Pohuwato Police Department, there are 2 efforts made, namely repressive efforts and preventive efforts. This study aims to determine the efforts made by the police in handling fraudulent investment cases in the jurisdiction of the Pohuwato Resort Police and to determine the police's obstacles in handling fraudulent investment cases 2 Citra Khairiyati, Astrie Krisnawati. “Analisi Pengaruh Literasi Keungan Terhadap Keputusan Investasi Pada Masyarakat Kota Bandung”.Jurnal Manajemen dan Bisnis.Vol. 3.No. 2 Agustus. 2019. Hal 301-302 3 Liffianisya Septi Alfarizty, Rani Apriani. “Upaya Penegakan Hukum Terhadap Korban Akibat Investasi Bodong Di Tinjau Dari Undang-undang Nomor 10 Tahun 1998 Tentang Perbankan”.Jurnal Ilmu Hukum dan Humaniora, Vol 9.No. 5 Tahun 2022.Hal. 2254. 4 Ardianto. 2022. “Pengaruh Literasi Keuangan dan Pola Pikir Terhadap Pencegahan Investasi Bodong Pada Nasabah Gis IAIN Palopo”. Hal 1 5 A.A.Angga Primantari,Kadek Sarna. 2014.“Upaya Menanggulangi Investasi Bodong Di Internet”. Kerta semaya: Jurnal Ilmu Hukum ,2(3), 1-5. Hal 1-2 PENDAHULUAN Era globalosasi, pasar di banjiri dengan berbagai jenis peluang investasi, tetapi perlu di ingat bahwa hal tersebut menciptakan peluang untuk eksploitasi kriminal. Maraknya penawaran investasi berbasis website ataupun aplikasi harus di waspadai karena pelakunya memanfaatkan ketidakpahaman masyarakat untuk menipu dengan cara iming-iming pemberi imbal hasil yang sangat tinggi dan tidak wajar namun terlebih dahulu masyarakat diminta menempatkan atau menyetorkan dananya. Beberapa peluang yang di tawarkan di rancang dengan cerdik untuk memanfaatkan ketidaktahuan publik. Akhir-akhir ini kasus investasi bodong marak terjadi di Indonesia. Kasus investasi bodong kerap muncul dan memakan korban. Disebut investasi bodong karena tidak memiliki izin dari instansi terkait.1 Pada era ekonomi seperti sekarang ini, banyak masyarakat yang semakin sadar akan pentingnya berinvestasi. Seseorang melakukan kegiatan investasi dengan harapan dapat memperoleh keuntungan besar dimasa yang akan datang. Akan tetapi masih banyak masyarakat yang masih kurang memahami cara berinvestasi yang baik dan benar sehingga banyak dari mereka yang tertipu oleh investasi dengan tawaran keuntungan bunga yang tak masuk akal dan pengelolaan investasi yang tidak jelas. Oleh karena itu, 1438 masyarakat harus lebih memahami jenis instrument investasi yang akan mereka gunakan agar tidak tertipu di kemudian hari.2 Investasi merupakan suatu kegiatan menghimpun dana atau menyimpan dana dengan tujuan mendapat keuntungan atau peningkatan nilai investasi di kemudian hari. Sedangkan investasi bodong merupakan kegiatan yang serupa dengan investasi pada umumnya namun hal ini berupa bohong belaka bahwa iming-iming investasi dengan return tinggi yang di janjikan kepada para investor sebenarnya tidak ada. Yang ada hanyalah oknum penipu akan membawa kabur uang tersebut. Masyarakat sebagai investor tanpa berfikir panjang turut serta berinvestasi karena kegiatan tersebut menawarkan keuntungan yang tinggi dari hasil kegiatan usaha tetap dan kemudian di dalamnya terdapat unsur-unsur yang di larang sehingga timbul menjadi suatu tindak kejahatan pidana. Dalam hal ini investasi bodong berkaitan dengan tindak pidana di bidang perbankan karena wujud dari salah satu kejahatan di bidang ekonomi.3 Investasi bodong masih marak terjadi ditengah masyarakat.Investasi dengan penawaran keuntungan besar sehingga menarik para calon korban untuk melakukan investasi. Masih maraknya kasus investasi bodong seharusnya menjadikan masyarakat lebih berhati-hati dalam memilih instrumen atau tempat berinvestasi. Penawaran investasi bodong ini sering berpura-pura sebagai penjualan langsung maupun bisnis dengan peluang imbal hasil yang tinggi.4 Investasi yang lazimnya disebut penanaman modal, kini menjadi istilah yang sudah tidak asing lagi di masyarakat. Investasi merupakan komitmen menanamkan sejumlah dana pada satu atau lebih aset selama beberapa periode pada masa mendatang. 6 Lihat UU No.10 Tahun 1998 tentang perubahan atas Undang-undang Republik Indonesia Nomor 7 Tahun 1992 Tentang perbankan 9 Al Vionita Vivin Novarina, ” Upaya Polisi Dalam Menanggulangi Tindak Pidana Penipuan Berkedok Investasi Melalui Sistem Online Di Polda DIY”, Universitas Negeri Yogyakarta. 2014. Hal 2 PENDAHULUAN Banyaknya keuntungan yang didapat dalam berinvestasi membuat banyak orang yang mengusahakan untuk melakukan investasi baik dalam bentuk tabungan di bank, investasi saham, properti, forex trading dan yang lainnya. Alasan seseorang lebih melakukan investasi adalah untuk mendapatkan kehidupan yang lebih layak di masa yang akan datang, mengurangi tekanan inflasi, dan dorongan untuk menghemat pajak.5 Tujuan investasi atau penanaman modal sangat bertolak belakang dengan praktik investasi bodong, karena justrun merugikan, merusak tatanan hukum investasi,hukum perbankan, serta menghambat kegiatan perekonomian nasional. Pada dasarnya, dampak negatif investasi bodong adalah tumbuh dan berkembangnya pelanggaran terhadap sistem hukum nasional, seperti pelanggaran terhadap ketentuan pasal 46 Undang-undang No.7 Tahun 1992 jo. Undang-undang No.10 Tahun 1998 tentang perbankan. Kesadaran hukum dan pemahaman hukum menjadi bagian penting karena dengan demikian terdapat kepatuhan dan perlindungan hukum terhadap para pihak. Hukum investasi dalam hal ini lebih berkaitan erat dengan aturan berdasarkan hukum perbankan yang secara tegas mengancam pidana penjara dan denda terhadap pelaku investasi bodong, yang bertitik tolak pada ancaman hukum terhadap pelaku yang menjalankan kegiatan usaha tanpa izin untuk menghimpun dana dari masyarakat, yang selain diatur dan di ancam pidana berdasarkan pasal 46 Undang-undang No.7 Tahun 1992 jo. Undang-undang No.10 Tahun 1998 tentang perbankan. Berdasarkan Undang-undang No.7 Tahun 1992 sebagaimana telah diubah dengan Undang-undang Republik Indonesia No.10 Tahun 1998 Tentang perbankan menyebutkan bahwa setiap pihak yang melakukan kegiatan menghimpun dana dari masyarakat dalam bentuk simpanan, wajib terlebih dahulu memperoleh izin usaha.6 Ketentuan mengenai hal ini dapat ditemukan dalam pasal 46 Undang-undang 1439 Republik Indonesia Nomor 10 Tahun 1998 tentang perubahan atas Undang-undang Republik Indonesia Nomor 7 Tahun 1992 Tentang perbankan “Pasal 46 ayat (1) yang menyatakan bahwa: Republik Indonesia Nomor 10 Tahun 1998 tentang perubahan atas Undang-undang Repu Republik Indonesia Nomor 10 Tahun 1998 tentang perubahan atas Undang-undang Republik Indonesia Nomor 7 Tahun 1992 Tentang perbankan “Pasal 46 ayat (1) yang menyatakan bahwa: Nomor 7 Tahun 1992 Tentang perbankan “Pasal 46 ayat (1) yang menyatakan bahwa: “Barang siapa menghimpun dana dari masyarakat dalam bentuk simpanan tanpa izin usaha dari pimpinan Bank Indonesia sebagaimana di maksud pasal 16 di ancam dengan pidana penjara sekurang-kurangnya 5(lima) tahun dan paling lama 15 (lima belas ) tahun serta dendan sekurang- kurangnya Rp 10.000.000.000,00 (sepuluh miliar rupiah) dan paling banyak 200.000.000.000,00 (dua ratus miliar rupiah)”.7 Hukum pidana menurut pasal 372 dan 378 KUHP mengancam pidana terhadap kegiatan investasi sebagai kejahatan penipuan investasi. PENDAHULUAN Pasal 372 KUHP menyebutkan “barang siapa dengan sengaja dan melawan hukum memiliki barang sesuatu yang seluruhnya atau sebagian adalah kepunyaan orang lain, tetapi yang ada dalam kekuasaanya bukan karena kejahatan di ancam karena penggelapan, dengan pidana penjara paling lama empat tahun atau pidana denda paling banyak Sembilan ratus rupiah”. Sedangkan dalam pasal 378 KUHP menyebutkan “barang siapa dengan maksud untuk menguntungkan diri sendiri atau orang lain secara melawan hukum, dengan memakai nama palsu atau martabat palsu, dengan tipu muslihat, ataupun rangkaian kebohongan, menggerakkan orang lain untuk menyerahkan baranag sesuatu kepadanya, atau supaya memberi hutang maupun menghapuskan piutang, di ancam karena penipuan dengan pidana penjara paling lama empat tahun”.8 Dalam upaya penanggulangan tindak pidana kasus investasi bodong yang di atur secara tegas Undang-Undang Nomor 2 Tahun 2002 tentang Kepolisian Negara Republik Indonesia dalam pasal 14 ayat (1) huruf g, memberi wewenang untuk melakukan penyelidikan dan penyidikan terhadap semua tindak pidana dalam hal ini terhadap kasus investasi bodong. Berdasarkan bunyi pasal 13 dalam undang-undang Nomor 2 Tahun 2002 tentang Kepolisian Negara Republik Indonesia yang menetapkan tugas di emban polisi berupa memelihara keamanan dan ketertiban dalam masyarakat, menegakkan hukum, dan memberikan perlindungan, pengayoman, dan pelayanan kepada masyarakat, maka seharusnya kejahatan berupa investasi bodong dapat di tanggulangi, diminimalisir atau bahkan di berantas.9 Namun pada kenyataan di Polres Pohuwato pada tahun 2022 terdapat 1 kasus 3 pelaku yang di proses secara pidana di Kepolisian Resor Pohuwato Provinsi Gorontalo. 1 pelaku sudah dilimpahkan ke kejaksaan sedangkan 2 pelaku lainnya masih dalam proses penyidikan. Hukum pidana menurut pasal 372 dan 378 KUHP mengancam pidana terhadap kegiatan investasi sebagai kejahatan penipuan investasi. Pasal 372 KUHP menyebutkan “barang siapa dengan sengaja dan melawan hukum memiliki barang sesuatu yang seluruhnya atau sebagian adalah kepunyaan orang lain, tetapi yang ada dalam kekuasaanya bukan karena kejahatan di ancam karena penggelapan, dengan pidana penjara paling lama empat tahun atau pidana denda paling banyak Sembilan ratus rupiah”. 8 Kitab undang-undang hukum pidana 7 Kristian, Yopi Gunawan. 2018. “Tindak Pidana Perbankan Dalam Proses Peradilan Di Indonesia”. Jakarta Timur: Prenedamedia Group. 10 Wawancara Dengan Bapak Sudirman Hippy Selaku Korban Investasi Bodong Pada Hari Rabu Tanggal 21 Desember 2022 11 Nando Mantulangi. 2017. “Kajian Hukum Investasi dan Perlindungan Terhadap Investasi Bodong”, Dalam Jurnal Lex Administratum, Vol. 1, No. 1, Jan-Feb 2017, Hal 108 PENDAHULUAN Sedangkan dalam pasal 378 KUHP menyebutkan “barang siapa dengan maksud untuk menguntungkan diri sendiri atau orang lain secara melawan hukum, dengan memakai nama palsu atau martabat palsu, dengan tipu muslihat, ataupun rangkaian kebohongan, menggerakkan orang lain untuk menyerahkan baranag sesuatu kepadanya, atau supaya memberi hutang maupun menghapuskan piutang, di ancam karena penipuan dengan pidana penjara paling lama empat tahun”.8 Dalam upaya penanggulangan tindak pidana kasus investasi bodong yang di atur secara tegas Dalam upaya penanggulangan tindak pidana kasus investasi bodong yang di atur secara tegas Undang-Undang Nomor 2 Tahun 2002 tentang Kepolisian Negara Republik Indonesia dalam pasal 14 ayat (1) huruf g, memberi wewenang untuk melakukan penyelidikan dan penyidikan terhadap semua tindak pidana dalam hal ini terhadap kasus investasi bodong. Berdasarkan bunyi pasal 13 dalam undang-undang Nomor 2 Tahun 2002 tentang Kepolisian Negara Republik Indonesia yang menetapkan tugas di emban polisi berupa memelihara keamanan dan ketertiban dalam masyarakat, menegakkan hukum, dan memberikan perlindungan, pengayoman, dan pelayanan kepada masyarakat, maka seharusnya kejahatan berupa investasi bodong dapat di tanggulangi, diminimalisir atau bahkan di berantas.9 Namun pada kenyataan di Polres Pohuwato pada tahun 2022 terdapat 1 kasus 3 pelaku yang di proses secara pidana di Kepolisian Resor Pohuwato Provinsi Gorontalo. 1 pelaku sudah dilimpahkan ke kejaksaan sedangkan 2 pelaku lainnya masih dalam proses penyidikan. Saat ini investasi semakin banyak di naungi oleh masyarakat Kabupaten Pohuwato yang bertujuan untuk mempertahankan kekayaan, atau meraih keuntungan mereka kepada pelaku bisnis, baik perusahaan maupun perorangan. Ketika telah terjun kedalam dunia investasi tujuan mereka tidak tercapai di karenakan menjadi korban dari pihak yang sangat tidak bertanggung jawab serta konsumen tidak mendapatkan keuntungan sedikitpun serta mengalami kerugian yang sangat besar. Umumnya masyarakat di Kabupaten Pohuwato masih tidak mengrti dunia investasi dan jenis instrument investasi apa saja yang sedang ada saat ini. Masyarakat harus memahami tentang perusahaan yang memfasilitasikegiatan investasi, karena jika tidak memiliki izin dari otoritas yang berwenang, maka penghimpunan dana tersebut menjadi ilegal. Tingkat kewaspadaan masyarakat Pohuwato yang di nilai sangat rendah karena kurangnya literasi dan informasi dari masyarakat itu sendiri menyebabkan masyarakat terjebak bisnis investasi bodong yang menggiurkan. Meningkatnya tingkatan kelas menengah di Kabupaten Pohuwato menjadi hal yang membuat banyaknya investasi bodong. Parahnya, yang menjadi korban adalah masyarakat yang berpendidikan yang seharusnya sudah mengerti investasi yang benar. y g y g Oleh karena itu, adanya oknum-oknum yang memanfaatkan hal tersebut dengan investasi bodong. PENDAHULUAN Adanya suatu perusahaan yang tidak memiliki izin melaksnakan kegiatan investasi bodong, yakni 1440 perusahaan tersebut menawarkan produk investasi yang akan menambahkan dana dalam beberapa waktu mendatang.10 Investasi bodong tentu bertentangan dengan tujuan utama investasi itu sendiri. Bukannya memperlancar pergerakan ekonomi negara, malah justru menjadi tembok tinggi yang menghambat ekonomi. Kepatuahan lahir dari adanya kepekaan dan wawasan masyarakat akan hukum. Hal ini tentu penting untuk mewujudkan perlindungan yuridis bagi pihak yang terlibat dalam kegiatan investasi. Minimnya kesadaran hukum masyarakat membuka celah bagi mafiah investasi untuk menghimpun dana tak berizin. Meskipun secara konstitusional investasi bodong telah diatur, banyak masyarakat awam yang tidak mengerti langkah yang harus di lakukan untuk membedakan investasi yang sah dan bodong.11 Tabel 1. 1 Julukan Pelaku dan Sanksi Tindak Pidana Tahun Nama Inves Sanksi Tindak Pidana 2022 Man Trader tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dalam pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan UU No.7 Tahun 1992 tentang perbankan dan atau pasal 378 da atau pasal 372 jo pasal 55 KUHP. Bintang Trader tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dala pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan atas UU No. 7 Tahun 1992 tentang perbankan dan atau pasal 377 atau pasal 372 jo. Smart Trader tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dalam pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan atas UU No. 7 Tahun 1992 tentang perbankan dan atau pasal 378 dan atau pasal 372 jo. 12 Mukti Fajar dan Yulianto Achmad, “Dualisme Penelitian Hukum Normatif dan Empiris”, (Yogyakarta : Pustaka Pelajar, 2010), Hal. 153 13 Lidya Ananda Putri. Dalam Skripsi “Peranan Kepolisian Dalam Menanggulangi Tindak Pidana Perjudian Sabung Ayam Di Kota Kotamobagu”. Universitas Negeri Gorontalo. Gorontalo. 2021. Hal 34-35 14Wawancara Dengan Aipda Sengly Manopo Selaku Penyidik Kasus Investasi Bodong Pada Tanggal 19 September 2023 15 Wawancara Dengan Brigpol Husnul Hamka Selaku Penyidik pada tanggal 19 september 2023 METODE PENELITIAN Jenis yang digunakan dalam penelitian ini adalah penelitian hukum empiris (sosiologi). Penelitian hukum sosiologis atau empiris, yang mencakup penelitian terhadap identifikasi hukum (tidak tertulis) dan penelitian terhadap efektivitas hukum.12 Penelitian hukum empiris yaitu penelitian dengan adanya data-data lapangan sebagai sumber data utama, seperti hasil wawancara dan observasi.Penelitian empiris digunakan untuk menganalisis hukum yang diihat sebagai perilaku masyarakat yang berpola dalam kehidupan masyarakat yang selalu berinteraksi dan berhubungan dalam aspek kemasyarakatan.13 HASIL DAN PEMBAHASAN 1. Upaya kepolisian dalam penanggulangan kasus investasi bodong yang terjadi di wilayah hukum kepolisian resor pohuwato j Sumber: Data di Polres Pohuwato 2022 j Sumber: Data di Polres Pohuwato 2022 Pada tahun 2022 sudah tercatat ada 1 kasus yang terdiri dari 3 pelaku yang di proses secara pidana di Kepolisian Resor Pohuwato Provinsi Gorontalo. Banyaknya aduan terhadap perusahaan investasi bodong yang tidak memiliki izin ini banyak di selesaikan ke ranah pidana.Ketiga pelaku investasi bodong tersebut di juluki Man Trader, Bintang Trader, dan Smart Trader. a. Untuk man trader tindak pidana menghimpun dana dari masyarakat dalam bentuk simpanan tanpa izin dari pimpinan Bank Indonesia, tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dalam pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan UU No.7 Tahun 1992 tentang perbankan dan atau pasal 378 da atau pasal 372 jo pasal 55 KUHP. b. Untuk bintang trader dugaan tindak pidana menhimpun dana dari masyarakat dalam bentuk simpanan tanpa izin bank Indonesia, tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dala pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan atas UU No. 7 Tahun 1992 tentang perbankan dan atau pasal 377 atau pasal 372 jo. b. Untuk bintang trader dugaan tindak pidana menhimpun dana dari masyarakat dalam bentuk simpanan tanpa izin bank Indonesia, tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dala pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan atas UU No. 7 Tahun 1992 tentang perbankan dan atau pasal 377 atau pasal 372 jo. c. Untuk smart trader tindak pidana menhimpun dana dalam bentuk simpanan tanpa izin bank Indonesia, tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dalam pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan atas UU No. 7 Tahun 1992 tentang perbankan dan atau pasal 378 dan atau pasal 372 jo. c. Untuk smart trader tindak pidana menhimpun dana dalam bentuk simpanan tanpa izin bank Indonesia, tindak pidana penipuan dan penggelapan sebagaimana yang di maksud dalam pasal 46 ayat (1) UU No. 10 Tahun 1998 tentang perubahan atas UU No. 7 Tahun 1992 tentang perbankan dan atau pasal 378 dan atau pasal 372 jo. 1441 Dampak dari investasi bodong ini bukan hanya masyarakat yang mengalami kerugian keuangan. Bahkan ada salah satu masyarakat yang menjadi korban investasi bodong yang melakukan bunuh diri di Kabupaten Pohuwato Provinsi Gorontalo. p Dengan adanya kasus ini peneliti tertarik untuk mencari tahu bagaimana UPAYA KEPOLISIAN DALAM PENYELESAIAN KASUS INVESTASI BODONG (STUDI KASUS WILAYAH HUKUM KEPOLISIAN RESOR POHUWATO)”. Selanjutnya untuk di angkat sebagai karya ilmiah. 1. Upaya kepolisian dalam penanggulangan kasus investasi bodong yang terjadi di wilayah hukum kepolisian resor pohuwato Berdasarkan wawancara penulis bersama Aipda Sengly Manopo,SH upaya kepolisian dalam menangani kasus ini adalah dengan memberian edukasi kepada masyarakat tentang cara-cara dari para pelaku menawarkan investasi. Yang pertama, mencari tahu lebih dulu siapa yang menawarkan investasi ini latar belakangnya apa, backgroundnya apa,usahanya apa, dengan cara dia menawarkan investasi ini dengan iming-iming atau keuntungan yang sudah tidak masuk akal maka di curigai kalau itu masuk dalam investasi ilegal. Kedua, melakukan penegakan hukum yang tegas kepada para pelaku investasi bodong ini dengan menjerat mereka dengan undang-undang perbankan pasal 46 Undang-undang Nomor 10 Tahun 1998 tentang menghimpun dana dari masyarakat tanpa izin maka di ancam dengan pidana penjara. Selain itu para pelaku juga akan di jerat dengan undang-undang dalam KUHP Pasal 378 dan 372 tentang penipuan dan penggelapan.14 Adapun hasil wawancara penulis bersama dengan bapak Brigpol Husnul Hamka selaku penyidik kasus investasi bodong , bapak mengatakan bahwa upaya kepolisian dalam menanggulangi kasus investasi bodong ada beberpa upaya yaitu: U tif Upaya dalam menanggulangi tindak pidana investasi bodong di Polres Pohuwato telah dilakukan oleh bagian Humas dengan melakukan sosialisasi kepada masyarakat dengan memberikan penjelasan kepada masyarakat melalui pemberitaan di media massa guna mengantisipasi merebaknya tindak pidana investasi bodong. Upaya Represif dalam menanggulangi tindak pidana investasi bodong di Polres Pohuwato dilakukan oleh Polisi Penyidik Kanit Tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Penyidik tersebut terdiri dari Ipda Yobtan R. Frans, SH, Aipda Sengly Manopo, SH, Brigpol Husnul Hamka, SH, Briptu Zulkarnain Darise, STP, dan Briptu Arief Alfitrah.15 Untuk memperoleh gambaran yang jelas mengenai upaya represif polisi dalam menanggulangi tindak pidana investasi bodong di Polres Pohuwato akan diuraikan sebagai berikut: 1442 16 Al Vionita Vivin Novariana. “Upaya Kepolisian Dalam Menanggulangi Tindak Pidana Penipuan Bekedok Investasi Melalui Sistem Online Di Polda DIY”. Universitas Negeri Yogyakarta. 2014. 17 Wawancara Dengan Briptu Zulkarnain Darise Selaku Penyidik pada tanggal 19 september 2023 a. Hambatan Internal Hambatan internal yang dimaksud adalah hambatan yang terdapat dalam lingkup upaya menanggulangi tindak pidana investasi bodong di Polres Pohuwato khususnya Kanit Tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Hambatan Internal dalam upaya menaggulangi tindak pidana investasi bodong di Polres Pohuwato yaitu: Hambatan internal yang dimaksud adalah hambatan yang terdapat dalam lingkup upaya menanggulangi tindak pidana investasi bodong di Polres Pohuwato khususnya Kanit Tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Hambatan Internal dalam upaya menaggulangi tindak pidana investasi bodong di Polres Pohuwato yaitu: a) Belum ada peraturan khusus yang mengatur tentang tindak pidana investasi bodong. Peraturan yang digunakan oleh Polisi Penyidik Kanit Tipidkor dan KBO Polres Pohuwato dalam menanggulangi tindak pidana investasi bodong adalah Pasal 378 KUHAP tentang penipuan secara umum. Tidak ada kriteria tentang investasi bodong di dalam Pasal 378 KUHAP. g g b) Peraturan Perundang-undangan Perbankan yang Berlaku (Undang-Undang Nomor 10 Tahun 1998 tentang Perubahan Atas Undang-Undang Nomor 7 Tahun 1992 tentang Perbankan) Penyelidikan kasus tindak pidana investasi bodong untuk menemukan bukti permulaan adanya tindak pidana investasi bodong mengalami hambatan karena ketika penyelidik membutuhkan petunjuk dari rekening yang diduga sebagai pelaku Pasal 42 Undang-Undang Nomor 10 Tahun 1998 tentang Perubahan Atas Undang-Undang Nomor 7 Tahun 1992 tentang Perbankan menghendaki status orang yang akan diaudit rekeningnya sudah merupakan tersangka, sehingga akan sulit bagi polisi penyelidik untuk melakukan penyelidikan yang lebih lanjut. g gy 17 Wawancara Dengan Briptu Zulkarnain Darise Selaku Penyidik pada tanggal 19 september 2023 16 Al Vionita Vivin Novariana. “Upaya Kepolisian Dalam Menanggulangi Tindak Pidana Penipuan Investasi Melalui Sistem Online Di Polda DIY”. Universitas Negeri Yogyakarta. 2014. c. Penyelidikan Kendala kepolisian dalam penanggulangan kasus investasi bodong di wilayah hukum kepolisian resor pohuwato Briptu Zukarnain Darise mengatakan banyak masyarakat yang menjadi korban ivestasi bodong ini sehingga banyaknya korban dan admin membuat kepolisian kesulitan dalam mengidentifikasi informasi dari mulut ke mulut masyrakat.17 a. Hambatan Internal Hambatan internal yang dimaksud adalah hambatan yang terdapat dalam lingkup upaya menanggulangi tindak pidana investasi bodong di Polres Pohuwato khususnya Kanit Tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Hambatan Internal dalam upaya menaggulangi tindak pidana investasi bodong di Polres Pohuwato yaitu: 1) Peraturan Perundang-Undangan a) Belum ada peraturan khusus yang mengatur tentang tindak pidana investasi bodong. Peraturan yang digunakan oleh Polisi Penyidik Kanit Tipidkor dan KBO Polres Pohuwato dalam menanggulangi tindak pidana investasi bodong adalah Pasal 378 KUHAP tentang penipuan secara umum. Tidak ada kriteria tentang investasi bodong di dalam Pasal 378 KUHAP. b) Peraturan Perundang-undangan Perbankan yang Berlaku (Undang-Undang Nomor 10 Tahun 1998 tentang Perubahan Atas Undang-Undang Nomor 7 Tahun 1992 tentang Perbankan) Penyelidikan kasus tindak pidana investasi bodong untuk menemukan bukti permulaan adanya tindak pidana investasi bodong mengalami hambatan karena ketika penyelidik membutuhkan petunjuk dari rekening yang diduga sebagai pelaku Pasal 42 Undang-Undang Nomor 10 Tahun 1998 tentang Perubahan Atas Undang-Undang Nomor 7 Tahun 1992 tentang Perbankan menghendaki status orang yang akan diaudit rekeningnya sudah merupakan tersangka, sehingga akan sulit bagi polisi penyelidik untuk melakukan penyelidikan yang lebih lanjut. b Hambatan Eksternal p y p p g 2. Kendala kepolisian dalam penanggulangan kasus investasi bodong di wilayah hukum kepolisian resor pohuwato 2. Kendala kepolisian dalam penanggulangan kasus investasi bodong di wilayah hukum kepolisian resor pohuwato Briptu Zukarnain Darise mengatakan banyak masyarakat yang menjadi korban ivestasi bodong ini 2. Kendala kepolisian dalam penanggulangan kasus investasi bodong di wilayah hukum kepolisian resor pohuwato Briptu Zukarnain Darise mengatakan banyak masyarakat yang menjadi korban ivestasi bodong ini sehingga banyaknya korban dan admin membuat kepolisian kesulitan dalam mengidentifikasi informasi dari mulut ke mulut masyrakat.17 resor pohuwato Briptu Zukarnain Darise mengatakan banyak masyarakat yang menjadi korban ivestasi bodong ini sehingga banyaknya korban dan admin membuat kepolisian kesulitan dalam mengidentifikasi informasi dari mulut ke mulut masyrakat.17 Briptu Zukarnain Darise mengatakan banyak masyarakat yang menjadi korban ivestasi bodong ini sehingga banyaknya korban dan admin membuat kepolisian kesulitan dalam mengidentifikasi informasi dari mulut ke mulut masyrakat.17 c. Penyelidikan y Penyelidikan terhadap tindak pidana investasi bodong di Polres Pohuwato dilakukan oleh Polisi Penyelidik Kanit Tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Sebelum dilakukan tindakan penyidikan, dilakukan dulu penyelidikan oleh pejabat penyelidik, dengan maksud dan tujuan mengumpulkan “bukti permulaan” atau “bukti yang cukup” agar dapat dilakukan tindak lanjut penyidikan. Setelah mendapatkan laporan adanya tindak pidana investasi bodong dilakukan tindakan penyelidikan. Pada tahap penyelidikan, polisi penyelidik melakukan serangkaian tindakan yaitu: Penyelidikan terhadap tindak pidana investasi bodong di Polres Pohuwato dilakukan oleh Polisi Penyelidik Kanit Tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Sebelum dilakukan tindakan penyidikan, dilakukan dulu penyelidikan oleh pejabat penyelidik, dengan maksud dan tujuan mengumpulkan “bukti permulaan” atau “bukti yang cukup” agar dapat dilakukan tindak lanjut penyidikan. Setelah mendapatkan laporan adanya tindak pidana investasi bodong dilakukan tindakan penyelidikan. Pada tahap penyelidikan, polisi penyelidik melakukan serangkaian tindakan yaitu: p y p p y p p y g 1) Menerima laporan atau pengaduan dari seseorang tentang adanya tindak pidana Inves 1) Menerima laporan atau pengaduan dari seseorang tentang adanya tindak pidana Investasi bodong 1) Menerima laporan atau pengaduan dari seseorang tentang adanya tindak pidana Investasi bodong 2) M i k t d l t b kti 2) Mencari keterangan dan alat bukti 3) Kewenangan Penyelidik Membuat dan Menyampaikan Laporan Hasil Pelaksanaan Tindakan Penyelidikan d. Penyidikan Penyidikan merupakan serangkaian tindakan penyidik dalam hal dan menurut cara yang diatur dalam undang-undang untuk mencari serta mengumpulkan bukti yang terjadi dan guna menemukan tersangkanya (Pasal 1 angka 2 KUHP). Dalam hal ini penyidikan tindak pidana investasi bodong di Polres Pohuwato di lakukan oleh Polisi Penyidik kanit tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Setelah dikeluarkan surat perintah penyidikan dan surat perintah tugas, polisi penyidik segera melakukan penyidikan terhadap tindak pidana investasi bodong.16 3) Kewenangan Penyelidik Membuat dan Menyampaikan Laporan Hasil Pelaksanaan Tindakan Penyelidikan d. Penyidikan Penyidikan merupakan serangkaian tindakan penyidik dalam hal dan menurut cara yang diatur dalam undang-undang untuk mencari serta mengumpulkan bukti yang terjadi dan guna menemukan tersangkanya (Pasal 1 angka 2 KUHP). Dalam hal ini penyidikan tindak pidana investasi bodong di Polres Pohuwato di lakukan oleh Polisi Penyidik kanit tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Setelah dikeluarkan surat perintah penyidikan dan surat perintah tugas, polisi penyidik segera melakukan penyidikan terhadap tindak pidana investasi bodong.16 p y p p g 2. b) Negara Lain b) Negara Lain Negara lain sebagai pusat penyedia layanan online trading sehingga polisi penyidik harus meminta data yang ada pada di penyedia layanan online trading di negara lain tersebut dengan mengajukan surat permohonan ke instansi di negara lain tersebut. Negara lain sebagai pusat penyedia layanan online trading sehingga polisi penyidik harus meminta data yang ada pada di penyedia layanan online trading di negara lain tersebut dengan mengajukan surat permohonan ke instansi di negara lain tersebut. Hal ini tentu saja akan membutuhkan waktu yang lama dan belum tentu mendapatkan respon dari instansi tersebut, karena saat ini belum ada kerjasama secara khusus antara pihak Kepolisian dengan negara lain tersebut terkait tindak pidana ini. Kerjasama yang ada masih secara umum hubungan internasional kenegaraan. g 2) Hambatan Kurangnya Kesadaran Masyarakat (Korban) untuk menjadi Saksi Tindak Pidana Investasi Bodong g 2) Hambatan Kurangnya Kesadaran Masyarakat (Korban) untuk menjadi Saksi Tindak Pidana Investasi Bodong Polisi beranggapan bahwa masyarakat yang menjadi korban tindak pidana investasi bodong masih kurang kooperatif untuk diajak kerjasama dengan polisi penyidik. Hal ini terlihat dari kurangnya kesadaran masyarakat untuk menjadi saksi dalam kasus tindak pidana investasi bodong.18 KESIMPULAN Berdasarkan hasil penelitian dan pembahasan tentang upaya kepolisian dalam penanggulangan kasus investasi bodong di wilayah kepolisian Polres Pohuwato dapat dikemukakan kesimpulan sebagai berikut: wawancara penulis bersama dengan bapak Brigpol Husnul Hamka selaku penyidik kasus investasi bodong , bapak mengatakan bahwa upaya kepolisian dalam menanggulangi kasus investasi bodong di lakukan Upaya preventif oleh bagian Humas Polres Pohuwato dengan melakukan sosialisasi kepada masyarakat dengan memberikan penjelasan kepada masyarakat melalui pemberitaan di media massa guna mengantisipasi merebaknya tindak pidana investasi bodong dan upaya represif dalam menanggulangi tindak pidana investasi bodong di Polres Pohuwato dilakukan oleh Polisi Penyidik Kanit Tipidkor dan KBO (Kaur Bin Opsnal) Polres Pohuwato. Dalam upaya kepolisian dalam penanggulangan kasus investasi bodong di Wilayah Hukum Kepolisian Resor Pohuwato terdapat kendala yaitu: Briptu Zukarnain Darise mengatakan banyak masyarakat yang menjadi korban ivestasi bodong ini sehingga banyaknya korban dan admin membuat kepolisian kesulitan dalam mengidentifikasi informasi dari mulut ke mulut masyrakat. 1) Hambatan Birokrasi Untuk mendukung upaya represif polisi dalam menanggulangi tindak pidana investasi bodong berupa penyelidikan dan penyidikan memerlukan kerjasama dengan lembaga atau instansi lain. Namun untuk mendapatkan data guna mencari alat bukti dalam penyelidikan dan penyidikan tindak pidana investasi bodong terdapat hambatan dari lembaga atau instansi lain, karena birokrasi yang berbelit-belit. Hambatan dari lembaga atau instansi lain tersebut diuraikan sebagai berikut: a) Bank a) Bank 1443 Proses polisi penyidik untuk mendapatkan data rekening pelaku tindak pidana investasi bodong pada bank yang digunakan untuk menghimpun dana dari para investor, membutuhkan waktu yang cukup lama yaitu kurang lebih enam bulan, sehingga hal tersebut menghambat polisi dalam penyidikan tindak pidana investasi bodong b) N L i BIBLIOGRAFI li, Zainudin., 2009. Metode Penelitian Hukum.Jakarta : Sinar Grafika Ali, Zainudin., 2009. Metode Penelitian Hukum.Jakarta : Sinar Grafika Arief Nawari Barda. 2014. Masalah Penegakan Hukum Dan Kebijakan Pidana Dalam Penaggulangan Kejahatan. Jakarta Arief Nawari Barda. 2014. Masalah Penegakan Hukum Dan Kebijakan Pidana Dalam Penaggulangan Kejahatan. Jakarta Kristia, Gunawan Yopi. 2018. Tindak Pidana Perbankan Dalam Proses Peradilan Di Indonesia. Jakarta Timur Kristia, Gunawan Yopi. 2018. Tindak Pidana Perbankan Dalam Proses Peradilan Di Indonesia. Jakarta Timur Moeljatno. 2009. Asas-asas Hukum Pidana Edisi Revisi. Jakarta: Rineka Cipta no. 2009. Asas-asas Hukum Pidana Edisi Revisi. Jakarta: Rineka Cipta Rusianto Agus. 2016. Tindak Pidana Dan Pertanggung Jawaban Pidana. Jakarta Rusianto Agus. 2016. Tindak Pidana Dan Pertanggung Jawaban Pidana. Jakarta Riza Faisal, Sibarani Anshari Fauzi. 2021. Prinsip The Best Interest Of The Child. Medan : Umsu Press Riza Faisal, Sibarani Anshari Fauzi. 2021. Prinsip The Best Interest Of The Child. Medan : Umsu Press Yulianto, Achmad dan Mukti Fajar., 2010.Dualisme Peneltian Hukum Normatif dan Empiris.Yogyakarta : Pustaka Belajar Yulianto, Achmad dan Mukti Fajar., 2010.Dualisme Peneltian Hukum Normatif dan Empiris.Yogyakarta : Pustaka Belajar Subagyo, Joko. 2015. Metode Penelitian Dalam Teori dan Praktik. Jakarta: Rineka Cipta Sedarmayanti, Hidayat Syarifudin. 2011. Metodologi Penelitian. Bandung : CV Bandar Maju ubagyo, Joko. 2015. Metode Penelitian Dalam Teori dan Praktik. Jakarta: Rineka Cipta edarmayanti Hidayat Syarifudin 2011 Metodologi Penelitian Bandung : CV Bandar M Subagyo, Joko. 2015. Metode Penelitian Dalam Teori dan Praktik. Jakarta: Rineka Cip Sedarmayanti, Hidayat Syarifudin. 2011. Metodologi Penelitian. Bandung : CV Bandar y , y y g g j Sunggono Bambang. 2016. Metodologi Penelitian Hukum. Jakarta: PT Rajagrafindo Persada Skripsi Sunggono Bambang. 2016. Metodologi Penelitian Hukum. Jakarta: PT Rajagrafindo Persada Skripsi 18 Ibid, Hal. 22-25 1444 Ardianto, A. (2022). Pengaruh Literasi Keuangan dan Pola Pikir Terhadap Pencegahan Investasi Bodong Pada Nasabah GIS IAIN PALOPO (Doctoral dissertation, Institut agama islam Negeri (IAIN Palopo). Ibrahim Alfian. 2018 “Peran Kepolisian Dalam Upaya Menaggulangi Tindak Pidana Kekerasan Pada Anak”.Fakultas Hukum Universitas Negeri Gorontalo. Gorontalo Ibrahim Rahmadawati. 2021 “Peran Kepolisian Dalam Penanggulangan Maraknya Perilaku Seks Di Luar Nikah Di Wilayah Hukum Polres Gorontalo Kota”. Fakultas Hukum Negeri Gorontalo. Gorontalo Ismail DJ. Regita. 2020. “Peran Kepolisian Dalam Penyelesaian Tindak Pidana Penganiyaan Yang Dilakukan Oleh Geng Motor Di Polda Gorontalo”. Fakultas Hukum Universitas Negeri Gorontalo. Gorontalo Lopiani Lori Anggun. 2018. “Penegakan Hukum Pidana Terhadap Pelaku Pencurian Dengan Kekerasan Yang Menggunakan Senjata Api”. Fakultas Hukum Universitas Sriwijaya. Palembang g Novarina Vivin Vionita Al. g gp y p gg p wancara Dengan Bapak Sudirman Hippy Selaku Korban Investasi Bodong Pada Hari Rabu BIBLIOGRAFI 2014.”Upaya Polisi Dalam Menanggulangi Tindak Pidana Penipuan Berkedok Investasi Melalui Sistem Online Di Polda DIY”, Universitas Negeri Yogyakarta. Putri Ananda Lidya. 2021. “Peranan Kepolisian Dalam Menanggulangi Tindak Pidana Perjudian Sabung Ayam Di Kota Kotamobagu”.Fakultas Hukum Universitas Negeri Gorontalo. 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(2014).Upaya Menanggulangi Investasi Bodong di Internet”.Kertha Semaya: Journal Ilmu Hukum, 2(3) 1-5 W Wawancara Dengan Aipda Sengly Manopo Selaku Penyidik Pada Tanggal 19 Septembe Wawancara Dengan Aipda Sengly Manopo Selaku Penyidik Pada Tanggal 19 September 2023 Wawancara Dengan Briptu Zulkarnain Darise Selaku Penyidik pada tanggal 19 september 2023 Wawancara Dengan Aipda Sengly Manopo Selaku Penyidik Pada Tanggal 19 September 2023 Wawancara Dengan Briptu Zulkarnain Darise Selaku Penyidik pada tanggal 19 september Wawancara Dengan Aipda Sengly Manopo Selaku Penyidik Pada Tanggal 19 September 2023 Wawancara Dengan Briptu Zulkarnain Darise Selaku Penyidik pada tanggal 19 september 2023 wancara Dengan Brigpol Husnul Hamka Selaku Penyidik pada tanggal 19 september 2023 Wawancara Dengan Brigpol Husnul Hamka Selaku Penyidik pada tanggal 19 september 2023 Wawancara Dengan Bapak Sudirman Hippy Selaku Korban Investasi Bodong Pada Hari Rabu Wawancara Dengan Bapak Sudirman Hippy Selaku Korban Investasi Bodong Pada Hari Rabu 1445 Tanggal 21 Desember 2022 Artikel https://www.topkarir.com/2/article/detail/12-rekomendasi-aplikasi-investasi-saham-terbaik- terdaftar-ojk di akses pada 13 Februari pukul 15.22 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. 1446
https://openalex.org/W3037345274	https://www.researchsquare.com/article/rs-26430/v1.pdf	English	null	Evaluation of the Etest and disk diffusion method for detection of the activity of ceftazidime-avibactam against Enterobacterales and Pseudomonas aeruginosa in China	BMC Microbiology	2,020	cc-by	4,677	Evaluation of Etests and the disk diffusion method for assessment of the activity of ceftazidime-avibactam against Enterobacterales and Pseudomonas aeruginosa in China Qi Wang Peking University People's Hospital Feifei Zhang Peking University People's Hospital Zhanwei Wang Peking University People's Hospital Hongbin Chen Peking University People's Hospital Xiaojuan Wang Peking University People's Hospital Yawei Zhang Peking University People's Hospital Shuguang Li Peking University People's Hospital Hui Wang (  whuibj@163.com ) Peking University People's Hospital https://orcid.org/0000-0001-9220-0357 Research article Keywords: broth microdilution, disk diffusion, Etest, ceftazidime-avibactam Posted Date: May 7th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-26430/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Microbiology on June 29th, 2020. See the published version at https://doi.org/10.1186/s12866-020-01870-z. Evaluation of Etests and the disk diffusion method for assessment of the activity of ceftazidime-avibactam against Enterobacterales and Pseudomonas aeruginosa in China Qi Wang Peking University People's Hospital Feifei Zhang Peking University People's Hospital Zhanwei Wang Peking University People's Hospital Hongbin Chen Peking University People's Hospital Xiaojuan Wang Peking University People's Hospital Yawei Zhang Peking University People's Hospital Shuguang Li Peking University People's Hospital Hui Wang (  whuibj@163.com ) Peking University People's Hospital Qi Wang Peking University People's Hospital Feifei Zhang Peking University People's Hospital Zhanwei Wang Peking University People's Hospital Hongbin Chen Peking University People's Hospital Xiaojuan Wang Peking University People's Hospital Yawei Zhang Peking University People's Hospital Shuguang Li Peking University People's Hospital Hui Wang (  whuibj@163.com ) Peking University People's Hospital Background Gram-negative bacilli, particularly carbapenem-resistant Enterobacterales (CRE) and Pseudomonas aeruginosa, exhibit major antimicrobial resistance worldwide, including in European countries, the United States of America (USA), and China [1–3]. The approval of ceftazidime-avibactam for clinical use in Europe and the USA has brought new treatment options to CRE-infected patients, particularly those with serine-carbapenemase resistance mechanisms [4, 5]. Ceftazidime- avibactam was approved for use in China in 2019. However, currently available commercial antimicrobial susceptibility test kits have not yet been developed for analysis of ceftazidime-avibactam resistance in China, and in patients with infections caused by CRE, which are resistant to multiple antimicrobials, the results of ceftazidime-avibactam susceptibility tests are urgently needed to facilitate appropriate targeted treatment. Although ceftazidime-avibactam has excellent in vitro activity against carbapenem-resistant Klebsiella pneumoniae (CRKP) and Pseudomonas aeruginosa strains, a small number of resistant strains will still appear during treatment [6–8]. Therefore, susceptibility test results for ceftazidime-avibactam are even more critical. In most of the laboratories in China, performing the standard broth microdilution method (BMD) is challenging. Therefore, other rapid, simple methods are required as alternatives to determine ceftazidime-avibactam susceptibility. In this study, we evaluated two antimicrobial susceptibility test methods for ceftazidime-avibactam, i.e., the Etest method and the disk diffusion method with the standard BMD, to evaluate whether these easy-to-use methods could replace standard methods when required in the clinical setting. Research article DOI: https://doi.org/10.21203/rs.3.rs-26430/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License ed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Microbiology on June 29th, 2020. See the published version at https://doi.org/10.1186/s12866-020-01870-z. Page 1/11 Abstract Background: Gram-negative bacilli, particularly Enterobacterales and Pseudomonas aeruginosa, often acquire antimicrobial resistance. Ceftazidime-avibactam was approved for use in China in 2019. However, currently available commercial antimicrobial susceptibility test kits have not yet been developed. Here, we evaluated the Etest and disk diffusion method for assessment of the efficacy of ceftazidime-avibactam against Enterobacterales and P. aeruginosa in China. Results: In total, 194 Enterobacterales and 77 P. aeruginosa isolates, which were divided into a random selection group (140 Enterobacterales and 54 P. aeruginosa isolates) and a stock group (46 Enterobacterales and 31 P. aeruginosa isolates), were assessed by the Etest, disk diffusion, and broth microdilution (BMD) methods. Minimum inhibitory concentrations (MICs) and zone diameters were interpreted according to the CLSI M100 30th edition. For all 271 Enterobacterales and P. aeruginosa isolates, no very major errors were found using Etests. The overall categorical agreement rates (CA%) of Etests for Enterobacterales and P. aeruginosa were 99.5% (193/194) and 96.1% (74/77), respectively. The overall essential agreement rates (EA%) of Etests for Enterobacterales and P. aeruginosa were 95.9% (186/194) and 94.8% (73/77), respectively. In both the random selection and stock groups, EA% and CA% values of Etests exceeded 90%. Overall CA% values of the disk diffusion method for Enterobacterales and P. aeruginosa were 98.5% (191/194) and 93.5% (71/77), respectively. There was no linear relationship between zone diameter and BMD MIC. Conclusions: For Enterobacterales and P. aeruginosa, Etests and the disk diffusion method could have better performance as alternative methods to meet the needs of clinical treatment interpretation. Application of the disk diffusion method in Enterobacterales was superior to that in P. aeruginosa. Materials And Methods Groups Page 2/11 Page 2/11 Isolates were divided into two groups, i.e., the random selection group and the stock group. For the random selection group, we randomly selected 140 Enterobacterales and 46 Pseudomonas aeruginosa strains from clinical nonrepeated isolates obtained at Peking University People’s Hospital. Among these samples, 59.3% (83/140) of Enterobacterales and 56.5% (26/46) of Pseudomonas aeruginosa strains were defined as fresh strains from clinical isolates obtained within 1 month prior to testing (November 2019 to March 2020). The remaining strains in the random selection group were obtained from the strain repository of Peking University People’s Hospital from January 2018 to October 2019. The 140 strains of Enterobacterales used in the testing included 13 species, i.e., 25 K. pneumoniae, 19 Escherichia coli, 18 Proteus mirabilis, 17 Enterobacter cloacae, 16 Serratia marcescens, 15 Citrobacter freundii, 14 K. oxytoca, four Proteus vulgaris, three Morganella morganii, three Providencia stuartii, two Providencia rettgeri, two K. aerogenes, and two Citrobacter koseri strains. For the stock group, we selected 54 strains of Enterobacterales from 15 hospitals in the CRE China-Network from January 2015 to October 2019 and requested that the minimum inhibitory concentration (MIC) of ceftazidime- avibactam be between 2 and 256 µg/mL. Among these strains, six strains (11.1%) showed MICs for ceftazidime- avibactam of between 8 and 16 µg/mL, and 15 strains (27.8%) showed MIC between 4 and 32 µg/mL. The carbapenem- resistance genes present in these strains were elucidated in previous studies [9]. The 54 strains of Enterobacterales used in this study included 29 K. pneumoniae (18 strains having the blaKPC gene and 10 strains having the blaNDM gene), 12 Escherichia coli (two strains having the blaKPC gene and seven strains having the blaNDM gene), eight Enterobacter cloacae (one strain having the blaKPC gene, five strains having the blaNDM gene, one strain having the blaIMP gene, and one strain having blaVIM gene), three K. oxytocai (two strains having the blaIMP gene and one strain having the blaNDM gene), and two Citrobacter freundii (one strain having the blaIMP gene and one strain having the blaNDM gene). We selected 31 strains of Pseudomonas aeruginosa from the eight hospitals involved in the CARES 2018 project as stock group strains and required the MIC of ceftazidime-avibactam to be between 2 and 256 µg/mL. Materials And Methods Groups Among these strains, 12 strains (38.7%) had MICs for ceftazidime-avibactam between 8 and 16 µg/mL, and 25 strains (80.6%) had MICs between 4 and 32 µg/mL. All strains were removed from a -80 °C ultra-low temperature refrigerator and transferred to Columbia blood agar twice before antimicrobial susceptibility testing. Antimicrobial susceptibility testing For the disk diffusion method, ceftazidime-avibactam disks were obtained from Oxoid (Thermo Scientific, China) The content of ceftazidime-avibactam in each disk was 30 µg/20 µg. The operation process was strictly tested following the requirements of the CLSI standardized method [10]. For the Etest gradient diffusion method, ceftazidime-avibactam Etest strips were obtained from BioMérieux (Marcy l’Etoile, France). The test was performed in strict accordance with the manufacturer’s instructions. The ceftazidime- avibactam concentration gradient ranged from 0.016 to 256 µg/mL. When the Etest MIC value was between the standard value and twice the standard value (0.016, 0.032, 0.064, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256), the high standard value will be read as the MIC. The MH agar used for both the disk diffusion method and the Etest gradient diffusion method for antimicrobial susceptibility testing was obtained from Oxoid (Thermo Scientific Inc). The MH agar used for both the disk diffusion method and the Etest gradient diffusion method for antimicrobial susceptibility testing was obtained from Oxoid (Thermo Scientific Inc). The BMD was performed strictly following CLSI guidelines [11]. Ceftazidime and avibactam powers were obtained from Pfizer (USA). The concentration ranged from the standard double dilution of 0.016 µg/mL to 256 µg/mL. The BMD was performed strictly following CLSI guidelines [11]. Ceftazidime and avibactam powers were obtained from Pfizer (USA). The concentration ranged from the standard double dilution of 0.016 µg/mL to 256 µg/mL. Quality control and colony counting are performed simultaneously for each batch of experiments. Escherichia coli ATCC 25922, K. pneumoniae ATCC 700603, Escherichia coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853 were Page 3/11 Page 3/11 used as experimental quality control strains. The experiment was considered valid only when the experimental values of all the quality control strains were within the acceptable range. used as experimental quality control strains. The experiment was considered valid only when the experimental values of all the quality control strains were within the acceptable range. The MICs and zone diameters of ceftazidime-avibactam for Enterobacterales and Pseudomonas aeruginosa were interpreted according to CLSI M100 30th edition [11]. Antimicrobial susceptibility testing Briefly, MICs of less than or equal to 8/4 µg/mL or a zone diameter of greater than or equal to 21 mm indicated that the strain was susceptible, whereas MICs of greater than or equal to 16/4 µg/mL or a zone diameter of less than or equal to 20 mm indicated that the strain was resistant. Essential agreement (EA) indicated that the difference between the MIC value measured by Etest and the BMD did not exceed one dilution factor. Categorical agreement (CA) indicated that the BMD method was consistent with the classification results from the disk diffusion method and Etest method based on the same CLSI breakpoints. Very major errors (VMEs) indicated that the strain was susceptible by Etest or the disk diffusion method but resistant by the BMD. Major errors (MEs) indicated that the strain was susceptible by the BMD but resistant by Etest or the disk diffusion method. Etest versus the BMD The Etest MICs of 22 (28.6%) strains were one dilution higher than those obtained by the BMD, whereas those of six (7.8%) strains were one dilution lower than the MICs obtained by the BMD. For one strain, the Etest MIC was two dilutions higher than those obtained by the BMD. Three MEs appeared in the stock group when the Etest method was used. Etest versus the BMD For 194 Enterobacterales strains, no VMEs were found using the Etest method. As shown in Table 1, the overall CA rate was 99.5%, and the overall EA rate was 95.9%. The CA rate of the stock group was 90.7%, and the CA rate in the random selection group was 97.9%. When comparing Etest results with BMD results, we found that the MICs of eight strains exceeded the two dilution factors. As shown in Fig. 1, the Etest MICs of 102 (52.5%) strains were consistent with the BMD MICs. The Etest MICs of 71 (36.6%) strains were a dilution multiple higher than the BMD MICs. Only 12 (6.2%) strains had Etest MICs that were a dilution multiple lower than the BMD MICs. Page 4/11 Table 1 Table 1 Table 1 Evaluation of essential and categorical agreement between the BMD method and Etests or the disk diffusion method for analysis of ceftazidime-avibactam antimicrobial susceptibility Evaluation of essential and categorical agreement between the BMD method and Etests or the disk diffusion method for analysis of ceftazidime-avibactam antimicrobial susceptibility Organisms No. of strains tested Etest Disk diffusion No. of EA EA% No. of CA CA% No. of VMEs No. of MEs No. of CA CA% No. of VMEs No. of MEs Enterobacterales Random selection Group 140 137 97.9% 140 100.0% 0 0 140 100.0% 0 0 Stock group: 54 49 90.7% 53 98.1% 0 1 51 94.4% 2 1 Total in Enterobacterales 194 186 95.9% 193 99.5% 0 1 191 98.5% 2 1 Pseudomonas aeruginosa Random selection Group 46 45 97.8% 46 100.0% 0 0 43 93.5% 0 3 Stock group: 31 28 90.3% 28 90.3% 0 3 29 93.5% 1 1 Total in Pseudomonas aeruginosa 77 73 94.8% 74 96.1% 0 3 72 93.5% 1 4 Total in all tested strains 271 259 95.6% 267 98.5% 0 4 263 97.0% 3 5 EA: essential agreement; CA: categorical agreement; VMEs: very major errors (false susceptible); MEs: major errors (false resistant) For 77 Pseudomonas aeruginosa strains, no VMEs were found using the Etest method. As shown in Table 1, the overall CA rate was 96.1%, and the overall EA rate was 94.8%. The CA rate in the stock group was 90.3%, and the CA rate in the random selection group was 100%. As shown in Fig. 2, the Etest MICs of 45 (58.4%) strains were consistent with the MICs obtained by the BMD. Discussion In the past ten years, the incidence of CRE, particularly CRKP, has increased significantly in China. The latest China Antimicrobial Resistance Surveillance System (CARSS) data show that the incidence of CRKP nationwide is as high as 10.1% (http://www.carss.cn/Report/Details?aId=648). However, few active antibacterial agents, such as tigecycline and colistin, are available to treat CRKP in the clinical setting [12, 13], resulting in high mortality worldwide. Previous molecular epidemiological data show that more than 70% of CRE isolated in China from 2012 to 2016 produce KPC-type carbapenemases [9]. Ceftazidime-avibactam, a drug with potent antibacterial activity against serine-carbapenemase, was approved for use in China in 2019. Recent retrospective studies have shown that ceftazidime-avibactam treatment of CRKP and Pseudomonas aeruginosa infection in patients who have undergone solid organ transplantation improves clinical success rates [14, 15]. Despite these promising findings, ceftazidime-avibactam antimicrobial susceptibility test results are essential for clinical use of this treatment. However, no ceftazidime-avibactam combination kits are available for assessing resistance in China. To the best of our knowledge, this is the first study comparing Etests, the disk diffusion method, and the BMD with regard to detection of ceftazidime-avibactam susceptibility in China. Compared with the standard BMD method, no VMEs were found using the Etest method. The results of Etest MICs and BMD MICs were reasonably well correlated for both Enterobacterales and Pseudomonas aeruginosa. The overall of EA% of 271 tested strains was 95.6%. Compared with the BMD method, the Etest method exhibited an excellent linear correlation, supporting the use of this approach as an alternative to the standard clinical method without considering economic costs. This result was similar to the previous research findings of other authors [16]. The disk diffusion method is easy to implement in the clinical setting from an economic standpoint. The CA% values of the ceftazidime-avibactam disk diffusion method against Enterobacterales and Pseudomonas aeruginosa were 98.5% and 93.5%, respectively, similar to the findings of Shields and other scholars [16, 17]. Indeed, our results showed that application of the disk diffusion method was more appropriate for Enterobacterales than for Pseudomonas aeruginosa. Notably, however, the disk diffusion method did not have an excellent linear correlation with BMD. Therefore, the zone diameters of strains near the breakpoints (± 2 mm) should be checked by the BMD carefully, consistent with other research results [17, 18]. Conclusions In conclusion, for Enterobacterales and Pseudomonas aeruginosa, Etests and the disk diffusion method showed good performance as alternative methods to meet the needs of clinical treatment interpretation. The application of the disk diffusion method was superior for Enterobacterales compared with Pseudomonas aeruginosa. as shown in Fig. 4, when the zone diameter was 20 mm, the MICs obtained by the BMD method were 4, 8, 16, or 64 µg/mL. as shown in Fig. 4, when the zone diameter was 20 mm, the MICs obtained by the BMD method were 4, 8, 16, or 64 µg/mL. as shown in Fig. 4, when the zone diameter was 20 mm, the MICs obtained by the BMD method were 4, 8, 16, or 64 µg/mL. Discussion This should also be considered when users refer to the new version of CLSI M100, which suggests using the disk diffusion method [11]. Declarations Ethics approval and consent to participate: Not applicable Disk diffusion method versus BMD A comparison of the disk diffusion method and BMD results for 194 Enterobacterales strains (Fig. 3) showed that there were no linear relationships between zone diameter and MIC. The overall CA rate in the 194 Enterobacterales strains was 98.5%. Two VMEs and one ME were found using the disk diffusion method in the stock group; all were for K. pneumoniae strains. No VMEs or MEs were found using the disk diffusion method in the random selection group. There were 22 strains of Enterobacterales with zone diameters of 2 mm (19–22 mm susceptibity breakpoint). Forty-one of the 45 resistant strains obtained by BMD had zone diameters in the range of 13–20 mm. In a comparison of the results of the disk diffusion method and BMD for 77 Pseudomonas aeruginosa strains, the number of MEs was four, and the number of VMEs was 1. As shown in Table 1, the overall CA rate was 93.5%. Moreover, Page 5/11 Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Competing interests: The authors declare that they have no competing interests Competing interests: The authors declare that they have no competing interests Funding: This work was supported by National Natural Science Foundation of China (81625014) Page 6/11 Authors' contributions: HW conceived and designed the study. QW, FZ, HC, XW, YZ and SL performed experiments described in this study. QW wrote the draft, and HW revised it. All authors approved the final version. Competing Interests: The authors declare no conflicts of interest in this work. Competing Interests: The authors declare no conflicts of interest in this work. Competing Interests: The authors declare no conflicts of interest in this work. ompeting Interests: The authors declare no conflicts of interest in this work. Acknowledgements: We would like to thank Editage (www.editage.cn) for English language editing. Acknowledgements: We would like to thank Editage (www.editage.cn) for English language editing. Acknowledgements: We would like to thank Editage (www.editage.cn) for English language editing. 5. Shirley M: Ceftazidime-Avibactam: A Review in the Treatment of Serious Gram-Negative Bacterial Infections. Drugs 2018, 78(6):675-692. 5. Shirley M: Ceftazidime-Avibactam: A Review in the Treatment of Serious Gram-Negative Bacterial Infections. Drugs 2018, 78(6):675-692. 6. Karlowsky JA, Kazmierczak KM, Bouchillon SK, de Jonge BLM, Stone GG, Sahm DF: In Vitro Activity of Ceftazidime- Avibactam against Clinical Isolates of Enterobacteriaceae and Pseudomonas aeruginosa Collected in Latin American Countries: Results from the INFORM Global Surveillance Program, 2012-2015. Antimicrob Agents Chemother 2019. 7. Giddins MJ, Macesic N, Annavajhala MK, Stump S, Khan S, McConville TH, Mehta M, Gomez-Simmonds A, Uhlemann AC: Successive Emergence of Ceftazidime-Avibactam Resistance through Distinct Genomic Adaptations in blaKPC-2- Harboring Klebsiella pneumoniae Sequence Type 307 Isolates. Antimicrob Agents Chemother 2018, 62(3). 8. Gaibani P, Campoli C, Lewis RE, Volpe SL, Scaltriti E, Giannella M, Pongolini S, Berlingeri A, Cristini F, Bartoletti M et al: In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment. The Journal of antimicrobial chemotherapy 2018, 73(6):1525-1529. 8. Gaibani P, Campoli C, Lewis RE, Volpe SL, Scaltriti E, Giannella M, Pongolini S, Berlingeri A, Cristini F, Bartoletti M et al: In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment. The Journal of antimicrobial chemotherapy 2018, 73(6):1525-1529. 9. Wang Q, Wang X, Wang J, Ouyang P, Jin C, Wang R, Zhang Y, Jin L, Chen H, Wang Z et al: Phenotypic and Genotypic Characterization of Carbapenem-resistant Enterobacteriaceae: Data From a Longitudinal Large-scale CRE Study in China (2012-2016). Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2018, 67(suppl_2):S196-S205. 9. Wang Q, Wang X, Wang J, Ouyang P, Jin C, Wang R, Zhang Y, Jin L, Chen H, Wang Z et al: Phenotypic and Genotypic Characterization of Carbapenem-resistant Enterobacteriaceae: Data From a Longitudinal Large-scale CRE Study in China (2012-2016). Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2018, 67(suppl_2):S196-S205. 10. Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial disk susceptibility tests; 12th ed. M02-A12. Clinical and Laboratory Standards Institute, Wayne, PA. 10. Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial disk susceptibility tests; 12th ed. M02-A12. Clinical and Laboratory Standards Institute, Wayne, PA. 11. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2020. 11. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2020. 12. References 1. Chinese XDRCWG, Guan X, He L, Hu B, Hu J, Huang X, Lai G, Li Y, Liu Y, Ni Y et al: Laboratory diagnosis, clinical management and infection control of the infections caused by extensively drug-resistant Gram-negative bacilli: a Chinese consensus statement. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 2016, 22 Suppl 1:S15-25. 2. Hu F, Zhu D, Wang F, Wang M: Current Status and Trends of Antibacterial Resistance in China. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2018, 67(suppl_2):S128-S134. 2. Hu F, Zhu D, Wang F, Wang M: Current Status and Trends of Antibacterial Resistance in China. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2018, 67(suppl_2):S128-S134. 3. Grundmann H, Glasner C, Albiger B, Aanensen DM, Tomlinson CT, Andrasević AT, Cantón R, Carmeli Y, Friedrich AW, Giske CG et al: Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE): a prospective, multinational study. The Lancet Infectious Diseases 2017, 17(2):153-163. 4. Pogue JM, Bonomo RA, Kaye KS: Ceftazidime/Avibactam, Meropenem/Vaborbactam, or Both? Clinical and Formulary Considerations. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2019, 68(3):519-524. 18. Sader HS, Rhomberg PR, Huband MD, Critchley IA, Stone GG, Flamm RK, Jones RN: Assessment of 30/20-Microgram Disk Content versus MIC Results for Ceftazidime-Avibactam Tested against Enterobacteriaceae and Pseudomonas aeruginosa. Journal of clinical microbiology 2018, 56(6). 5. Shirley M: Ceftazidime-Avibactam: A Review in the Treatment of Serious Gram-Negative Bacterial Infections. Drugs 2018, 78(6):675-692. Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL: Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clinical microbiology reviews 2012, 25(4):682- 707. Page 7/11 Page 7/11 13. Gutiérrez-Gutiérrez B, Salamanca E, de Cueto M, Hsueh P-R, Viale P, Paño-Pardo JR, Venditti M, Tumbarello M, Daikos G, Cantón R et al: Effect of appropriate combination therapy on mortality of patients with bloodstream infections due to carbapenemase-producing Enterobacteriaceae (INCREMENT): a retrospective cohort study. The Lancet Infectious Diseases 2017, 17(7):726-734. 14. Chen W, Sun L, Guo L, Cao B, Liu Y, Zhao L, Lu B, Li B, Chen J, Wang C: Clinical outcomes of ceftazidime-avibactam in lung transplant recipients with infections caused by extensively drug-resistant gram-negative bacilli. Annals of translational medicine 2020, 8(3):39. 15. Sternbach N, Leibovici Weissman Y, Avni T, Yahav D: Efficacy and safety of ceftazidime/avibactam: a systematic review and meta-analysis. The Journal of antimicrobial chemotherapy 2018, 73(8):2021-2029. 16. Shields RK, Clancy CJ, Pasculle AW, Press EG, Haidar G, Hao B, Chen L, Kreiswirth BN, Nguyen MH: Verification of Ceftazidime-Avibactam and Ceftolozane-Tazobactam Susceptibility Testing Methods against Carbapenem-Resistant Enterobacteriaceae and Pseudomonas aeruginosa. Journal of clinical microbiology 2018, 56(2). 17. Sader HS, Rhomberg PR, Chandrasekaran S, Trejo M, Fedler KA, Boyken LD, Diekema DJ: Correlation between Broth Microdilution and Disk Diffusion Results when Testing Ceftazidime-Avibactam against a Challenge Collection of Enterobacterales Isolates: Results from a Multilaboratory Study. Journal of clinical microbiology 2020, 58(4). 17. Sader HS, Rhomberg PR, Chandrasekaran S, Trejo M, Fedler KA, Boyken LD, Diekema DJ: Correlation between Broth Microdilution and Disk Diffusion Results when Testing Ceftazidime-Avibactam against a Challenge Collection of Enterobacterales Isolates: Results from a Multilaboratory Study. Journal of clinical microbiology 2020, 58(4). 18. Sader HS, Rhomberg PR, Huband MD, Critchley IA, Stone GG, Flamm RK, Jones RN: Assessment of 30/20-Microgram Disk Content versus MIC Results for Ceftazidime-Avibactam Tested against Enterobacteriaceae and Pseudomonas aeruginosa. Journal of clinical microbiology 2018, 56(6). 18. Sader HS, Rhomberg PR, Huband MD, Critchley IA, Stone GG, Flamm RK, Jones RN: Assessment of 30/20-Microgram Disk Content versus MIC Results for Ceftazidime-Avibactam Tested against Enterobacteriaceae and Pseudomonas aeruginosa. Journal of clinical microbiology 2018, 56(6). Figures Figures Page 8/11 Page 8/11 Figure 1 Scatter plot of ceftazidime-avibactam Etest MICs versus BMD MICs against Enterobacterales. Dotted lines represent the susceptibility breakpoint for ceftazidime-avibactam. VMEs: very major errors (false susceptible); MEs: major errors (false resistant). The gray background indicates that the MIC of the Etest did not satisfy the essential agreement compared with the MIC of the BMD; the yellow background indicates that a major error occurred in the MIC result of the Etest compared with the MIC of the BMD. Scatter plot of ceftazidime-avibactam Etest MICs versus BMD MICs against Enterobacterales. Dotted lines represent the susceptibility breakpoint for ceftazidime-avibactam. VMEs: very major errors (false susceptible); MEs: major errors (false resistant). The gray background indicates that the MIC of the Etest did not satisfy the essential agreement compared with the MIC of the BMD; the yellow background indicates that a major error occurred in the MIC result of the Etest compared with the MIC of the BMD. Page 9/11 Figure 2 Scatter plot of ceftazidime-avibactam Etest MICs versus BMD MICs against Pseudomonas aeruginosa. Dotted lines represent the susceptibility breakpoint for ceftazidime-avibactam. VMEs: very major errors (false susceptible); MEs: major errors (false resistant). The gray background indicates that the MIC of the Etest did not satisfy the essential agreement compared with the MIC of the BMD; the yellow background indicates that three major errors occurred in the MIC results of the Etest compared with the MIC of the BMD. Figure 2 Scatter plot of ceftazidime-avibactam Etest MICs versus BMD MICs against Pseudomonas aeruginosa. Dotted lines represent the susceptibility breakpoint for ceftazidime-avibactam. VMEs: very major errors (false susceptible); MEs: major errors (false resistant). The gray background indicates that the MIC of the Etest did not satisfy the essential agreement compared with the MIC of the BMD; the yellow background indicates that three major errors occurred in the MIC results of the Etest compared with the MIC of the BMD. Scatter plot of ceftazidime-avibactam Etest MICs versus BMD MICs against Pseudomonas aeruginosa. Dotted lines represent the susceptibility breakpoint for ceftazidime-avibactam. VMEs: very major errors (false susceptible); MEs: major errors (false resistant). The gray background indicates that the MIC of the Etest did not satisfy the essential agreement compared with the MIC of the BMD; the yellow background indicates that three major errors occurred in the MIC results of the Etest compared with the MIC of the BMD. Page 10/11 Figure 3 Scatter plot of ceftazidime-avibactam zone diameters versus BMD MICs against Enterobacterales. Dotted lines represent the susceptibility breakpoint for ceftazidime-avibactam. VMEs: very major errors (false susceptible); MEs: major errors (false resistant). The yellow background indicates that a major error occurred when comparing the results of the disk diffusion method with the results of the BMD. The red background indicates that two very major errors occurred when comparing the results of the disk diffusion method with the results of the BMD. Figure 4 Figure 4 Scatter plot of ceftazidime-avibactam zone diameters versus BMD MICs against Pseudomonas aeruginosa. Dotted lines represent the susceptibility breakpoint for ceftazidime-avibactam. VMEs: very major errors (false susceptible); MEs: major errors (false resistant). The yellow background indicates that four major errors occurred when comparing the results of the disk diffusion method with the results of the BMD. The red background indicates that a very major error occurred when comparing the results of the disk diffusion method with the results of the BMD. Page 11/11 Page 11/11
https://openalex.org/W2227574826	https://journals.iucr.org/e/issues/2009/07/00/at2800/at2800.pdf	English	null	<i>rac</i>-7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid–2-amino-1,3,4-thiadiazole–water (1/1/1)	Acta crystallographica. Section E	2,009	cc-by	3,082	organic compounds b = 18.4267 (15) A˚ c = 12.7546 (11) A˚ = 101.336 (6) V = 1329.1 (2) A˚ 3 Z = 4 Mo K radiation = 0.27 mm1 T = 296 K 0.30 0.16 0.09 mm Data collection Bruker APEXII area-detector diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.949, Tmax = 0.977 10820 measured reﬂections 2995 independent reﬂections 2026 reﬂections with I > 2(I) Rint = 0.040 Reﬁnement R[F 2 > 2(F 2)] = 0.046 wR(F 2) = 0.133 S = 1.05 2995 reﬂections 187 parameters 3 restraints H atoms treated by a mixture of independent and constrained reﬁnement max = 0.30 e A˚ 3 min = 0.29 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Mo K radiation = 0.27 mm1 T = 296 K 0.30 0.16 0.09 mm Related literature 7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (nor- cantharidin) is a lower toxicity anticancer drug, see: Shimi & Zaki (1982). Experimental Crystal data C8H10O5C2H3N3SH2O Mr = 305.31 Monoclinic, P21=n a = 5.7678 (5) A˚ The authors thank the Natural Science Foundation of Zhejiang Province, China (grant No. Y407301) for ﬁnancial support. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: AT2800). Table 1 Key indicators: single-crystal X-ray study; T = 296 K; mean (C–C) = 0.003 A˚; R factor = 0.046; wR factor = 0.133; data-to-parameter ratio = 16.0. D—H A D—H H A D A D—H A N1—H1A O4i 0.86 2.11 2.930 (3) 160 N1—H1C N3ii 0.86 2.15 2.994 (3) 166 N1—H1C N2ii 0.86 2.69 3.519 (3) 161 O2—H2A O1Wiii 0.82 1.81 2.626 (2) 176 O5—H5B N2iv 0.82 1.85 2.664 (2) 172 O1W—H1WA O3v 0.859 (17) 1.910 (17) 2.766 (2) 175 (3) O1W—H1WB O4vi 0.819 (17) 2.51 (3) 3.151 (3) 137 (3) O1W—H1WB O1vi 0.819 (17) 2.55 (3) 3.061 (3) 122 (3) Symmetry codes: (i) x þ 1 2; y 1 2; z þ 1 2; (ii) x þ 1; y; z; (iii) x þ 1 2; y 1 2; z þ 1 2; (iv) x 1 2; y 1 2; z 1 2; (v) x 1 2; y þ 1 2; z þ 1 2; (vi) x; y; z. The title compound, C8H10O5C2H3N3SH2O, was synthesized by the reaction of 2-amino-1,3,4-thiadiazole with norcanthar- idin. The crystal structure is stabilized by N—H O, N— H N, O—H O and O—H N hydrogen bonds. In addition, weak – interactions are observed between symmetry-related thiadiazole ring systems [centroid–centroid distance = 3.9110 (3) A˚ , interplanar spacing = 3.4845 A˚ ]. Data collection: APEX2 (Bruker, 2006); cell reﬁnement: SAINT Data collection: APEX2 (Bruker, 2006); cell reﬁnement: SAINT (Bruker, 2006); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97. Na Wang, Qiu-Yue Lin* and Yan-Jun Wang Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces, Institute of Physical Chemistry, Zhejiang Normal University, Jinhua, Zhejiang 321004, People’s Republic of China, and, College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, Zhejiang, People’s Republic of China Correspondence e-mail: sky51@zjnu.cn Received 27 May 2009; accepted 9 June 2009 References Bruker (2006). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Shimi, I. R. & Zaki, Z. (1982). Eur. J. Cancer Clin. Oncol. 18, 785–793. Bruker (2006). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Experimental Bruker (2006). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Shimi, I. R. & Zaki, Z. (1982). Eur. J. Cancer Clin. Oncol. 18, 785–793. S2. Experimental 7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride and 2-amino-1,3,4-thiadiazole were dissolved in tetrahydrofuran and the mixture was stirred for 6 h at room temperature. The clear solution was left undisturbed for days to give colourless crystals of the compound. S1. Comment 7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin) derived from cantharidin is a lower toxicity anticancer drug (Shimi & Zaki, 1982). The title compound was synthesized by the reaction of 2-amino-1,3,4-thiadiazole with 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin). In this paper, we reports its structure. X-ray crystallography measurement confirmed the molecular structure and the atom connectivity for the title compound (Fig. 1). The crystal structure is stabilized by N—H···O, N—H···N, O—H···O and O—H···N hydrogen bonds (Table 1). Further, weak π–π interactions are observed between symmetry related thiadiazole ring systems [centroid-centroid distance of 3.9110 (3)Å and interplanar spacing of 3.4845 Å]. 7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin) derived from cantharidin is a lower toxicity anticancer drug (Shimi & Zaki, 1982). The title compound was synthesized by the reaction of 2-amino-1,3,4-thiadiazole with 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin). In this paper, we reports its structure. X-ray crystallography measurement confirmed the molecular structure and the atom connectivity for the title compound (Fig. 1). The crystal structure is stabilized by N—H···O, N—H···N, O—H···O and O—H···N hydrogen bonds (Table 1). 7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin) derived from cantharidin is a lower toxicity anticancer drug (Shimi & Zaki, 1982). The title compound was synthesized by the reaction of 2-amino-1,3,4-thiadiazole with 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (norcantharidin). In this paper, we reports its structure. X-ray crystallography measurement confirmed the molecular structure and the atom connectivity for the title compound (Fig. 1). The crystal structure is stabilized by N—H···O, N—H···N, O—H···O and O—H···N hydrogen bonds (Table 1). Further, weak π–π interactions are observed between symmetry related thiadiazole ring systems [centroid-centroid distance of 3.9110 (3)Å and interplanar spacing of 3.4845 Å]. Experimental Monoclinic, P21=n a = 5.7678 (5) A˚ o1590 Wang et al. Acta Cryst. (2009). E65, o1590 doi:10.1107/S1600536809021825 supporting information supporting information Acta Cryst. (2009). E65, o1590 [doi:10.1107/S1600536809021825] Acta Cryst. (2009). E65, o1590 [doi:10.1107/S1600536809021825] Acta Cryst. (2009). E65, o1590 [doi:10.1107/S1600536809021825] S3. Refinement The H atoms bonded to C and N atoms were positioned geometrically and refined using ariding model [C—H =0.93- 0.98 Å, N—H = 0.86 Å and O—H = 0.82 Å and Uiso(H) = 1.2 or 1.5Ueq(C,N,O)]. The H atoms of the water molecule were located in a difference Fourier maps and refined with O—H distance restraints of 0.85 (2) and Uiso(H) = 1.5Ueq(O). sup-1 Acta Cryst. (2009). E65, o1590 supporting information e 1 Figure 1 g A view of the molecule of (I) showing the atom-labelling scheme with displacement ellipsoids drawn at the 30% probability. rac-7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid–2-amino-1,3,4-thiadiazole–water (1/1/1) -7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid–2-amino-1,3,4-thiadiazole–water (1/1/1 Crystal data C8H10O5·C2H3N3S·H2O Mr = 305.31 Monoclinic, P21/n Hall symbol: -P 2yn a = 5.7678 (5) Å b = 18.4267 (15) Å c = 12.7546 (11) Å β = 101.336 (6)° V = 1329.1 (2) Å3 Z = 4 F(000) = 640 Dx = 1.526 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 1905 reflections θ = 2.0–27.6° µ = 0.27 mm−1 T = 296 K Block, colourless 0.30 × 0.16 × 0.09 mm Data collection Bruker APEXII area-detector diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.949, Tmax = 0.977 10820 measured reflections 2995 independent reflections 2026 reflections with I > 2σ(I) Rint = 0.040 θmax = 27.6°, θmin = 2.0° h = −6→7 k = −24→23 l = −16→15 sup-2 Acta Cryst. (2009). E65, o1590 supporting information supporting information Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.046 wR(F2) = 0.133 S = 1.05 2995 reflections 187 parameters 3 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.062P)2 + 0.3174P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.29 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.062P)2 + 0.3174P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.30 e Å−3 Δρmin = −0.29 e Å−3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq C1 −0.2976 (5) 0.05357 (14) 0.3265 (2) 0.0537 (7) H1B −0.3880 0.0947 0.3053 0.064 C2 −0.0094 (4) −0.03835 (13) 0.37115 (19) 0.0401 (6) C3 −0.7049 (4) −0.14263 (12) −0.02775 (19) 0.0371 (5) H3A −0.7478 −0.1570 −0.1031 0.044* C4 −0.4729 (4) −0.10527 (12) 0.11367 (19) 0.0378 (5) H4A −0.3228 −0.0881 0.1563 0.045* C5 −0.5597 (4) −0.17663 (11) 0.15371 (18) 0.0308 (5) H5A −0.6555 −0.1649 0.2069 0.037* C6 −0.7289 (4) −0.20482 (11) 0.05131 (18) 0.0320 (5) H6A −0.8916 −0.2080 0.0630 0.038* C7 −0.8388 (4) −0.07593 (13) −0.0002 (2) 0.0436 (6) H7A −0.9932 −0.0888 0.0135 0.052* H7B −0.8573 −0.0399 −0.0566 0.052* C8 −0.6732 (4) −0.04919 (12) 0.1016 (2) 0.0451 (6) H8A −0.7506 −0.0499 0.1626 0.054* H8B −0.6158 −0.0006 0.0925 0.054* C9 −0.3734 (4) −0.23059 (12) 0.20304 (18) 0.0339 (5) C10 −0.6556 (4) −0.27519 (12) 0.00717 (19) 0.0357 (5) S1 −0.00230 (12) 0.04890 (3) 0.32282 (6) 0.0514 (2) N1 0.1766 (3) −0.08184 (12) 0.39063 (18) 0.0534 (6) H1A 0.1631 −0.1250 0.4145 0.064* H1C 0.3110 −0.0670 0.3794 0.064* N2 −0.2197 (3) −0.05840 (10) 0.38689 (17) 0.0431 (5) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ue l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) / l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2009). Special details E65, o1590 supporting information N3 −0.3854 (4) −0.00394 (11) 0.36078 (19) 0.0511 (6) O1 −0.4621 (3) −0.12182 (8) 0.00484 (13) 0.0399 (4) O1W 0.3491 (3) 0.19399 (12) 0.19534 (16) 0.0573 (5) O2 −0.1540 (3) −0.20904 (9) 0.21051 (15) 0.0470 (5) H2A −0.0641 −0.2405 0.2405 0.071* O3 −0.4251 (3) −0.28854 (9) 0.23715 (14) 0.0453 (4) O4 −0.4568 (3) −0.28772 (9) −0.00388 (16) 0.0527 (5) O5 −0.8343 (3) −0.31955 (9) −0.02355 (17) 0.0575 (5) H5B −0.7870 −0.3568 −0.0474 0.086* H1WA 0.213 (4) 0.1986 (18) 0.212 (2) 0.086* H1WB 0.317 (5) 0.2024 (19) 0.1311 (15) 0.086* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 C1 0.0577 (16) 0.0398 (14) 0.064 (2) 0.0017 (11) 0.0119 (14) 0.0069 (13) C2 0.0431 (13) 0.0412 (13) 0.0358 (14) −0.0098 (10) 0.0075 (10) −0.0022 (10) C3 0.0419 (13) 0.0369 (12) 0.0311 (13) 0.0077 (9) 0.0038 (10) −0.0009 (10) C4 0.0383 (12) 0.0342 (12) 0.0394 (14) −0.0035 (9) 0.0041 (10) 0.0000 (10) C5 0.0310 (11) 0.0334 (11) 0.0289 (12) 0.0019 (8) 0.0081 (9) 0.0004 (9) C6 0.0280 (11) 0.0316 (11) 0.0371 (14) 0.0013 (8) 0.0081 (9) −0.0036 (9) C7 0.0443 (13) 0.0368 (12) 0.0486 (16) 0.0118 (10) 0.0062 (11) 0.0015 (11) C8 0.0606 (16) 0.0306 (12) 0.0448 (16) 0.0032 (10) 0.0119 (12) −0.0033 (11) C9 0.0339 (12) 0.0390 (12) 0.0300 (13) 0.0015 (9) 0.0094 (9) −0.0002 (10) C10 0.0366 (13) 0.0334 (12) 0.0365 (14) 0.0016 (9) 0.0058 (10) −0.0027 (9) S1 0.0575 (4) 0.0399 (4) 0.0583 (5) −0.0095 (3) 0.0150 (3) 0.0073 (3) N1 0.0475 (12) 0.0419 (12) 0.0697 (17) −0.0015 (9) 0.0089 (11) 0.0107 (11) N2 0.0455 (11) 0.0354 (10) 0.0480 (13) −0.0044 (8) 0.0085 (9) 0.0048 (9) N3 0.0462 (12) 0.0434 (12) 0.0625 (15) 0.0023 (9) 0.0083 (11) 0.0072 (11) O1 0.0417 (9) 0.0391 (9) 0.0425 (10) 0.0032 (7) 0.0170 (7) 0.0082 (7) O1W 0.0381 (10) 0.0837 (14) 0.0524 (12) −0.0079 (9) 0.0140 (9) −0.0121 (11) O2 0.0315 (9) 0.0499 (10) 0.0589 (12) 0.0004 (7) 0.0068 (8) 0.0103 (8) O3 0.0374 (9) 0.0435 (9) 0.0558 (12) 0.0049 (7) 0.0108 (8) 0.0171 (8) O4 0.0406 (10) 0.0447 (10) 0.0747 (14) 0.0032 (7) 0.0163 (9) −0.0214 (9) O5 0.0411 (10) 0.0362 (9) 0.0954 (16) −0.0057 (7) 0.0137 (9) −0.0213 (10) H1WB 0.317 (5) 0.2024 (19) 0.1311 (15) 0.086* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 C1 0.0577 (16) 0.0398 (14) 0.064 (2) 0.0017 (11) 0.0119 (14) 0.0069 (13) C2 0.0431 (13) 0.0412 (13) 0.0358 (14) −0.0098 (10) 0.0075 (10) −0.0022 (10) C3 0.0419 (13) 0.0369 (12) 0.0311 (13) 0.0077 (9) 0.0038 (10) −0.0009 (10) C4 0.0383 (12) 0.0342 (12) 0.0394 (14) −0.0035 (9) 0.0041 (10) 0.0000 (10) C5 0.0310 (11) 0.0334 (11) 0.0289 (12) 0.0019 (8) 0.0081 (9) 0.0004 (9) C6 0.0280 (11) 0.0316 (11) 0.0371 (14) 0.0013 (8) 0.0081 (9) −0.0036 (9) C7 0.0443 (13) 0.0368 (12) 0.0486 (16) 0.0118 (10) 0.0062 (11) 0.0015 (11) C8 0.0606 (16) 0.0306 (12) 0.0448 (16) 0.0032 (10) 0.0119 (12) −0.0033 (11) C9 0.0339 (12) 0.0390 (12) 0.0300 (13) 0.0015 (9) 0.0094 (9) −0.0002 (10) C10 0.0366 (13) 0.0334 (12) 0.0365 (14) 0.0016 (9) 0.0058 (10) −0.0027 (9) S1 0.0575 (4) 0.0399 (4) 0.0583 (5) −0.0095 (3) 0.0150 (3) 0.0073 (3) N1 0.0475 (12) 0.0419 (12) 0.0697 (17) −0.0015 (9) 0.0089 (11) 0.0107 (11) N2 0.0455 (11) 0.0354 (10) 0.0480 (13) −0.0044 (8) 0.0085 (9) 0.0048 (9) N3 0.0462 (12) 0.0434 (12) 0.0625 (15) 0.0023 (9) 0.0083 (11) 0.0072 (11) O1 0.0417 (9) 0.0391 (9) 0.0425 (10) 0.0032 (7) 0.0170 (7) 0.0082 (7) O1W 0.0381 (10) 0.0837 (14) 0.0524 (12) −0.0079 (9) 0.0140 (9) −0.0121 (11) O2 0.0315 (9) 0.0499 (10) 0.0589 (12) 0.0004 (7) 0.0068 (8) 0.0103 (8) O3 0.0374 (9) 0.0435 (9) 0.0558 (12) 0.0049 (7) 0.0108 (8) 0.0171 (8) O4 0.0406 (10) 0.0447 (10) 0.0747 (14) 0.0032 (7) 0.0163 (9) −0.0214 (9) O5 0.0411 (10) 0.0362 (9) 0.0954 (16) −0.0057 (7) 0.0137 (9) −0.0213 (10) Geometric parameters (Å, º) C1—N3 1.287 (3) C6—H6A 0.9800 C1—S1 1.715 (3) C7—C8 1.534 (3) C1—H1B 0.9300 C7—H7A 0.9700 C2—N2 1.320 (3) C7—H7B 0.9700 C2—N1 1.323 (3) C8—H8A 0.9700 C2—S1 1.725 (2) C8—H8B 0.9700 C3—O1 1.433 (3) C9—O3 1.212 (3) C3—C7 1.529 (3) C9—O2 1.311 (3) C3—C6 1.550 (3) C10—O4 1.205 (3) C3—H3A 0.9800 C10—O5 1.313 (3) Atomic displacement parameters (Å2) Geometric parameters (Å, º) sup-4 Acta Cryst. supporting information supporting informa 1.434 (3) N1—H1A 0.8600 1.530 (3) N1—H1C 0.8600 1.535 (3) N2—N3 1.381 (3) 0.9800 O1W—H1WA 0.859 (17) 1.508 (3) O1W—H1WB 0.819 (17) 1.558 (3) O2—H2A 0.8200 0.9800 O5—H5B 0.8200 1.507 (3) 115.3 (2) C5—C6—H6A 110.7 122.4 C3—C7—C8 101.18 (17) 122.4 C3—C7—H7A 111.5 122.5 (2) C8—C7—H7A 111.5 113.71 (18) C3—C7—H7B 111.5 123.80 (18) C8—C7—H7B 111.5 103.14 (18) H7A—C7—H7B 109.4 102.49 (17) C7—C8—C4 101.53 (18) 109.34 (19) C7—C8—H8A 111.5 113.6 C4—C8—H8A 111.5 113.6 C7—C8—H8B 111.5 113.6 C4—C8—H8B 111.5 102.67 (17) H8A—C8—H8B 109.3 102.77 (18) O3—C9—O2 122.9 (2) 108.84 (18) O3—C9—C5 121.68 (19) 113.8 O2—C9—C5 115.39 (19) 113.8 O4—C10—O5 123.7 (2) 113.8 O4—C10—C6 123.5 (2) 116.94 (17) O5—C10—C6 112.67 (18) 114.05 (17) C1—S1—C2 86.76 (12) 101.48 (17) C2—N1—H1A 120.0 108.0 C2—N1—H1C 120.0 108.0 H1A—N1—H1C 120.0 108.0 C2—N2—N3 111.89 (19) 108.99 (18) C1—N3—N2 112.4 (2) 115.12 (17) C3—O1—C4 96.42 (15) 100.22 (17) H1WA—O1W—H1WB 101 (2) 110.7 C9—O2—H2A 109.5 110.7 C10—O5—H5B 109.5 90.1 (2) C6—C5—C9—O3 −62.0 (3) −161.49 (19) C4—C5—C9—O2 2.8 (3) −34.65 (19) C6—C5—C9—O2 120.9 (2) 73.8 (2) C3—C6—C10—O4 65.8 (3) −86.2 (2) C5—C6—C10—O4 −45.8 (3) 164.91 (18) C3—C6—C10—O5 −110.5 (2) 35.06 (19) C5—C6—C10—O5 137.9 (2) −73.9 (2) N3—C1—S1—C2 0.4 (2) −10.1 (3) N2—C2—S1—C1 −0.1 (2) C4—O1 1.434 (3) N1—H1A 0.8600 C4—C5 1.530 (3) N1—H1C 0.8600 C4—C8 1.535 (3) N2—N3 1.381 (3) C4—H4A 0.9800 O1W—H1WA 0.859 (17) C5—C9 1.508 (3) O1W—H1WB 0.819 (17) C5—C6 1.558 (3) O2—H2A 0.8200 C5—H5A 0.9800 O5—H5B 0.8200 C6—C10 1.507 (3) N3—C1—S1 115.3 (2) C5—C6—H6A 110.7 N3—C1—H1B 122.4 C3—C7—C8 101.18 (17) S1—C1—H1B 122.4 C3—C7—H7A 111.5 N2—C2—N1 122.5 (2) C8—C7—H7A 111.5 N2—C2—S1 113.71 (18) C3—C7—H7B 111.5 N1—C2—S1 123.80 (18) C8—C7—H7B 111.5 O1—C3—C7 103.14 (18) H7A—C7—H7B 109.4 O1—C3—C6 102.49 (17) C7—C8—C4 101.53 (18) C7—C3—C6 109.34 (19) C7—C8—H8A 111.5 O1—C3—H3A 113.6 C4—C8—H8A 111.5 C7—C3—H3A 113.6 C7—C8—H8B 111.5 C6—C3—H3A 113.6 C4—C8—H8B 111.5 O1—C4—C5 102.67 (17) H8A—C8—H8B 109.3 O1—C4—C8 102.77 (18) O3—C9—O2 122.9 (2) C5—C4—C8 108.84 (18) O3—C9—C5 121.68 (19) O1—C4—H4A 113.8 O2—C9—C5 115.39 (19) C5—C4—H4A 113.8 O4—C10—O5 123.7 (2) C8—C4—H4A 113.8 O4—C10—C6 123.5 (2) C9—C5—C4 116.94 (17) O5—C10—C6 112.67 (18) C9—C5—C6 114.05 (17) C1—S1—C2 86.76 (12) C4—C5—C6 101.48 (17) C2—N1—H1A 120.0 C9—C5—H5A 108.0 C2—N1—H1C 120.0 C4—C5—H5A 108.0 H1A—N1—H1C 120.0 C6—C5—H5A 108.0 C2—N2—N3 111.89 (19) C10—C6—C3 108.99 (18) C1—N3—N2 112.4 (2) C10—C6—C5 115.12 (17) C3—O1—C4 96.42 (15) C3—C6—C5 100.22 (17) H1WA—O1W—H1WB 101 (2) C10—C6—H6A 110.7 C9—O2—H2A 109.5 C3—C6—H6A 110.7 C10—O5—H5B 109.5 O1—C4—C5—C9 90.1 (2) C6—C5—C9—O3 −62.0 (3) C8—C4—C5—C9 −161.49 (19) C4—C5—C9—O2 2.8 (3) O1—C4—C5—C6 −34.65 (19) C6—C5—C9—O2 120.9 (2) C8—C4—C5—C6 73.8 (2) C3—C6—C10—O4 65.8 (3) O1—C3—C6—C10 −86.2 (2) C5—C6—C10—O4 −45.8 (3) C7—C3—C6—C10 164.91 (18) C3—C6—C10—O5 −110.5 (2) O1—C3—C6—C5 35.06 (19) C5—C6—C10—O5 137.9 (2) C7—C3—C6—C5 −73.9 (2) N3—C1—S1—C2 0.4 (2) C9—C5—C6—C10 −10.1 (3) N2—C2—S1—C1 −0.1 (2) sup-5 Acta Cryst. Special details (2009). E65, o1590 supporting information supporting information (2009). E65, o1590 supporting information supporting information C4—C5—C6—C10 116.51 (19) N1—C2—S1—C1 179.7 (2) C9—C5—C6—C3 −126.86 (18) N1—C2—N2—N3 −179.9 (2) C4—C5—C6—C3 −0.22 (19) S1—C2—N2—N3 −0.1 (3) O1—C3—C7—C8 −34.6 (2) S1—C1—N3—N2 −0.5 (3) C6—C3—C7—C8 73.9 (2) C2—N2—N3—C1 0.4 (3) C3—C7—C8—C4 0.3 (2) C7—C3—O1—C4 56.17 (19) O1—C4—C8—C7 33.9 (2) C6—C3—O1—C4 −57.40 (18) C5—C4—C8—C7 −74.5 (2) C5—C4—O1—C3 57.35 (18) C4—C5—C9—O3 179.9 (2) C8—C4—O1—C3 −55.63 (18) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A N1—H1A···O4i 0.86 2.11 2.930 (3) 160 N1—H1C···N3ii 0.86 2.15 2.994 (3) 166 N1—H1C···N2ii 0.86 2.69 3.519 (3) 161 O2—H2A···O1Wiii 0.82 1.81 2.626 (2) 176 O5—H5B···N2iv 0.82 1.85 2.664 (2) 172 O1W—H1WA···O3v 0.86 (2) 1.91 (2) 2.766 (2) 175 (3) O1W—H1WB···O4vi 0.82 (2) 2.51 (3) 3.151 (3) 137 (3) O1W—H1WB···O1vi 0.82 (2) 2.55 (3) 3.061 (3) 122 (3) Symmetry codes: (i) x+1/2, −y−1/2, z+1/2; (ii) x+1, y, z; (iii) −x+1/2, y−1/2, −z+1/2; (iv) x−1/2, −y−1/2, z−1/2; (v) −x−1/2, y+1/2, −z+1/2; (vi) −x, −y, −z. Hydrogen-bond geometry (Å, º) sup-6 Acta Cryst. (2009). E65, o1590
https://openalex.org/W2890413967	https://pure.eur.nl/ws/files/48131039/REPUB_111856-OA.pdf	English	null	Bloody Zebrafish: Novel Methods in Normal and Malignant Hematopoiesis	Frontiers in cell and developmental biology	2,018	cc-by	8,809	Bloody Zebraﬁsh: Novel Methods in Normal and Malignant Hematopoiesis Emma de Pater1* and Eirini Trompouki2* 1 Department of Hematology, Erasmus MC, Rotterdam, Netherlands, 2 Department of Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg im Breisgau, Germany Hematopoiesis is an optimal system for studying stem cell maintenance and lineage differentiation under physiological and pathological conditions. In vertebrate organisms, billions of differentiated hematopoietic cells need to be continuously produced to replenish the blood cell pool. Disruptions in this process have immediate consequences for oxygen transport, responses against pathogens, maintenance of hemostasis and vascular integrity. Zebraﬁsh is a widely used and well-established model for studying the hematopoietic system. Several new hematopoietic regulators were identiﬁed in genetic and chemical screens using the zebraﬁsh model. Moreover, zebraﬁsh enables in vivo imaging of hematopoietic stem cell generation and differentiation during embryogenesis, and adulthood. Finally, zebraﬁsh has been used to model hematopoietic diseases. Recent technological advances in single-cell transcriptome analysis, epigenetic regulation, proteomics, metabolomics, and processing of large data sets promise to transform the current understanding of normal, abnormal, and malignant hematopoiesis. In this perspective, we discuss how the zebraﬁsh model has proven beneﬁcial for studying physiological and pathological hematopoiesis and how these novel technologies are transforming the ﬁeld. Hematopoiesis is an optimal system for studying stem cell maintenance and lineage differentiation under physiological and pathological conditions. In vertebrate organisms, billions of differentiated hematopoietic cells need to be continuously produced to replenish the blood cell pool. Disruptions in this process have immediate consequences for oxygen transport, responses against pathogens, maintenance of hemostasis and vascular integrity. Zebraﬁsh is a widely used and well-established model for studying the hematopoietic system. Several new hematopoietic regulators were identiﬁed in genetic and chemical screens using the zebraﬁsh model. Moreover, zebraﬁsh enables in vivo imaging of hematopoietic stem cell generation and differentiation during embryogenesis, and adulthood. Finally, zebraﬁsh has been used to model hematopoietic diseases. Recent technological advances in single-cell transcriptome analysis, epigenetic regulation, proteomics, metabolomics, and processing of large data sets promise to transform the current understanding of normal, abnormal, and malignant hematopoiesis. In this perspective, we discuss how the zebraﬁsh model has proven beneﬁcial for studying physiological and pathological hematopoiesis and how these novel technologies are transforming the ﬁeld. Edited by: Masatake Osawa, Gifu University, Japan Edited by: Masatake Osawa, Gifu University, Japan Reviewed by: Veronica Ramos-Mejia, Centro Pﬁzer-Universidad de Granada-Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), Spain Toshiyuki Yamane, Mie University, Japan Keywords: zebraﬁsh, hematopoiesis, next generation sequencing, hematopoietic (stem) cells, technology *Correspondence: Emma de Pater e.depater@erasmusmc.nl Eirini Trompouki trompouki@ie-freiburg.mpg.de INTRODUCTION Over the past three decades, zebraﬁsh has been established as an important model to study various biological processes during development and homeostasis, including hematopoiesis. Many attractive features underpin the success of zebraﬁsh as a model for vertebrate hematopoiesis. Cell-intrinsic and -extrinsic signaling mechanisms in hematopoiesis are well conserved between zebraﬁsh and mammals, with the exception of a few hematopoietic niche components (Liao et al., 1998; Murayama et al., 2006; Bertrand and Traver, 2009; Paik and Zon, 2010; Goessling and North, 2011; Zhang and Liu, 2011; Zhang et al., 2013; Frame et al., 2017; Nik et al., 2017; Gore et al., 2018). Moreover, zebraﬁsh embryos are small and transparent so they are ideal for imaging and easy to manipulate, at low cost. Additionally, genetic manipulation is easy and population studies can be easily performed in zebraﬁsh. Thus, zebraﬁsh have become invaluable vertebrate models for robust large-scale genetic screens (Mullins et al., 1994; Driever et al., 1996; Amsterdam et al., 1999) and, more recently, high-throughput chemical compound screens (North et al., 2007; Yeh et al., 2009). However, there are certain disadvantages in the zebraﬁsh model. For example, zebraﬁsh is not a mammal, but rather a poikilothermic animal in which the development of embryos occurs outside of the animal body and without placenta. That may lead to many metabolic and other diﬀerences between zebraﬁsh and mammals, including drug action and utilization. Finally, the Specialty section: This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology Received: 16 July 2018 Accepted: 10 September 2018 Published: 15 October 2018 Citation: de Pater E and Trompouki E (2018) Bloody Zebraﬁsh: Novel Methods in Normal and Malignant Hematopoiesis. Front. Cell Dev. Biol. 6:124. doi: 10.3389/fcell.2018.00124 Specialty section: This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology Received: 16 July 2018 Accepted: 10 September 2018 Published: 15 October 2018 PERSPECTIVE published: 15 October 2018 doi: 10.3389/fcell.2018.00124 PERSPECTIVE Keywords: zebraﬁsh, hematopoiesis, next generation sequencing, hematopoietic (stem) cells, technology Lineage Differentiation: The Impact of Single-Cell RNA Sequencing The development of single-cell RNA-sequencing (scRNA- seq) revolutionized the way we understand hematopoiesis. As most cellular compartments have a certain degree of heterogeneity, with bulk RNA-seq one cannot distinguish between a small transcriptional diﬀerence in many cells, and a large transcriptional diﬀerence in a few cells. Several insightful reviews describe the diﬀerent methods used for single-cell RNA- seq (Kolodziejczyk et al., 2015; Ziegenhain et al., 2017; Dal Molin and Di Camillo, 2018). Zebraﬁsh has been extensively used for modeling human hematopoietic disease, including anemia, thrombocytopenia, bone marrow failure syndromes, leukemia, and lymphoma (Taylor and Zon, 2011; Kwan and North, 2017; Potts and Bowman, 2017; Gore et al., 2018). The ﬁrst transplantable leukemia modeled in zebraﬁsh was T-cell acute lymphoblastic leukemia (T-ALL), which was induced by T cell-speciﬁc c-Myc overexpression (Langenau et al., 2003). Thereafter, several models of myelodysplastic syndromes and myeloproliferative neoplasms have been described (Le et al., 2007; He et al., 2014; Gjini et al., 2015; Peng et al., 2015). In zebraﬁsh, one of the ﬁrst methods used to characterize the transcriptome of single cells was massive parallel qPCR, where up to 96 transcripts could be analyzed in great sequencing depth using the Fluidigm system. This method revealed two distinct sub-populations of HSPCs in the CD41-GFP low-expressing stem cell compartment of the adult kidney marrow. Moreover, by using this technique and genetic ablation of T cells, a previously uncharacterized hematopoietic cytotoxic T/NK cell population in zebraﬁsh was uncovered (Moore et al., 2016). Although for many years zebraﬁsh was mainly used to study embryonic and larval developmental hematopoiesis, recent technological advances have transformed the ﬁeld. In this perspective, we will brieﬂy discuss how research using zebraﬁsh genetic models in combination with chemical screens, high- end imaging, and genome-wide molecular, metabolics and proteomics approaches has contributed to our understanding of hematopoiesis. Recent technological advances in scRNA-seq have enabled analyses without restriction to speciﬁc transcripts. A re- examination of the CD41-GFPlow population revealed four HSPC sub-populations with diﬀerent cellular characteristics and potential novel markers for HSCs were uncovered. Citation: de Pater E and Trompouki E (2018) Bloody Zebraﬁsh: Novel Methods in Normal and Malignant Hematopoiesis. October 2018 \| Volume 6 \| Article 124 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 1 Zebraﬁsh Hematopoiesis Meets Technology de Pater and Trompouki zebraﬁsh genome is duplicated and thus many genes have paralogs and homologs that make the otherwise easy genetic manipulation complicated (Glasauer and Neuhauss, 2014). hematopoiesis (Travnickova et al., 2015). Where transient and rapid cell-interactions occur, light-sheet microscopy, SPIM (selective plane illumination) or spinning disk microscopy can be used to visualize these processes in vivo in embryos and adults, because these systems record time-lapse 3D ﬂuorescent images 100–1000x faster than conventional confocal microscopy (Inoue and Inoue, 1996; Huisken et al., 2004; Arrenberg et al., 2010). Moreover, transparent adult zebraﬁsh models (White et al., 2008) have enabled the imaging of adult hematopoiesis, thereby opening the way to research exploring diﬀerent HSC niche components and HSC-niche interactions in the adult kidney marrow, thymus, and spleen. Embryonic hematopoiesis in zebraﬁsh is a multistep process occurring in a spatially restricted manner in three distinct waves. During the intraembryonic primitive wave, the medial and anterior lateral mesoderm give rise to erythroid and myeloid cells, respectively. Erythro-myeloid progenitors (EMPs) form in the posterior blood island (PBI) during a transient intermediate wave. Finally, during the deﬁnitive wave, hematopoietic stem cells (HSC) with multilineage capacity originate in the aorta- gonad-mesonephros (AGM) region. The HSCs then translocate to and expand in the caudal hematopoietic tissue (CHT), which is followed by the colonization of the kidney and the thymus (Figure 1). Interestingly, it was recently discovered that HSC- independent T-cells can originate from the AGM and PBI during the embryonic and larval stages of development (Tian et al., 2017). Lineage Differentiation: The Impact of Single-Cell RNA Sequencing Importantly, some cells in these subpopulations expressed the thrombocyte diﬀerentiation program long before they would have been characterized as thrombocytes, showing that there is an early lineage bias (Guo et al., 2013; Buenrostro et al., 2015; Paul et al., 2015; Drissen et al., 2016; Grover et al., 2016; Nestorowa et al., 2016; Olsson et al., 2016; Alberti-Servera et al., 2017; Velten et al., 2017; Villani et al., 2017; Buenrostro et al., 2018; Dahlin et al., 2018). In addition, scRNA-seq analyses in various transgenic lines revealed that ribosomal genes and lineage regulators control hematopoietic diﬀerentiation (Athanasiadis et al., 2017) and uncovered several novel hematopoietic populations, including two new types of NK cells (Tang et al., 2017). Finally, elegant comparative evolutionary studies on LCK-GFP transgenic zebraﬁsh and mammals showed that membrane proteins are less conserved in NK cells than in T cells (Carmona et al., 2017). In the pathological context, scRNA-seq analysis of Myc-induced T-ALLs demonstrated that few cells expressed an immature stem cell program, suggesting that only a small proportion of leukemia Imaging the Origin of Hematopoiesis cells promote the disease. This is remarkable as a single transgenic approach was used to initiate leukemogenesis and all leukemia cells overexpress Myc (Moore et al., 2016). Imaging the Origin of Hematopoiesis The transparency and accessibility of zebraﬁsh embryos was pivotal to collect evidence showing that hematopoietic stem and progenitor cells (HSPCs) emerge from the ventral wall of the dorsal aorta in vivo (Bertrand et al., 2010; Kissa and Herbomel, 2010). Moreover, high-end imaging techniques in zebraﬁsh embryos uncovered the mechanisms of thymus development (Hess and Boehm, 2012) and revealed that HSPCs are ampliﬁed and interact with endothelial cells in the CHT (Tamplin et al., 2015). Multiple signaling pathways and cell-interactions aﬀect HSPC emergence. For instance, inﬂammatory signaling provided by neutrophils is required for HSC generation (Espin-Palazon et al., 2014; Li et al., 2014; Sawamiphak et al., 2014; He et al., 2015). These unique properties of zebraﬁsh allowed to uncover the role of macrophages in HPSC mobilization and deﬁnitive October 2018 \| Volume 6 \| Article 124 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 2 Zebraﬁsh Hematopoiesis Meets Technology de Pater and Trompouki FIGURE 1 \| (A) Overview of deﬁnitive hematopoietic sites in the developing embryo where HSPCs are born from hemogenic endothelial cells of the dorsal aorta (DA). HSPCs are ampliﬁed in the caudal hematopoietic tissue (CHT) and migrate to the kidney and thymus. (B) Endothelial-to-hematopoietic transition (EHT) event imaged in a Tg(ﬂi:GFP) embryo between 32 and 40 hpf. (C) CHT region in Tg(ﬂt:RFP)/Tg(CD41:GFP) embryo at 56 hpf indicating erythroid myeloid progenitors (EMP) in green and deﬁnitive HSPCs in yellow as they originate from the artery and retain RFP at this timepoint. (D) Sagittal section through an adult zebraﬁsh where the head kidney is indicated with enlargement showing the hematopoietic cells in between the kidney tubules. FIGURE 1 \| (A) Overview of deﬁnitive hematopoietic sites in the developing embryo where HSPCs are born from hemogenic endothelial cells of the dorsal aorta (DA). HSPCs are ampliﬁed in the caudal hematopoietic tissue (CHT) and migrate to the kidney and thymus. (B) Endothelial-to-hematopoietic transition (EHT) event imaged in a Tg(ﬂi:GFP) embryo between 32 and 40 hpf. (C) CHT region in Tg(ﬂt:RFP)/Tg(CD41:GFP) embryo at 56 hpf indicating erythroid myeloid progenitors (EMP) in green and deﬁnitive HSPCs in yellow as they originate from the artery and retain RFP at this timepoint. (D) Sagittal section through an adult zebraﬁsh where the head kidney is indicated with enlargement showing the hematopoietic cells in between the kidney tubules. a method that combines traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction (Junker et al., 2014). Lineage Tracing Additionally, labeling with CRISPR/Cas9 scarring in embryos and tracing of unique hematopoietic clones into adulthood has revealed that the hematopoietic system is only generated from a handful of cells present at dome stage (Alemany et al., 2018). This study claimed that all clones contribute to all blood lineages, a subject that is controversial in mammalian studies (Yamamoto et al., 2013; Notta et al., 2016; Pei et al., 2017). Chemical Screens to Identify Regulators of Normal and Abnormal Hematopoiesis Zebraﬁsh is an ideal vertebrate model system to conduct bio- reactive compound screens (Zon and Peterson, 2005; Cusick et al., 2012; Tamplin et al., 2012; Veinotte et al., 2014; Rennekamp and Peterson, 2015; Deveau et al., 2017). The animals are small-sized and lay hundreds of eggs that develop very rapidly, thereby allowing the monitoring of compound activity and biotoxicity in vivo across development. Such screens have led to the identiﬁcation of prostaglandin E2 as a compound that increases HSC production (North et al., 2007). Prostaglandin E2 is currently being investigated for HSC expansion applications in human and non-human primates (Goessling et al., 2011; Cutler et al., 2013). A diﬀerent approach for lineage-tracing cells consists of performing high-throughput scRNAseq at various developmental stages and then mapping similarities in transcriptional proﬁles across a pseudo timescale of diﬀerentiation (Macosko et al., 2015). By using this method in early embryogenesis, two independent studies have described gradually divergent diﬀerentiation patterns for speciﬁc lineages and uncovered signaling networks required for zebraﬁsh development (Farrell et al., 2018; Wagner et al., 2018). Important insights into the molecular regulation of T-ALL came from zebraﬁsh studies where immature T cells served as models for T-ALL cells. By screening small molecules for an eﬀect on immature T cells using LCK-GFP transgenic zebraﬁsh, a novel compound, 1H-indole-3-carbaldehyde quinolin-8-yl-hydrazone, named Lenaldekar, was identiﬁed with the potential to speciﬁcally attack T-ALL cells (Ridges et al., 2012). Lenaldekar also has a potential eﬀect against autoimmune diseases such as multiple sclerosis, as they are caused by an oﬀ-target activity of T cells (Cusick et al., 2012). Currently there are ongoing clinical trials to study the eﬀectiveness of this promising compound. These examples highlight the power of zebraﬁsh models for screening novel chemical compounds aﬀecting normal, abnormal or malignant hematopoiesis (Shaﬁzadeh et al., 2004; Yeh et al., 2009; Future studies combining scRNAseq with lineage tracing will be paramount to advance our understanding of the developmental origins of hematopoietic populations. Lineage Tracing g g Zebraﬁsh has traditionally been utilized to lineage-trace diﬀerentiation during embryonic stages by labeling single cells with dyes and following them throughout development, until the dye fades or dilutes. However, the recent development of complex genetic models has removed this time restriction and enabled lineage tracing from the embryo into adulthood. For instance, HSPCs generated from the hemogenic endothelium of the aorta have been lineage-traced by using the multicolor transgenic labeling system “blood bow” (Henninger et al., 2017) in combination with high-end imaging and ﬂuorescence-activated cell sorting (FACS). Additionally, labeling with CRISPR/Cas9 scarring in embryos and tracing of unique hematopoietic clones into adulthood has revealed that the hematopoietic system is only generated from a handful of cells present at dome stage (Alemany et al., 2018). This study claimed that all clones contribute to all blood lineages, a subject that is controversial in mammalian studies (Yamamoto et al., 2013; Notta et al., 2016; Pei et al., 2017). A diﬀerent approach for lineage-tracing cells consists of performing high-throughput scRNAseq at various developmental stages and then mapping similarities in transcriptional proﬁles across a pseudo timescale of diﬀerentiation (Macosko et al., 2015). By using this method in early embryogenesis, two independent studies have described gradually divergent diﬀerentiation patterns for speciﬁc lineages and uncovered signaling networks required for zebraﬁsh development (Farrell et al., 2018; Wagner et al., 2018). Future studies combining scRNAseq with lineage tracing will be paramount to advance our understanding of the developmental origins of hematopoietic populations. However, this approach has the important caveat that scRNA-seq does not provide topographic information for each individual cell. To overcome this limitation, the Van Oudernaarden and Bakkers laboratories have developed RNA-tomography (TOMOSEQ), Zebraﬁsh has traditionally been utilized to lineage-trace diﬀerentiation during embryonic stages by labeling single cells with dyes and following them throughout development, until the dye fades or dilutes. However, the recent development of complex genetic models has removed this time restriction and enabled lineage tracing from the embryo into adulthood. For instance, HSPCs generated from the hemogenic endothelium of the aorta have been lineage-traced by using the multicolor transgenic labeling system “blood bow” (Henninger et al., 2017) in combination with high-end imaging and ﬂuorescence-activated cell sorting (FACS). Frontiers in Cell and Developmental Biology \| www.frontiersin.org Effects of Perturbations in Embryonic HSC Generation and Adult Hematopoiesis Several acute myeloid leukemia (AML) predisposition syndromes are caused by innate mutations in transcription factors that aﬀect embryonic hematopoiesis, such as Gata2 and Runx1 (Babushok et al., 2016), suggesting that perturbations in embryonic hematopoiesis aﬀect the adult HSC compartment. As the eﬀects of alterations in embryonic hematopoiesis can be easily monitored in zebraﬁsh throughout development, as well as during adulthood, this is an excellent system to study AML predisposition syndromes. Until the recent development of targeted gene editing, manipulating the zebraﬁsh genome to create speciﬁc mutations for making knockout and knockin animals was challenging. Although TILLING (Targeting Induced Local Lesions in Genomes) was a signiﬁcant advancement, this is a costly method that requires thousands of ﬁsh to search for a STOP codon in the gene of interest. Moreover, TILLING is rather limiting as it does not allow to induce speciﬁc mutations (Wienholds et al., 2003; Draper et al., 2004). Targeting the zebraﬁsh genome with zinc-ﬁnger nucleases was the beginning of a new era in zebraﬁsh biology, as selected genes could ﬁnally be speciﬁcally targeted for genome editing (Amacher, 2008; Foley et al., 2009). Shortly after this technology was introduced, TALENS (Dahlem et al., 2012; Hwang et al., 2014; Huang et al., 2016; Liu et al., 2016), and more recently, CRISPR/Cas9 (Hruscha et al., 2013; Irion et al., 2014; Shah et al., 2015; Li et al., 2016; Liu et al., 2017) were developed. Whilst it is relatively easy to generate knockouts and large deletions with these gene-targeting techniques, making knockin animals remains challenging. Nevertheless, several laboratories have successfully created knockin animals by using CRISPR/Cas9 and co-injecting a repair template to facilitate homology-directed repair (Hruscha et al., 2013; Auer et al., 2014; Albadri et al., 2017; Kesavan et al., 2017). Additionally, Cre/lox, Flp/FRT, and 8C31 systems are also currently being used in zebraﬁsh for precise genome editing (Mosimann et al., 2013; Felker and Mosimann, 2016; Carney and Mosimann, 2018). Importantly, tissue-speciﬁc expression of Cas9 in the hematopoietic system can be performed in zebraﬁsh to enable conditional manipulation of hematopoietic cells (Ablain et al., 2015). A major caveat in both perturbing the zebraﬁsh genome and comparing the zebraﬁsh with the mammalian transcriptome in the context of clinical translation, is, as previously mentioned, the Teleost genome duplication (Glasauer and Neuhauss, 2014). As a result most genes are present twice with (partially) redundant biological roles. Lineage Tracing However, this approach has the important caveat that scRNA-seq does not provide topographic information for each individual cell. To overcome this limitation, the Van Oudernaarden and Bakkers laboratories have developed RNA-tomography (TOMOSEQ), October 2018 \| Volume 6 \| Article 124 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3 Zebraﬁsh Hematopoiesis Meets Technology de Pater and Trompouki Epigenetic Regulation of the Hematopoietic System Paik et al., 2010; Gutierrez et al., 2014; Arulmozhivarman et al., 2016). Paik et al., 2010; Gutierrez et al., 2014; Arulmozhivarman et al., 2016). Future studies addressing malignancy heterogeneity may combine chemical screens with scRNAseq to identify therapy- resistant cells and explore the mechanisms underpinning resistance to treatment in individual cells, a fundamental unresolved question in the cancer research ﬁeld. Chromatin conformation is essential for controlling gene expression, and deregulation of this process may cause malignant transformation (Groschel et al., 2014). Zebraﬁsh is an excellent system to explore the mechanisms underlying chromatin regulation and to evaluate the eﬀects of chromatin-modifying drugs in vivo. Gene regulatory elements can be identiﬁed in zebraﬁsh using chromatin immunoprecipitation combined with sequencing (ChIP-seq), however, the technique is limited by the low number of zebraﬁsh-speciﬁc antibodies currently available and the large amount of input material required (Havis et al., 2006; Trompouki et al., 2011; Bogdanovic et al., 2013). ChIP- seq has been mostly used in early zebraﬁsh embryos (Paik et al., 2010; Vastenhouw et al., 2010; Bogdanovic et al., 2012; Xu et al., 2012; Winata et al., 2013; Nelson et al., 2017; Meier et al., 2018). Antibodies against histone marks, which are highly conserved between species, have been successfully utilized in zebraﬁsh erythrocytes to describe the potential locus control region (LCR) regulating globin expression (Ganis et al., 2012). Moreover, given the functional conservation of these genes, zebraﬁsh is useful to functionally validate enhancers identiﬁed in mouse and/or human models (Tijssen et al., 2011; Chiang et al., 2017). Effects of Perturbations in Embryonic HSC Generation and Adult Hematopoiesis This means that for a complete perturbation of a mammalian gene, the zebraﬁsh counterparts have to be removed both, complicating genetic crossings and analyses. Other techniques for identifying gene regulatory elements are based on the detection of open chromatin, for instance, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). ATAC-seq requires much less input material than ChIP-seq, and has even been used successfully with single cells (Fernandez-Minan et al., 2016; Doganli et al., 2017). This method allowed the identiﬁcation of endothelial enhancers (Quillien et al., 2017) and revealed the role of cohesin in rearranging the genomic architecture during the transition of maternal to zygotic transcription in early embryos (Meier et al., 2018). Combining scRNA-seq with ATAC-seq and immune phenotypic analysis is a powerful approach to integrate our understanding of lineage diﬀerentiation with the regulatory elements involved in that process (Buenrostro et al., 2018). DNA methylation studies can also be used to understand chromatin accessibility, although more material is needed in these methods. Methylation experiments have been conducted in zebraﬁsh albeit not speciﬁcally in the hematopoietic system (Lee et al., 2015; Kaaij et al., 2016). Since many tissue-speciﬁc ﬂuorescent lines exist in zebraﬁsh, future research should aim to identify enhancers and promoters in speciﬁc cell types, rather than using whole embryos. Despite the advantages of ATAC-seq and methylation analyses, these approaches cannot oﬀer the same information as ChIP-seq. Thus, improved ChIP-seq protocols, such as the high sensitivity indexing-ﬁrst chromatin immunoprecipitation approach (iChIP) developed in Ido Amit’s laboratory, should be adapted to zebraﬁsh (Gury-BenAri et al., 2016). Moreover, it would be important to unravel chromatin interactions in active enhancer and promoter regions during hematopoiesis. However, although chromatin conformation has been studied in early zebraﬁsh embryos (Gomez-Marin et al., 2015; Fernandez-Minan et al., 2016), to date no studies have addressed this question speciﬁcally in zebraﬁsh hematopoiesis. It is important to mention Frontiers in Cell and Developmental Biology \| www.frontiersin.org October 2018 \| Volume 6 \| Article 124 4 Zebraﬁsh Hematopoiesis Meets Technology de Pater and Trompouki FIGURE 2 \| Graph indicating different methods used to study zebraﬁsh hematopoiesis. FIGURE 2 \| Graph indicating different methods used to study zebraﬁsh hematopoiesis. has proven useful to understand the neurological damage resulting from chemical perturbations in zebraﬁsh embryos (Ong et al., 2009; Rabinowitz et al., 2017; Roy et al., 2017). 1https://danio-code.zﬁn.org Proteomics and Metabolomics Studies Proteomics and Metabolomics Studies In the era of genome-wide technology, gene expression studies should be complemented with proteomic studies, as transcriptional and translational outcomes can sometimes diﬀer. Additionally, the extension of these analyses to metabolomics may uncover another layer of regulation critical for hematopoiesis. Indeed, it was recently shown that dormant stem cell populations have low metabolic activity, and this is required to maintain the hematopoietic system during aging and periods of intense stress (Cabezas-Wallscheid et al., 2017). Although proteomics and metabolomics methods have not yet been extensively explored in zebraﬁsh, particularly in the hematopoietic system, some studies have reported diﬀerences between transcript and protein levels in multiple genes by using proteomic analyses either in whole zebraﬁsh embryos or in speciﬁc cell populations during regeneration (Alli Shaik et al., 2014; Baral et al., 2014; Rabinowitz et al., 2017). Metabolomics Effects of Perturbations in Embryonic HSC Generation and Adult Hematopoiesis Finally, as mass spectrometry analyses are constantly improving, the sensitivity of these methods will likely overcome the current problem of heterogeneous and low cell-number populations. the combined eﬀort of many groups to collate all available genome-wide data in zebraﬁsh in the DANIO-CODE Data Coordination Center1 (Tan et al., 2016). This recently launched database will provide an easy access to high-quality genome data to all scientists. 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CONCLUSION The zebraﬁsh has become an invaluable model system for understanding how HSCs form and are maintained, and how hematopoietic cell diﬀerentiation is regulated during embryogenesis and in adulthood. The unique advantages oﬀered by this model system over traditional mouse models regarding the use in chemical screens and the accessibility during embryonic stages allowing easy manipulation and visualization and tracing into adult stages, in combination with recent new technologies (Figure 2), have opened the way for novel exciting hypotheses on the mechanisms promoting hematopoietic diseases, the role of the niche in normal and malignant hematopoiesis, and the eﬀect of chemical compounds on malignant cells. The high conservation between the zebraﬁsh and human hematopoietic systems means that discoveries in ﬁsh may have strong translational potential and important clinical implications for the treatment of hematopoietic diseases. October 2018 \| Volume 6 \| Article 124 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 5 Zebraﬁsh Hematopoiesis Meets Technology de Pater and Trompouki FUNDING We thank Dr. I. P. Touw for careful reading of the manuscript, Dr. van Royen, E. Gioacchino, J. Peulen for graphical contributions and Dr. T. Clapes for producing the second ﬁgure. We thank Dr. I. P. Touw for careful reading of the manuscript, Dr. van Royen, E. Gioacchino, J. Peulen for graphical contributions and Dr. T. Clapes for producing the second ﬁgure. EdP was supported by EHA junior non-clinical research fellowship and by KWF/Alpe’dHuzes (SK10321). ET was supported by the Max Planck Society, a Marie Curie Career Integration Grant (631432 Bloody Signals), the Deutsche EdP was supported by EHA junior non-clinical research fellowship and by KWF/Alpe’dHuzes (SK10321). 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Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464, 112–115. doi: 10. 1038/nature08761 Gore, A. V., Pillay, L. M., Venero Galanternik, M., and Weinstein, B. M. (2018). The zebraﬁsh: a ﬁntastic model for hematopoietic development and disease. Wiley Interdiscip. Rev. Dev. Biol. 7:e312. doi: 10.1002/wdev.312 Kolodziejczyk, A. A., Kim, J. K., Svensson, V., Marioni, J. C., and Teichmann, S. A. (2015). The technology and biology of single-cell RNA sequencing. Mol. Cell. 58, 610–620. doi: 10.1016/j.molcel.2015.04.005 Groschel, S., Sanders, M. A., Hoogenboezem, R., de Wit, E., Bouwman, B. A. M., Erpelinck, C., et al. (2014). A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia. Cell 157, 369–381. doi: 10.1016/j.cell.2014.02.019 Kwan, W., and North, T. E. (2017). Netting novel regulators of hematopoiesis and hematologic malignancies in zebraﬁsh. Curr. Top. Dev. Biol. 124, 125–160. doi: 10.1016/bs.ctdb.2016.11.005 Langenau, D. M., Traver, D., Ferrando, A. A., Kutok, J. L., Aster, J. C., Kanki, J. P., et al. (2003). Myc-induced T cell leukemia in transgenic zebraﬁsh. Science 299, 887–890. doi: 10.1126/science.1080280 Grover, A., Sanjuan-Pla, A., Thongjuea, S., Carrelha, J., Giustacchini, A., Gambardella, A., et al. (2016). Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells. Nat. Commun. 7:11075. doi: 10.1038/ncomms11075 Le, X., Langenau, D. M., Keefe, M. D., Kutok, J. L., Neuberg, D. S., and Zon, L. I. (2007). Heat shock-inducible Cre/Lox approaches to induce diverse types of October 2018 \| Volume 6 \| Article 124 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 7 Zebraﬁsh Hematopoiesis Meets Technology de Pater and Trompouki tumors and hyperplasia in transgenic zebraﬁsh. Proc. Natl. Acad. Sci. U.S.A. 104, 9410–9415. doi: 10.1073/pnas.0611302104 Paik, E. J., de Jong, J. L., Pugach, E., Opara, P., and Zon, L. I. (2010). A chemical genetic screen in zebraﬁsh for pathways interacting with cdx4 in primitive hematopoiesis. Zebraﬁsh 7, 61–68. REFERENCES R., Cvejic, A., Joshi, A., Hannah, R. L., Ferreira, R., Forrai, A., et al. (2011). Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL binding in megakaryocytes identiﬁes hematopoietic regulators. Dev. Cell 20, 597–609. doi: 10.1016/j.devcel.2011.04.008 Ong, E. S., Chor, C. F., Zou, L., and Ong, C. N. (2009). A multi-analytical approach for metabolomic proﬁling of zebraﬁsh (Danio rerio) livers. Mol. Biosyst. 5, 288–298. doi: 10.1039/b811850g October 2018 \| Volume 6 \| Article 124 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 8 Zebraﬁsh Hematopoiesis Meets Technology de Pater and Trompouki Travnickova, J., Tran Chau, V., Julien, E., Mateos-Langerak, J., Gonzalez, C., Lelievre, E., et al. (2015). Primitive macrophages control HSPC mobilization and deﬁnitive haematopoiesis. Nat. Commun. 6:6227. doi: 10.1038/ncomms7227 Xu, C., Fan, Z. P., Muller, P., Fogley, R., DiBiase, A., Trompouki, E., et al. (2012). Nanog-like regulates endoderm formation through the Mxtx2-Nodal pathway. Dev. Cell 22, 625–638. doi: 10.1016/j.devcel.2012.01.003 Yamamoto, R., Morita, Y., Ooehara, J., Hamanaka, S., Onodera, M., Rudolph, K. L., et al. (2013). Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells. Cell 154, 1112–1126. doi: 10.1016/j.cell.2013.08.007 Trompouki, E., Bowman, T. V., Dibiase, A., Zhou, Y., and Zon, L. I. (2011). Chromatin immunoprecipitation in adult zebraﬁsh red cells. Methods Cell Biol. 104, 341–352. doi: 10.1016/B978-0-12-374814-0.00019-7 Yeh, J. R., Munson, K. M., Elagib, K. E., Goldfarb, A. N., Sweetser, D. A., and Peterson, R. T. (2009). Discovering chemical modiﬁers of oncogene- regulated hematopoietic diﬀerentiation. Nat. Chem. Biol. 5, 236–243. doi: 10. 1038/nchembio.147 Vastenhouw, N. L., Zhang, Y., Woods, I. G., Imam, F., Regev, A., Liu, X. S., et al. (2010). Chromatin signature of embryonic pluripotency is established during genome activation. Nature 464, 922–926. doi: 10.1038/nature08866 Veinotte, C. J., Dellaire, G., and Berman, J. N. (2014). Hooking the big one: the potential of zebraﬁsh xenotransplantation to reform cancer drug screening in the genomic era. Dis. Model. Mech. 7, 745–754. doi: 10.1242/dmm.015784 Zhang, C., Patient, R., and Liu, F. (2013). Hematopoietic stem cell development and regulatory signaling in zebraﬁsh. Biochim. Biophys. Acta 1830, 2370–2374. doi: 10.1016/j.bbagen.2012.06.008 Velten, L., Haas, S. F., Raﬀel, S., Blaszkiewicz, S., Islam, S., Hennig, B. P., et al. (2017). Human haematopoietic stem cell lineage commitment is a continuous process. Nat. Cell Biol. 19, 271–281. doi: 10.1038/ncb3493 Zhang, P., and Liu, F. (2011). In vivo imaging of hematopoietic stem cell development in the zebraﬁsh. Front. Med. 5:239–247. doi: 10.1007/s11684-011- 0123-0 Villani, A. Frontiers in Cell and Developmental Biology \| www.frontiersin.org October 2018 \| Volume 6 \| Article 124 REFERENCES C., Satija, R., Reynolds, G., Sarkizova, S., Shekhar, K., Fletcher, J., et al. (2017). Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science 356:eaah4573. doi: 10.1126/science. aah4573 Ziegenhain, C., Vieth, B., Parekh, S., Reinius, B., Guillaumet-Adkins, A., Smets, M., et al. (2017). Comparative analysis of single-cell RNA sequencing methods. Mol. Cell. 65, 631–643e4. doi: 10.1016/j.molcel.2017.01.023 Cell. 65, 631–643e4. doi: 10.1016/j.molcel.2017.01.023 Wagner, D. E., Weinreb, C., Collins, Z. M., Briggs, J. A., Megason, S. G., and Klein, A. M. (2018). Single-cell mapping of gene expression landscapes and lineage in the zebraﬁsh embryo. Science 360, 981–987. doi: 10.1126/science.aar4362 Zon, L. I., and Peterson, R. T. (2005). In vivo drug discovery in the zebraﬁsh. Nat. Rev. Drug Discov. 4, 35–44. doi: 10.1038/nrd1606 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. 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. White, R. M., Sessa, A., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., et al. (2008). Transparent adult zebraﬁsh as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183–189. doi: 10.1016/j.stem.2007.11.002 Wienholds, E., van Eeden, F., Kosters, M., Mudde, J., Plasterk, R. H., and Cuppen, E. (2003). Eﬃcient target-selected mutagenesis in zebraﬁsh. Genome Res. 13, 2700–2707. doi: 10.1101/gr.1725103 Copyright © 2018 de Pater and Trompouki. 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. Winata, C. L., Kondrychyn, I., Kumar, V., Srinivasan, K. G., Orlov, Y., Ravishankar, A., et al. (2013). Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage speciﬁc developmental genes in zebraﬁsh. PLoS Genet. 9:e1003852. doi: 10.1371/journal.pgen.1003852 October 2018 \| Volume 6 \| Article 124 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 9
https://openalex.org/W2090243137	https://europepmc.org/articles/pmc3912888?pdf=render	English	null	Isolated Fracture of the Coracoid Process	Case reports in orthopedics	2,014	cc-by	1,807	Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2014, Article ID 482130, 3 pages http://dx.doi.org/10.1155/2014/482130 Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2014, Article ID 482130, 3 pages http://dx.doi.org/10.1155/2014/482130 Ali Güleç,1 Harun Kütahya,2 Recep Gani Göncü,3 and Serdar Toker4 1 Department of Orthopedics and Traumatology, Konya Training and Research Hospital, Konya, Turkey 2 Department of Orthopedics and Traumatology, Konya Beyhekim State Hospital, Selc¸uklu, 42100 Konya, Turkey 3 Department of Orthopedics and Traumatology, Mevlana University Medical Faculty, Konya, Turkey 4Department of Orthopedics and Traumatology, Meram Medical School, Necmettin Erbakan University, Konya, Turkey Correspondence should be addressed to Harun K¨utahya; drharunkutahya@gmail.com Received 8 November 2013; Accepted 4 December 2013; Published 6 January 2014 Academic Editors: T. Tsurumoto and T. Yasuda Copyright © 2014 Ali G¨ulec¸ 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. Coracoid fractures are rarely seen fractures. In the shoulder girdle, coracoid process fractures generally accompany dislocation of the acromioclavicular joint or glenohumeral joint, scapula corpus, clavicula, humerus fracture, or rotator cuff tear. Coracoid fractures can be missed and the treatment for coracoid process fractures is still controversial. In this paper, a 34-year-old male manual labourer presented to the emergency department with complaints of pain and restricted movement in the left shoulder following a traffic accident. On direct radiographs and computerised tomography images a fragmented fracture was observed on the base of the coracoid process. In addition to the coracoid fracture, a mandibular fracture was determined. The patient was admitted for surgery on both fractures. After open reduction, fixation was made with a 3.5 mm cannulated screw and washer. At the postoperative 6th week, bone union was determined. The patient returned to his previous occupation pain-free and with a full range of joint movement. In conclusion, in the current case of isolated fragmented coracoid process fracture showing minimal displacement in a patient engaged in heavy manual work, surgery was preferred as it was thought that nonunion might be encountered particularly because of the effect of forces around the coracoid. 1. Introduction the physical examination, ecchymosis and sensitivity in the left shoulder, restricted shoulder movements, and sensitivity in the jaw were determined. The results of the neurovascular examination were normal. On direct radiographs and com- puterised tomography (CT) images a fragmented fracture was observed on the base of the coracoid process (Figures 1 and 2). In addition to the coracoid fracture, a mandibular fracture was determined. The patient was admitted for surgery on both fractures. After making the incision along the Langer’s line on the coracoid process, the fracture line was reached. After open reduction, fixation was made with a 3.5 mm cannulated screw and washer. Postoperatively, the patient was followed up for 2 weeks with the application of a simple shoulder sling. Passive joint exercises were allowed in the first 2 weeks; from the 3rd postoperative week, active joint movement exercises were started and from the 5th week, shoulder strengthening exercises. At the postoperative 6th Coracoid fractures are rarely seen fractures [1]. In the shoul- der girdle, coracoid process fractures generally accompany dislocation of the acromioclavicular (AC) joint or gleno- humeral joint, scapula corpus fracture, clavicular fracture, humerus proximal end fracture, or rotator cuff tear [2]. Coracoid fractures can be missed and the treatment for coracoid process fractures is still controversial. The case presented here is of an isolated coracoid process fracture treated surgically. 2. Case Report A 34-year-old male manual labourer presented at the Emer- gency Department with complaints of pain and restricted movement in the left shoulder following a traffic accident. In 2 Case Reports in Orthopedics (a) (a) (b) Figure 3: Postoperative 6th week X-ray images (fracture fixed bicortical). Figure 1: Preoperative computerised tomography (CT) image. (a) (b) Figure 1: Preoperative computerised tomography (CT) image. Figure 2: Preoperative 3-dimensional CT image. (b) Figure 3: Postoperative 6th week X-ray images (fracture fixed bicortical). Figure 2: Preoperative 3-dimensional CT image. An important point related to coracoid fractures is the neurological injuries which may accompany the fracture. Neer stated that in fractures involving the coracoid process in particular, there may be brachial plexus pressure and suprascapular nerve paralysis and therefore evaluation with electromyography prior to exploration is recommended [10]. week, bone union was determined (Figure 3). The patient returned to his previous occupation pain free and with a full range of joint movement. 3. Discussion Treatment of coracoid process fractures has not yet achieved clarity. The majority is preferably treated conser- vatively [6]. Previous studies on this subject have mostly been in the form of a case report or series of no relevance to others. The most extensive study in literature related to coracoid process fractures was conducted by Anavian et al. Surgery was applied to 14 coracoid process fractures of 26 patients including scapula process fractures and successful results were obtained for all patients [11]. Isolated coracoid fractures are seen extremely rarely. All coracoid process fractures constitute approximately 1% of all fractures and 2–13% of scapula fractures [3–5]. Fractures are often seen on the base of the coracoid process and are generally minimally displaced and together with AC joint injuries [6]. Coracoid fractures may be easily missed. Lal et al., in a case report where surgery had not been applied and Vaienti and Pogliacomi in a series of 9 cases with delayed diagnosis, applied conservative treatment and clearly demonstrated this situation [7, 8]. In cases which cannot be determined with direct radiographs, the use of CT may be necessary. In a study by Botchu et al. of 7 cases, it was shown that coracoid process fractures can be diagnosed with ultrasonography [9]. In the case presented here, diagnosis was made with CT. Indications for surgical treatment were accepted as painful nonunion, >1 cm displacement, concomitant scapula fracture on the same side and the presence of superior shoul- der suspensory complex injuries. In a study by Lal and Bansal of 22 patients, all with coracoid fractures and various shoul- der girdle injuries, 10 patients were treated conservatively and nonunion was encountered in 1 patient [8]. Spormann 3 Case Reports in Orthopedics fracture: a case report,” Journal of Orthopaedic Surgery, vol. 20, no. 1, pp. 121–125, 2012. et al. operated on 3 cases of isolated coracoid process fracture and obtained successful results [12]. Again successful results were obtained from surgical treatment applied by Subramanian et al. of an isolated coracoid fracture in an unstable shoulder [13]. Garcia-Elias and Salo applied excision following shoulder dislocation and reported nonunion of the coracoid process [14]. In studies by Guttentag and Rechtine and Goos, conservative treatment was applied to coracoid fractures in athletes and patients engaged in heavy manual work and poor results were obtained [15, 16]. 3. Discussion In the current case, as the patient was a construction worker, surgery was preferred despite the minimal displacement and successful results were obtained.i [9] R. Botchu, K. J. Lee, and S. Bianchi, “Radiographically unde- tected coracoid fractures diagnosed by sonography. Report of seven cases,” Skeletal Radiology, vol. 41, no. 6, pp. 693–698, 2012. [10] C. S. Neer II, “Fractures about the shoulder,” in Fractures, C. A. Rockwood and D. P. Green, Eds., pp. 713–721, Lippincott, Philadephia, Pa, USA, 1984. [11] J. Anavian, C. A. Wijdicks, L. K. Schroder, S. Vang, and P. A. Cole, “Surgery for scapula process fractures: good outcome in 26 patients,” Acta Orthopaedica, vol. 80, no. 3, pp. 344–350, 2009. [12] C. Spormann, P. Holzach, and C. Ryf, “Open reduction and internal fixation of isolated fractures of the coracoid-process— presentation of three cases and review of the literature,” Swiss Surgery, vol. 4, no. 4, pp. 198–202, 1998. In coracoid fractures, surgical fixation can be applied with open reduction and with screws [12]. Even though the most frequently used method is the anterior approach, indirect reduction and fixation may be applied with a posterior approach [11]. In a study by Bhatia, fluoroscopy-guided percutaneous fixation was applied to a coracoid process fracture which was accompanied by AC joint dislocation [17]. In the current case, fixation was achieved with 1 screw and washer following open reduction with an anterior approach. [13] A. S. Subramanian, M. A. Khalik, and M. M. Shah, “Isolated fracture of the coracoid process associated with unstable shoul- der,” ANZ Journal of Surgery, vol. 77, no. 3, pp. 188–189, 2007. [14] M. Garcia-Elias and J. M. Salo, “Non-union of a fractured coracoid process after dislocation of the shoulder. A case report,” Journal of Bone and Joint Surgery B, vol. 67, no. 5, pp. 722–723, 1985. In conclusion, in the current case of isolated fragmented coracoid process fracture showing minimal displacement in a patient engaged in heavy manual work, surgery was preferred as it was thought that nonunion might be encountered par- ticularly because of the effect of forces around the coracoid. Although this is a rarely seen fracture, further multicentre, randomised controlled studies would give clearer ideas about the choice of treatment alternatives. [15] I. J. Guttentag and G. R. Rechtine, “Fractures of the scapula. A review of the literature,” Orthopaedic Review, vol. 17, no. 2, pp. 147–158, 1988. [16] T. P. 3. Discussion Goss, “The scapula: coracoid, acromial, and avulsion fractures,” American Journal of Orthopedics, vol. 25, no. 2, pp. 106–115, 1996. [17] D. N. Bhatia, “Orthogonal biplanar fluoroscopy-guided per- cutaneous fixation of a coracoid base fracture associated with acromioclavicular joint dislocation,” Techniques in Hand and Upper Extremity Surgery, vol. 16, no. 1, pp. 56–59, 2012. References [1] O. H. Petty, “Fracture of the coracoid process of the scapula caused by muscular action,” Annals of Surgery, vol. 45, no. 3, pp. 427–430, 1907. [2] K. Ogawa, A. Yoshida, M. Takahashi, and M. Ui, “Fractures of the coracoid process,” Journal of Bone and Joint Surgery B, vol. 79, no. 1, pp. 17–19, 1997. [3] J. R. Ada and M. E. Miller, “Scapular fractures: analysis of 113 cases,” Clinical Orthopaedics and Related Research, no. 269, pp. 174–180, 1991. [4] R. J. Imatani, “Fractures of the scapula: a review of 53 fractures,” Journal of Trauma, vol. 15, no. 6, pp. 473–478, 1975. [5] M. C. Wilber and E. B. Evans, “Fractures of the scapula. An analysis of forty cases and a review of the literature,” Journal of Bone and Joint Surgery A, vol. 59, no. 3, pp. 358–362, 1977. [6] K. P. Butters, “Fractures and dislocations of the scapula,” in Fractures in Adults, C. A. Rockwood Jr., D. P. Green, R. W. Bucholz, and J. D. Heckman, Eds., pp. 1163–1164, Lippincott- Raven, Philadephia, Pa, USA, 4th edition, 1996. [7] E. Vaienti and F. Pogliacomi, “Delayed diagnosis of isolated coracoid process fractures: results of 9 cases treated conserva- tively,” Acta Biomedica, vol. 83, no. 2, pp. 138–146, 2012. [8] H. Lal, P. Bansal, V. K. Sabharwal, L. Mawia, and D. Mittal, “Recurrent shoulder dislocations secondary to coracoid process
https://openalex.org/W2146559977	https://storage.googleapis.com/jnl-up-j-bha-files/journals/1/articles/250/submission/proof/250-1-1052-1-10-20111029.pdf	English	null	Bulletin of the Texas Archeological Society, Volume 71 (2000), Austin, Paper.	Bulletin of the history of archaeology	2,000	cc-by	618	Bulletin of the Texas Archeological Society, Volume 71 (2000), Austin, Paper. Bulletin of the Texas Archeological Society, Volume 71 (2000), Austin, Paper. Reviewed by Larry D. Banks Reviewed by Larry D. Banks This annual bulletin of the Texas Archeological Society is a unique contribution specifically focused upon the history of Texas archaeology in a fonnat that no others have done previollsly. The volume contains 150 pages, the majority of which consists of interviews (146 pages) conducted by the first State Archaeologist of Texas, Curtis Tunnel!. In 1968 Tunnell conceived of the idea of obtaining personal interviews from individuals whom he considered his heroes for their pioneering efforts in Texas archeol ogy. This volume entails the first publication of such infonnation, but morc will certainly follow. The remaining four pages comprise two different reviews of other publications important in their own right to those interested in Southern Plains archeology of Texas. These two reviews by Timothy K Pertulla and David T. Hughes, respectively, are of The Coronado Expedition to Tierra Nueva: The 1540·1542 Route Across the Southwest by Richard Flint and Shirley Cushing flint, and Gaff Creek: Artifact Collec· tion Strategy and Occupation Prehistory on the Southern High Plains, Texas County. Oklahoma. The section by TunneU titled "In Their Own Words: Stories from Some Pioneer Texas Arcbeologists" con· tains numerous previously unpublished photographs of people, sites and artifacts referred to the texts. The interviews are from twenty-three different people whose individual names are synonomous with the history of Texas archeology and with archeology on a national and international level. The list reads like a "Whose Who" for the development of archeology in Texas. Others of more recent influence include Fred Wended, Edward B. JeJks and Dee Ann Storey. The personal interviews are complemented in most cases with copies of newspaper articles, correspondence with others they considered important, and personal reminiscences of their contemporaries as well as stories about archeology itself. The period of time represented in the stories ranges from 1914 to the I99Os; but, of the twenty·three people represented. most of tbe interviews are from now deceased archaeologists, which makes these published stories that much more important. It provides an enlightenment of activities and roles of people in Texas archaeology that is not duplicated elsewhere. I highly recommend this volume for anyone interested in the history of archaeology and especially, of course, for those interested in Texas events. Reviewed by Robert C. Arkansas Archaeology. Essays in Honor of Dan and Phyllis Morse, edited by Robert C. Mainfort and Marvin D. Jeter, Fayetleville, University of Arkansas Press, 1999. xi, 324pp., paperback. ISBN 1-55728· 571-3. V. Book/Journal Article Reviews DOI: http://dx.doi.org/10.5334/bha.10203 Bulletin of the Texas Archeological Society, Volume 71 (2000), Austin, Paper. Dunnell This collection of ten essays honors the retirement of Dan and Phyllis Morse from the Northeast Re gional Office of the Arkansas Archeological Survey and the University of Arkansas system. A brief recounting of the honorees' professional lives is the subject of a well-written essay by Mary Kwas accompanied by a "selected" bibliography and a short collection of anecdotes. An environmental ac count of the central Mississippi valley from 16,000 BP to 1000 BP (radiocarbon or caJendric is signifi cant but not stated) by Paul and Hazel Delcourt and the late Roger Saucier follows. Constructed around Saucier's 1994 mapped reconstructions of hydrology/geology, it is f1eshed out by paleovegetation derived from eleven sites in the region, four new (but not described bere). The Holocene central valley is as well known now as any area south of the glacial border. Unfortunately. since the article is closely keyed to a series of maps, two of the maps, 2.2b and 2.2c, are mislabeled and likely to cause some initial confusion. -5-
https://openalex.org/W4296908073	https://biblio.ugent.be/publication/01GKRBYJ14Q9NR7GGXCXYC75Y4/file/01GKRBZMK4SZ3SVY8312H383Q2.pdf	English	null	Social network heterogeneity benefits individuals at the expense of groups in the creation of innovation	Journal of physics. Complexity	2,022	cc-by	8,847	This content was downloaded from IP address 78.23.3.4 on 08/12/2022 at 07:57 Journal of Physics: Complexity Journal of Physics: Complexity You may also like The openness condition for a coadjoint orbit projection of the semidirect product Lie group M((n, p), ) GL (n, ) E Kurniadi - Novel technologies and configurations of superconducting magnets for MRI Yuri Lvovsky, Ernst Wolfgang Stautner and Tao Zhang - Does trade openness explain the deviation of purchasing power parity and exchange rate movement? A L Ang, Y T Thum and S K Sek - PAPER • OPEN ACCESS PAPER • OPEN ACCESS Abstract Innovation is fundamental for development and provides a competitive advantage for societies. It is the process of creating more complex technologies, ideas, or protocols from existing ones. While innovation may be created by single agents (i.e. individuals or organisations), it is often a result of social interactions between agents exchanging and combining complementary expertise and perspectives. The structure of social networks impacts this knowledge exchange process. To study the role of social network structures on the creation of new technologies, we design an evolutionary mechanistic model combining self-creation and social learning. We ﬁnd that social heterogeneity allows agents to leverage the beneﬁts of diversity and to develop technologies of higher complexity. Social heterogeneity, however, reduces the group ability to innovate. Not only the social structure but also the openness of agents to collaborate affect innovation. We ﬁnd that interdisciplinary interactions lead to more complex technologies beneﬁting the entire group but also increase the inequality in the innovation output. Lower openness to interdisciplinary collaborations may be compensated by a higher ability to collaborate with multiple peers, but low openness also neutralises the intrinsic beneﬁts of network heterogeneity. Our ﬁndings indicate that social network heterogeneity has contrasting effects on microscopic (local) and macroscopic (group) levels, suggesting that the emergence of innovation leaders may suppress the overall group performance. Supplementary material for this article is available online Supplementary material for this article is available online Innovation becomes increasingly difﬁcult if no new added to the toolbox of scientists, artists, entrepreneurs, and other innovators, since it requires © 2022 The Author(s). Published by IOP Publishing Ltd Social network heterogeneity benefits individuals at the expense of groups in the creation of innovation F Zarei1,2,∗ , J Ryckebusch2, K Schoors1 and L E C Rocha1,2 1 Department of Economics, Ghent University, Ghent, Belgium 2 Department of Physics and Astronomy, Ghent University, Ghent, Belgium ∗ Author to whom any correspondence should be addressed. F Zarei1,2,∗ , J Ryckebusch2, K Schoors1 and L E C Rocha1,2 1 Department of Economics, Ghent University, Ghent, Belgium 2 Department of Physics and Astronomy, Ghent University, Ghent, Belgium ∗ Author to whom any correspondence should be addressed. Keywords: innovation, social networks, social heterogeneity, agent based modelling, creation of ideas, collaboration Social network heterogeneity benefits individuals at the expense of groups in the creation of innovation To cite this article: F Zarei et al 2022 J. Phys. Complex. 3 045002 To cite this article: F Zarei et al 2022 J. Phys. Complex. 3 045002 View the article online for updates and enhancements. https://doi.org/10.1088/2632-072X/ac9447 J.Phys.Complex. 3 (2022) 045002 (11pp) J.Phys.Complex. 3 (2022) 045002 (11pp) OPEN ACCESS RECEIVED 9 June 2022 REVISED 11 September 2022 ACCEPTED FOR PUBLICATION 22 September 2022 PUBLISHED 12 October 2022 Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. PAPER Social network heterogeneity benefits individuals at the expense of groups in the creation of innovation F Zarei1,2,∗ , J Ryckebusch2, K Schoors1 and L E C Rocha1,2 1 Department of Economics, Ghent University, Ghent, Belgium 2 Department of Physics and Astronomy, Ghent University, Ghent, Belgium ∗ Author to whom any correspondence should be addressed. E-mail: Fatemeh.Zarei@UGent.be Keywords: innovation, social networks, social heterogeneity, agent based modelling, creation of ideas, collaboration Supplementary material for this article is available online Abstract Innovation is fundamental for development and provides a competitive advantage for so is the process of creating more complex technologies, ideas, or protocols from existing o innovation may be created by single agents (i.e. individuals or organisations), it is often social interactions between agents exchanging and combining complementary expertise perspectives. The structure of social networks impacts this knowledge exchange process. the role of social network structures on the creation of new technologies, we design an ev mechanistic model combining self-creation and social learning. We ﬁnd that social hete allows agents to leverage the beneﬁts of diversity and to develop technologies of higher c Social heterogeneity, however, reduces the group ability to innovate. Not only the social but also the openness of agents to collaborate affect innovation. We ﬁnd that interdiscip interactions lead to more complex technologies beneﬁting the entire group but also incr inequality in the innovation output. Lower openness to interdisciplinary collaborations compensated by a higher ability to collaborate with multiple peers, but low openness als neutralises the intrinsic beneﬁts of network heterogeneity. Our ﬁndings indicate that soc network heterogeneity has contrasting effects on microscopic (local) and macroscopic ( levels, suggesting that the emergence of innovation leaders may suppress the overall grou performance. 1. Introduction Innovation is an idea, method, technology, or process, that is perceived as novel [1]. It is an process that typically relates to improvements over existing ideas or solutions. The importance the generative mechanism of innovation lies in understanding the infrastructure necessary to system’s performance and efﬁciency [2]. 1. Introduction Innovation is an idea, method, technology, or process, that is perceived as novel [1]. It is an evolutionary process that typically relates to improvements over existing ideas or solutions. The importance of studying the generative mechanism of innovation lies in understanding the infrastructure necessary to improve the system’s performance and efﬁciency [2]. Innovation becomes increasingly difﬁcult if no new elements are added to the toolbox of scientists, artists, entrepreneurs, and other innovators, since it requires a combina- tion of expertise, skills, and ideas to create novel knowledge and products. In recent years, interdisciplinarity has been encouraged across organisations to leverage the potential beneﬁts of complementary expertise, aim- ing to solve increasingly complex social and economic problems [3–7]. In this context, social interactions and collaboration between individuals or organisations (i.e. agents) generating complex social networks are funda- mental to provide means to exchange and combine information from diverse sources. The non-linear beneﬁts of social interactions are known to have an impact on innovation [8, 9]. Models of innovation have mostly focussed on studying the adoption of innovative ideas or products by groups of agents [10–13]. Diffusion of innovation is a social process governed by the impact of media and social interactions [14]. The relationship between agents, the number of fragmented or disconnected clusters, population density, and population heterogeneity are among environmental factors affecting information J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al exchange [15, 16]. Modelling innovation in networks makes it possible to understand the impact of social interactions on diffusion. Recent studies have shown how ideas spread and how diffusion depends on the network structure [17–19]. A simple model was proposed to study how the trade-off between acquiring new skills and improving existing skills shapes social networks [20], while the relation between network structure and product and process innovation was analysed using conﬁgurational terms [21]. The impact of the network structure in respect to population size and connectivity was studied by simulating innovation and diffusion of cultural traits in populations with stereotyped social structures [22]. These studies show that small, seemingly insigniﬁcant idiosyncrasies of their structures can heavily impact innovation’s diffusion among members of a social network [23, 24]. The creation of innovation is different from the spread of innovation. 1. Introduction The creation of innovation con- cerns the evolution of agent and group knowledge or the evolutionary process of increasing the complexity of ideas, products, or services by building up simpler existing solutions [20, 25]. Previous studies on social sciences hypothesise that social contacts within groups constrain the information ﬂow and create knowledge holes (structural holes) across the network [26, 27]. Individuals or organisations connecting different social groups are believed to have higher potential of innovation because they can leverage knowledge from differ- ent groups. Empirical studies have provided evidence on the correlation between social network structure and creativity [21, 25, 28, 29]. Correlation studies are however unable to explain the mechanisms connecting the network structure to the potential of innovation creation, and the interplay of social network and innovation dynamics at various structural scales. In this paper, we build upon the hypothesis that network structure regulates innovation dynamics via social interactions, and study the impact of social network structures, on the process of creating innova- tion. We assume that more complex technologies (i.e. innovation) result from simpler technologies, either via self-creation when sufﬁcient knowledge is available to an agent, or via social learning when there is exchange of knowledge between agents. We devise a mechanistic model incorporating the creation of tech- nologies and social structure where information exchange only occurs between connected agents with a sufﬁciently similar knowledge base. We study the effect of microscopic (local or agent level) and macro- scopic (global or group level) network structures of both theoretical and empirical networks on the creation of new technologies and the ability of agents and groups of agents to create technologies with higher com- plexity. We ﬁnd a paradoxical effect of social network structure. While agents beneﬁt from local network heterogeneity, this heterogeneity affects negatively the overall innovation ability of the group. Furthermore, reducing the openness to interdisciplinary collaboration removes the beneﬁt of network heterogeneity on innovation. 2.1. Innovation model In our model, we assume a population of N agents where agents i and j are connected via a social tie (i, j). Each agent i has a set Si(t) of technologies at time t. A technology is deﬁned as a combination of letters A and B such that the complexity level Ch of a technology h is given by the number of letters rep- resenting the respective technology, e.g. for technology {AAB}, Ch = 3. The complexity of an agent Ci(t) is deﬁned by the technology with the highest complexity in the set Si(t). The size of Si(t) (\|Si(t)\| = Di(t)) gives the diversity of technologies known by agent i. New technologies are created via a branching process (ﬁgure 1(A)). The simplest technologies are either A or B (Ch = 1). An agent can create one out of two new technologies AA or AB (C = 2) from A (or BB or BA from B). The same process is valid for more complex technologies (C > 2). This evolutionary process means that innovation only happens when an agent cre- ates a new technology with a complexity higher than the complexity of its current technologies. In other words, an agent only innovates on top of its most complex technologies because it aims to increase com- plexity. If an agent has more than one technology with the highest level of complexity, one of them is selected. This mechanism creates a historical dependency of innovation with various possible trajectories for Si(t) [25]. Initially, all agents have Si(t = 0) = {A} or Si(t = 0) = {B} technologies distributed uniformly at random (i.e. Di(t = 0) = \|Si(t = 0)\| = 1). An agent can increase the size of Si(t) through two mechanisms: (i) create one new technology based on its own knowledge of simpler technologies (self-creation) or (ii) acquire one technology via social learning. At each time step t, we select an agent uniformly at random to create a new technology with probability pi. This probability is adjusted to impose that agents must know a sufﬁcient num- ber of accumulated knowledge to be able to create one that is more complex than those in its own portfolio. The sigmoid function shows the mathematical equivalent of this probability 2 J.Phys.Complex. 3 (2022) 045002 (11pp) J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al F Zarei et al Figure 1. Innovation process. 2.1. Innovation model (A) Self-creation: a new and more complex technology can be created from a simpler technology (e.g. C = 2 from C = 1). (B) Social learning: (i) mutual learning, both agents learn the most complex technology of each other (both conditions are met); (ii) asymmetrical learning, one agent—blue here—has a complexity level larger than the red agent and θij > θ; thus only the blue agent learns; (iii) no learning, both agents have the same level of complexity and similarity (θij > θ) but there is an overlap of the most complex technologies, hence no learning for both agents. (iv) No learning, the similarity of the two agents is lower than the threshold (θij < θ). ( ) lf d l h l b d f l h l Figure 1. Innovation process. (A) Self-creation: a new and more complex technology can be created from a simpler technology (e.g. C = 2 from C = 1). (B) Social learning: (i) mutual learning, both agents learn the most complex technology of each other (both conditions are met); (ii) asymmetrical learning, one agent—blue here—has a complexity level larger than the red agent and θij > θ; thus only the blue agent learns; (iii) no learning, both agents have the same level of complexity and similarity (θij > θ) but there is an overlap of the most complex technologies, hence no learning for both agents. (iv) No learning, the similarity of the two agents is lower than the threshold (θij < θ). pi(t) = 1 1 + e−β(Di(t)−αCi(t)) . (1) (1) In our simulation we have set α = 1.2 to balance the rate of creation of innovation. A lower α ∼1 leads to a dynamics where the average complexity continuously increases without reaching stationarity. A higher α, on the other hand, causes the system to quickly reach the stationary state and thus results on relatively low levels of average complexity. We set β = 100 to obtain a Heaviside step function, such that there is a large probability of creating a new technology if Di(t) > αCi(t), whereas this probability is small if Di(t) < αCi(t). If there is no self-innovation (i.e. with probability 1 −pi(t)) at time t, the agent i interacts with one of its social contacts j (chosen uniformly at random) to collaborate and share knowledge. 2.2. Social networks To study the effect of social network structure on the creation of technologies, we use theoretical network models and real-world network data. A network is deﬁned by a set of N nodes in which nodes i and j are connected by a link (i, j). In our model, a node i corresponds to an agent i (an individual or an organisation). The number of social contacts of i is the degree κi. The clustering coefﬁcient cci = 2ei/(κi(κi −1)) (where ei is the number of links between common neighbours of i) gives the fraction of triangles connected to node i. The betweenness centrality bi gives the fraction of all shortest-paths, between any two nodes j and k in the network (σjk), passing through node i, i.e. bi = i̸=j̸=k σjk(i) σjk . j jk The reference theoretical network model is the Erd¨os–R´enyi (ER) random model in which links are made between pairs of nodes with a ﬁxed probability q. The emerging structure is homogeneous with a charac- teristic node degree [30]. The second network model contains heterogeneity in the clustering coefﬁcient of nodes, to capture different levels of clustering in the network. We use a conﬁguration model to generate ran- dom networks where the distribution of clustering coefﬁcients is controlled and the rest of the structure is randomised for a given average degree [31, 32]. The third theoretical network uses the conﬁguration model with a ﬁxed degree distribution p(κ) ∝κ−β, with β = 2.5 for ﬁnite mean and variance [33]. In our simula- tions, we ﬁrst generate ten realisations of each network model with the same set of parameters, and run ten times the innovation model to obtain averages over m = 100 simulations. g We also apply our innovation model to three data sets of social collaboration. The ﬁrst data set is a co- authorship network of scientists (COA). This network is a one-mode projection from the bipartite graph of authors and their scientiﬁc publications. The network is formed by nodes representing authors connected by links (i, j) if there is at least one joint publication [34]. The second empirical data set corresponds to cur- rent and previous afﬁliation of researchers at 206 computer science departments at universities in the USA (COM). 2.1. Innovation model For the exchange between the speciﬁc agents i and j to be successful, two conditions have to be met, otherwise nothing happens at this 3 J.Phys.Complex. 3 (2022) 045002 (11pp) Table 1. Summary of the network models used in our study. Code Network model N ⟨κ⟩ ER Erd¨os–R´enyi random model 1000 10 RC Random clustered model 1000 10 SF Scale-free random model 1000 10 COA Collaborations between authors 1461 4 COM Current and previous afﬁliation of researchers 206 27 SCOP Collaborations between institutions 1511 6 CSCOP Randomised version of SCOP with ﬁxed clustering 1511 6 DSCOP Randomised version of SCOP with ﬁxed degree 1511 6 time step t: (i) the technological similarity of these two nodes θij(t), θij(t) = \|Si(t) ∩Sj(t)\| \|Si(t) ∪Sj(t)\| (2) ime step t: (i) the technological similarity of these two nodes θij(t), θij(t) = \|Si(t) ∩Sj(t)\| \|Si(t) ∪Sj(t)\| (2) θij(t) = \|Si(t) ∩Sj(t)\| \|Si(t) ∪Sj(t)\| (2) (2) should be higher than the threshold θ (which is a hyperparameter of the model and constant for all agents); and (ii) an agent i can learn a technology with complexity level Ch only if Ch ⩽Ci(t). The complexity level of an agent does not increase through social learning, whereas its set of technologies, i.e. its diversity of knowledge (Di(t)), does. Therefore, higher diversity increases the agent’s chance of self-innovation at the next time it is selected. Figure 1(B) illustrates the potential social learning scenarios when both conditions are combined for θ = 0.2. In the ﬁrst scenario, θij = 0.33 and agents learn from each other because they are at the same level of complexity (mutual learning). In the second scenario, θij = 0.5 and only agent blue learns because the most complex technology of the blue agent is ABA (Cblue = 3) whereas the red agent has complexity Cred = 2 (asymmetrical learning). In the third scenario, θij = 0.75 but no learning is observed because the most complex technologies of both agents are similar (no learning). In the last scenario, the similarity is not sufﬁciently large (θij = 0.17) for social learning (no learning). This innovation process is repeated until the system reaches the stationary state, deﬁned as the time when d⟨C⟩ dt < 10−6. 2.2. Social networks Each link represents that a professor working at university i got a PhD at university j, therefore, this network represents the ﬂow of human capital between universities [35]. The third data set comes from Scopus and represents scientiﬁc collaborations between institutions in New Zealand in the period from 2010 to 2015 (SCOP). The network is formed by nodes representing institutions (e.g. universities, organisations), connected by links if there is at least one joint publication with authors from both institutions [36]. Relevant structural properties of these networks are shown in the SI. Finally, to study the correlations in the network structure, 4 J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al F Zarei et al Figure 2. Temporal evolution of the group complexity ⟨C⟩for different thresholds of similarity θ and networks (see table 1). The averages are taken over m = 100 realisations of the network and random starting conditions. Vertical bars represent standard errors. Figure 2. Temporal evolution of the group complexity ⟨C⟩for different thresholds of similarity θ and networks (see table 1). The averages are taken over m = 100 realisations of the network and random starting conditions. Vertical bars represent standard errors. we randomise the links of SCOP to create random networks with the same average degree but ﬁxed cluster- ing (CSCOP) and ﬁxed degree (DSCOP) distributions. Table 1 shows a summary of all networks used in our study. we randomise the links of SCOP to create random networks with the same average degree but ﬁxed cluster- ing (CSCOP) and ﬁxed degree (DSCOP) distributions. Table 1 shows a summary of all networks used in our study. 3. Results In this section, we will study the impact of social network structure on innovation at the macroscopic (network level) and microscopic (agent level) scales. We ﬁrst analyse the evolution of innovations on random network models to study speciﬁc structures and then apply the model on real-world collaboration networks to under- stand the effect of real structures on the innovation dynamics. Finally, we analyse correlations in real networks by studying randomised versions of real networks with a chosen ﬁxed structure. 3.1. Group innovation The averages are taken over m = 10 realisations of random starting conditions. Vertical bars represent standard errors. Figure 3. Temporal evolution of the group complexity ⟨C⟩on empirical structures (table 1). The averages are taken over m = 10 realisations of random starting conditions. Vertical bars represent standard errors. Figure 4. Temporal evolution of the number of times that agents in the network (A) self-create (SC) and (B) social learn (SL) a new technology; (C) the relative importance of SC to SL, χ = 100 SC−SL SC+SL , for the ER network model. The averages are taken over m = 100 realisations of the network and random starting conditions. Vertical bars represent standard errors. Figure 4. Temporal evolution of the number of times that agents in the network (A) self-create (SC) and (B) social learn (SL) a new technology; (C) the relative importance of SC to SL, χ = 100 SC−SL SC+SL , for the ER network model. The averages are taken over m = 100 realisations of the network and random starting conditions. Vertical bars represent standard errors. by encouraging collaborations between agents with substantially different know-hows or backgrounds) is an effective means to avoid saturation of knowledge and generate more complex new technologies. For higher θ, the social network structure becomes less relevant because the chance of having similar agents decreases and the connectivity does not affect social learning. In other words, a high θ means that agents are less open to social learning, independently of the number and structure of social contacts; too low openness (θ = 0.6) leads to approximately the same average complexity level irrespective of the social structure. On the other hand, the effect of lowering θ is relatively higher in denser networks because of the non-linear beneﬁt of increasing opportunities of social learning (see SI). pp g Figures 2(D), (E), and (F) shows the temporal evolution of ⟨C⟩for the empirical networks, that are highly heterogeneous in terms of clustering, degree, and betweenness centrality (see SI). In all cases, the complexity increases relatively fast in the beginning, and eventually reaches stationarity. Social learning increases with an increase of θ, also leading to an increase in innovation and thus on ⟨C⟩. Increasing θ causes a relatively higher increase in group innovation in the COM network in comparison to the other studied empirical networks. 3.1. Group innovation Figures 2(A), (B), and (C) show the temporal evolution of the average complexity ⟨C⟩for all N agents for three network models: ER, random clustered (RC), and scale-free (SF). In all cases, a fast increase of ⟨C⟩in the beginning is followed by a slower increase until the system reaches a nearly-stationary state. This happens because in the early stages, creating new complex technologies is relatively easier since agents have a small set of simple technologies and diversity grows faster than complexity (Di(t) > Ci(t)), providing the condi- tions for self-creation (see methods and equation (1)). Furthermore, there is higher overlap of knowledge of contacts (because Di(t) is small during early times) and thus exchanges via social learning are facilitated. As the dynamics evolves, the difference between diversity and complexity decreases and innovation becomes less likely. The diversity of the set of technologies also increases and less overlap of knowledge is observed between social contacts, reducing the speed of innovation, until it eventually converges. The average complexity ⟨C⟩is higher for homogeneous networks (ER, the reference case) in comparison to heterogeneous structures. The heterogeneous clustering reduces ⟨C⟩(RC) however the degree heterogeneity (SF) has a higher effect on lim- iting group innovation (ﬁgures 2(A), (B), and (C)). When the network has structural heterogeneity, the input information is not the same for all agents. Therefore the agents achieve different levels of complexity, and this disparity decreases social learning and consequently ⟨C⟩. The results also show the importance of the similarity between social contacts to promote group innovation. A lower level of expected similarity of existing technologies between social contacts (θ = 0.2, i.e. a higher level of interdisciplinarity) generates on average more complex technologies (ﬁgure 2(A)) in comparison to a higher level of similarity (θ = 0.6, i.e. less interdisciplinarity) (ﬁgure 2(C)). This result means that innovation is less likely if social learning occurs between too similar agents. On one hand, a high level of similarity is necessary for two agents to communicate effectively and exchange knowledge. On the other hand, too similar agents are unable to ﬁll gaps and complement each other’s knowledge, limiting their ability to create new complex technologies. Our model indicates that reducing the threshold θ (i.e. promoting interdisciplinarity, for example 5 J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al F Zarei et al Figure 3. Temporal evolution of the group complexity ⟨C⟩on empirical structures (table 1). 3.1. Group innovation The COM network is denser (and relatively smaller) than the other networks. A denser network provides more opportunities for social learning with different social contacts which increases the diversity Di(t) of agents, that in turn leads to the creation of technologies with higher complexity. Figure 3 compares the evolution of inno- vation using one empirical network (SCOP) and the randomised versions (CSCOP and DSCOP, see table 1) to show the impact of correlated structures in the dynamics. The result shows that, besides heterogeneity, the correlation between edges in a network also reduces group innovation. Figure 4 shows the temporal evolution of the cumulative number of times per agents that they increase their number of technologies via self-creation (SC), and social learning (SL). Also and the relative difference between SC and SL (χ = 100 SC−SL SC+SL), for a random network structure (ER model) with various similarity thresholds θ, (results are qualitatively similar for other network conﬁgurations). In the early stages, agents can social learn and self-create new complex technologies. After a while, the difference in the complexity level of agents increases, and agents with higher complexity cannot teach but only learn new technologies from their social contacts (ﬁgure 1(B)), leading to a decrease in social learning. Once these agents learn all the complex technologies of their social contacts, only self-creation is possible. At the later stage, diversity is not sufﬁciently large for self-creation and the system reaches stationarity. The results also show that for a larger 6 J.Phys.Complex. 3 (2022) 045002 (11pp) J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al Figure 5. Individual complexity and network structures. Agent complexity Ci(tf ) and local network structures for various network models (table 1) at stationarity. Panels (A), (D), (G) and (J) correspond to degree κi; (B), (E), (H) and (K) to betweenness centrality bi; and (C), (F), (I) and (L) to clustering coefﬁcient cci. The coefﬁcient γ indicates the slope of the relation between Ci(tf ) and log(κi), log(bi) and log(cci). The averages are taken over m = 100 realisations of the network and random starting conditions. Vertical bars represent standard errors. Figure 5. Individual complexity and network structures. Agent complexity Ci(tf ) and local network structures for various network models (table 1) at stationarity. 3.1. Group innovation Panels (A), (D), (G) and (J) correspond to degree κi; (B), (E), (H) and (K) to betweenness centrality bi; and (C), (F), (I) and (L) to clustering coefﬁcient cci. The coefﬁcient γ indicates the slope of the relation between Ci(tf ) and log(κi), log(bi) and log(cci). The averages are taken over m = 100 realisations of the network and random starting conditions. Vertical bars represent standard errors. similarity threshold, social learning is less important to transfer new technologies but important to increase the diversity of the agents. Both social learning and self-creation are affected by the degree of the agents, with self-creation being, relatively to social learning, larger for low-degree agents in comparison to high-degree agents (see SI, section 3). This happens possibly because a higher degree provides more diverse information for an agent but also decreases the similarity between the agent and its social contacts. The similarity between the pairs of interacting agents ﬂuctuates but the average similarity decreases over time. The cumulative pro- portion of failures due to dissimilarity between agents thus increases monotonically as the system evolves (see SI, section 4). 3.2. Microscopic dynamics (d(C)) at stationarity (tf ) for (A) random networks (tf = 107), and (B) the empirical network (SCOP) and its randomised versions (CSCOP and DSCOP), (tf = 2.5 × 107). We used θ = 0.2 in these simulations and simulations are repeated for m = 50 starting conditions. Figure 6. Distribution of individual complexity level. (d(C)) at stationarity (tf ) for (A) random networks (tf = 107), and (B) the empirical network (SCOP) and its randomised versions (CSCOP and DSCOP), (tf = 2.5 × 107). We used θ = 0.2 in these simulations and simulations are repeated for m = 50 starting conditions. centrality measures the brokerage potential of an agent connecting socially diverse groups of network nodes, and therefore, its potential to leverage knowledge from multiple groups of agents. The clustering coefﬁcient is a local measure of embeddedness in a social group. Agents that are overly embedded in a social group are less exposed to diverse views and knowledge because clustering tends to homogenous the information circulating within the group. The similarity threshold θ affects social learning. If θ is high, agents must be highly similar for interactions to result in social learning of new complex technologies. Figure 5 shows that the lack of openness or willingness to inter-disciplinary collaboration (i.e. high θ) not only reduces the agent level of complexity but also the relevance of social heterogeneity in the dynamics of social learning. Openness to collaborate with contacts with different knowledge (low θ), on the other hand, leverages the diversity provided by social heterogeneity to increase innovation. This can be seen in the values ofγ which is the regression coefﬁcient of the complexity level Ci(tf ) to the logarithm of the network measure log(xi ∈{κi, bi, cci}). Data are binned for visualisation but γ is calculated using the raw data. The importance of openness is highlighted by the fact that very open agents with few contacts may achieve the same level of complexity as less open agents with many contacts. Table 2 shows the range of γ for three random network structure (ER, RC, and SF) which have the same number of nodes and average degree. By decreasing θ, the effect of network structure on innovation increases. The coefﬁcient γ is not ﬁxed and depends on the network structure at both local and global scales. However, it is always inversely related to θ. 3.2. Microscopic dynamics The group dynamics is a result of microscopic interactions between the agents. Social networks have different levels of at the individual (i.e. local) level heterogeneity that can be captured by various network measures. We study three local structures: (i) the degree κi; (ii) the betweenness centrality bi, and (iii) the clustering coefﬁcient cci of agent i (see methods). Figure 5 shows the connection between the complexity levelof individual agents and their network features. Independently of the network, we observe that agent the complexity increases with its degree (ﬁgures 5(A), (D), (G) and (J)) and betweenness centrality (ﬁgures 5(B), (E), (H) and (K)) whereas it decreases with its clustering (ﬁgures 5(C), (F), (I) and (L)). The effect of the degree is stronger in the SF model because of the degree heterogeneity. Similarly, the RC model has stronger clustering heterogeneity than the other models and thus the effect on the agent complexity level is higher. Empirical networks have different network structures (see SI, section 1) but also for those networks, we observe a similar relation between the local connectivity and individual complexity level (ﬁgures 5(J), (K) and (L)). The degree indicates the local level of connectivity of an agent and thus its potential to be inﬂuenced by multiple social contacts, or network neighbours. A higher social degree thus increases the probability of getting new technologies because of higher exposure. The betweenness 7 J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al Table 2. Dependency between the network measures xi ∈{κi, bi, cci} and the complexity levels Ci(tf ) of the individual agents in the stationary regime as ﬁtted by the function Ci(tf ) = C0 + γ log(xi). The range of the ﬁtted γ values is given for the three random networks (ER, RC, and SF). θ x κi bi cci 0.2 [8.3, 9.6] [3.2, 4.5] [−5.4, −3.6] 0.4 [2.6, 3.4] [1.1, 1.5] [−2.0, −1.4] 0.6 [0.9, 1.5] [0.5, 0.6] [−0.8, −0.5] Figure 6. Distribution of individual complexity level. (d(C)) at stationarity (tf ) for (A) random networks (tf = 107), and (B) the empirical network (SCOP) and its randomised versions (CSCOP and DSCOP), (tf = 2.5 × 107). We used θ = 0.2 in these simulations and simulations are repeated for m = 50 starting conditions. Figure 6. Distribution of individual complexity level. 3.3. Knowledge normalisation The diversity of technologies Di(t) of each agent has to be larger than the complexity Ci(t) (equation (1)) to create a new technology. Social learning only happens when a sufﬁcient variety of knowledge exists between social contacts, otherwise, existing knowledge is reinforced and new technologies are not created. Social learn- ing is a dynamic process that promotes innovation but also normalises (i.e. homogeneous) local knowledge. This normalisation can be measured by the similarity Ωi(t) in the set of technologies between an agent and its social contacts κi Ωi(t) = 1 κi κi j=1 θij(t). (3) (3) Figure 7 shows Ωi(tf ) in different networks in comparison to local network measures. Data are binned for visualisation but γ is calculated using the raw data. There is anti-correlation between the level of complexity of each agent and the similarity with their social contacts. Over time, agents become more similar which even- tually saturates the possibilities to generate more complex technologies. Although too similar agents do not exchange new technologies, an agent that is too different from its social contacts (θij ∼0) cannot beneﬁt from social learning either. An agent that innovates more becomes less similar to its social contacts. Therefore, the similarity shows an inverse relationship with complexity, meaning that an agent with higher degree or higher betweenness centrality has a lower level of similarity to its social contacts. On the other hand, high clustering makes social learning inside the cluster more frequent than between clusters. Increasing θ leads to higher sim- ilarity between social contacts because only agents with high similarity can learn from each other whereas the openness given by low θ promotes diversity. 3.2. Microscopic dynamics Network heterogeneity not only distributes the ability of agents to be innovative but also affects top per- formers. Figure 6 shows the distribution density of the agent complexity level in the stationary state. The fraction of agents with low complexity is lower in homogeneous networks (ER) in comparison to heteroge- neous networks (RC and SF). As the complexity increases, only small differences are observed for different networks. However, the highest levels of individual complexity are achieved with the degree (SF) and clustered (RC) heterogeneous models. The SF structure promotes the emergence of some top performers and relatively many lower performers (i.e. higher inequality) followed by the clustered networks. In contrast, a random network structure promotes more equal social opportunities and innovation potential (i.e. less inequality). Figure 6(B) shows the distribution density of complexity at stationarity for the SCOP, CSCOP and DSCOP networks. The result indicates that homogeneity causes top performers to disappear. In the network with the highest average complexity (DSOP) the number of top performers is lower than in other networks and the distribution of the individual agent complexity is more homogeneous. 8 J.Phys.Complex. 3 (2022) 045002 (11pp) J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al Figure 7. Knowledge similarity and network structure. The similarity Ωi(tf ) of an agent i and local network structure, (A) κi (SF network); (B) bi (RC network); (C) cci (RC network); (D) κi (SCOP network); (E) bi (SCOP network); (F) cci (SCOP network). Vertical bars represent standard errors. Figure 7. Knowledge similarity and network structure. The similarity Ωi(tf ) of an agent i and local network structure, (A) κi (SF network); (B) bi (RC network); (C) cci (RC network); (D) κi (SCOP network); (E) bi (SCOP network); (F) cci (SCOP network). Vertical bars represent standard errors. 4. Conclusion from agents with different sets of knowledge), the overall level of complexity of the group is negatively affected and decreases. Furthermore, the heterogeneity of the social structure is only beneﬁcial at the microscopic level when there is sufﬁcient openness to interdis- ciplinarity. If agents avoid interdisciplinarity, the requested level of similarity between contacts is too high and the social network structure becomes irrelevant. Although network heterogeneity may be advantageous for individual agents, this is not true for the group. According to our model, promoting more collaboration of less connected agents or reducing the centrality of highly central agents increases not only the overall performance of the social network but also the overall diversity of technologies generated by the collaborative network. This model has a limitation on the mechanism of social learning, due to the assumption that high complexity agents are not able to teach social contacts who are at a lower complexity level (e.g. a master-pupil relationship), rendering them less useful for fostering group innovation. This assumption was made to focus on collaboration, requiring a sufﬁciently similar level of complexity coupled with a certain level of similarity between agents, in contrast to training where agents may learn or teach technologies with different levels of complexity within speciﬁc disciplines. Another limitation is that agents always learn the most complex technology rather than less complex technologies. Whereas these assumptions are motivated by agents trying to optimize and leverage the available individual capabilities to maximise innovation via social interactions [37], relaxing these constraints would increase the possibilities of social learning and thus increase the diversity of technologies of each agent, which in turn, would lead to a continuous increase in the complexity of the novel technologies. One consequence of our ﬁndings is that innovation may increase if organisations and governments provide more support to reduce heterogeneity, for example by supporting less central agents to improve their position in the network or by reducing ‘the-rich- get-richer’ schemes [38] that beneﬁt well-connected agents (i.e. by making research funding less dependent on candidate track record, signalled by past funding or past publications, or a more random distribution of resources). Our model does not account for individual characteristics of the agents, as for example, abilities, exper- tise, and resources to exploit social learning. We assume that all agents have the same capacity, and are able to process information and create technologies at the same speed. 4. Conclusion Innovation is a result of combining different ideas, skills, or knowledge to create new technologies, ideas, or solutions. To increase the innovation potential, individuals and organisations may interact and exchange infor- mation via social learning. The structure of the social networks thus affects the rate of innovation. In this paper, we devised and tested a mechanistic model, that includesan evolutionary process of innovation whereby agents interact via social networks and create more complex technologies from simpler ones. Within the model we studied the interplay between network structure and the creation of technologies at the individual and group levels. We found that local structural heterogeneity increases the ability of individual agents to generate more complex technologies. There is a strong correlation between degree and betweenness centrality and the level of agent complexity. Being embedded in social clusters reduces the ability of an agent to innovate. This happens because more central agents collaborate with various agents or social groups, and thus are inﬂu- enced by diverse sources of different knowledge whereas clusters tend to homogeneous knowledge within the group, thus reducing opportunities to leverage the advantages of diversity. We found however a contrasting 9 J.Phys.Complex. 3 (2022) 045002 (11pp) J.Phys.Complex. 3 (2022) 045002 (11pp) F Zarei et al effect at different scales. While such inequality beneﬁts individual agents, the same structural heterogeneity reduces the innovation potential of the group. This happens because heterogeneity implies that only a few agents are sufﬁciently exposed to diverse knowledge in detriment of the majority that are exposed to similar knowledge, either because of less connectivity or because it is not bridging clusters of knowledge. Once central agents develop high levels of complexity, those connected to them become less similar to the central agents and thus have lower chances to innovate via social learning. Our results suggest that heterogeneity may not be good for the group but top-performers in the het- erogeneous networks are better than the average innovation ability in homogeneous cases. In other words, heterogeneity in the network structure on one hand causes heterogeneity in the innovation of agents, which reduces their average innovation relative to the homogeneous network, but on the other hand causes some agents to have much higher performance than the average of the homogeneous group. If there is no willing- ness or ability to learn from interdisciplinary collaborations (i.e. 4. Conclusion High performers may achieve higher levels of innovation with fewer contacts and vice-versa. By adding a temporal dimension to our model and het- erogeneity of individual characteristics, we might be able to include such mechanisms to better describe the agent and social processes necessary to adapt the network structure to maximise innovation [39, 40]. Our model for innovation does also not account for shocks and paradigm shift. Agents could suddenly create a technology that has a very higher level in comparison to their knowledge. By adding a jump term to our model for self-creation, we might be able to include such sudden innovation and adapt our model to the real contexts. Acknowledgments LECR thanks Matthew Smith for pointing out helpful references. Data availability statement The data that support the ﬁndings of this study are openly available at the following URL/DOI: https:// networks.skewed.de/net/netscience, https://aaronclauset.github.io/facultyhiring/ and https://networks.ske- wed.de/net/new_zealand_collab 10 F Zarei et al J.Phys.Complex. 3 (2022) 045002 (11pp) References 48 531 [12] Kuandykov L and Sokolov M 2010 Impact of social neighborhood on diffusion of innovation s-curve Decis. Support Syst. 48 531–5 [13] Ting Z, Baojun G and Huiyu X 2009 Simulate the effects of advertising on the diffusion of innovation with cellular automata i d T h l d li l [12] Kuandykov L and Sokolov M 2010 Impact of social neighborhood on diffusion of innovation s-curve Decis. 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https://openalex.org/W3100065348	https://www.frontiersin.org/articles/10.3389/fnagi.2020.585988/pdf	English	null	Effectiveness of an Innovative Cognitive Treatment and Telerehabilitation on Subjects With Mild Cognitive Impairment: A Multicenter, Randomized, Active-Controlled Study	Frontiers in aging neuroscience	2,020	cc-by	12,694	CLINICAL TRIAL published: 16 November 2020 doi: 10.3389/fnagi.2020.585988 Effectiveness of an Innovative Cognitive Treatment and Telerehabilitation on Subjects With Mild Cognitive Impairment: A Multicenter, Randomized, Active-Controlled Study Rosa Manenti1, Elena Gobbi1, Francesca Baglio2, Ambra Macis3, Clarissa Ferrari3, Ilaria Pagnoni1, Federica Rossetto2, Sonia Di Tella2, Federica Alemanno4, Vincenzo Cimino5, Giuliano Binetti6, Sandro Iannaccone4, Placido Bramanti5, Stefano F. Cappa7,8 and Maria Cotelli1 1 Neuropsychology Unit, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy, 2 IRCCS, Fondazione Don Carlo Gnocchi – ONLUS, Milan, Italy, 3 Service of Statistics, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy, 4 Department of Rehabilitation and Functional Recovery, IRCCS San Raffaele Hospital and Scientiﬁc Institute, Vita-Salute San Raffaele University, Milan, Italy, 5 IRCCS Centro Neurolesi “Bonino Pulejo,” Messina, Italy, 6 MAC Memory Clinic and Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy, 7 NEtS, Scuola Universitaria Superiore IUSS-Pavia, Pavia, Italy, 8 IRCCS Fondazione Mondino, Pavia, Italy Edited by: Yang Jiang, University of Kentucky, United States Edited by: Yang Jiang, University of Kentucky, United States Edited by: Yang Jiang, University of Kentucky, United States Reviewed by: Juan F. Cardona, University of Valle, Colombia Gabriela Spulber, Karolinska Institutet (KI), Sweden Correspondence: Maria Cotelli mcotelli@fatebenefratelli.eu Rosa Manenti rmanenti@fatebenefratelli.eu Reviewed by: Juan F. Cardona, University of Valle, Colombia Gabriela Spulber, Karolinska Institutet (KI), Sweden Background: In recent years, the potential usefulness of cognitive training procedures in normal aging and mild cognitive impairment (MCI) have received increased attention. Background: In recent years, the potential usefulness of cognitive training procedures in normal aging and mild cognitive impairment (MCI) have received increased attention. Correspondence: Maria Cotelli mcotelli@fatebenefratelli.eu Rosa Manenti rmanenti@fatebenefratelli.eu Correspondence: Maria Cotelli mcotelli@fatebenefratelli.eu Rosa Manenti rmanenti@fatebenefratelli.eu Objective: The main aim of this study was to evaluate the efﬁcacy of the face-to-face cognitive virtual reality rehabilitation system (VRRS) and to compare it to that of face-to- face cognitive treatment as usual for individuals with MCI. Moreover, we assessed the possibility of prolonging the effects of treatment with a telerehabilitation system. Received: 22 July 2020 Accepted: 19 October 2020 Published: 16 November 2020 Methods: A total of 49 subjects with MCI were assigned to 1 of 3 study groups in a randomized controlled trial design: (a) those who received face-to-face cognitive VRRS (12 sessions of individualized cognitive rehabilitation over 4 weeks) followed by telerehabilitation (36 sessions of home-based cognitive VRRS training, three sessions for week); (b) those who received face-to-face cognitive VRRS followed by at-home unstructured cognitive stimulation (36 sessions of home-based unstructured cognitive stimulation, three sessions for week); and (c) those who received face-to-face cognitive treatment as usual (12 sessions of face-to-face cognitive treatment as usual). CLINICAL TRIAL published: 16 November 2020 doi: 10.3389/fnagi.2020.585988 INTRODUCTION A recent systematic review showed the eﬃcacy of telerehabilitation on cognitive abilities in individuals with MCI and in patients with neurodegenerative diseases associated with cognitive impairment (Cotelli et al., 2019). MCI is a condition associated with risk of progression to dementia, and represents a well-suited target for prevention studies (Petersen et al., 1999, 2014; Petersen, 2004; Livingston et al., 2017). However, these treatments are delivered in several ways and there is not a clinical consensus about content-design of telerehabilitation. A ﬁxed schedule approach has proved to be eﬀective in the treatment of elderly people with high risk of conversion in dementia resulting in a signiﬁcant improvement in global cognitive functioning, memory and processing speed (Lampit et al., 2014). In other studies, participants were given the opportunity to choose free among the activities available in each session of training (Medalia and Freilich, 2008; Gooding et al., 2016). A third alternative consisted in the user-centered approach, which customized the choice of rehabilitative contents based on the performance obtained by the individual to the set up tests implemented in the software at the beginning of the rehabilitation path (Solana et al., 2015; Vance et al., 2018). So far, the majority of studies are feasibility or pilot studies with small-medium sample size and are very heterogeneous in terms of intensity and duration of treatment (Burdea et al., 2015; Espay et al., 2016; Dodakian et al., 2017). For this reason, in the light of this ongoing deep transformation of health care, it is of great relevance the eﬀort to harmonize intervention protocols and randomized controlled trials (RCTs) are strongly needed to demonstrate the eﬀectiveness of these home-based technology-enhanced treatment protocols with respect to the gold-standard, named the usual face-to-face care (Linden et al., 2016; Fetta et al., 2017; Topol, 2019). In recent years, there has been growing interest in the use of telerehabilitation methods in patients with neurodegenerative diseases (Cherney and van Vuuren, 2012; Cotelli et al., 2019). Given the limited eﬀectiveness of pharmacological treatments, there is a critical need to develop novel interventions aimed at preventing or delaying the onset of Alzheimer’s disease (AD), and mild cognitive impairment (MCI) might represent a potential target for intervention trials (Kidd, 2008; Hong et al., 2015; Janoutova et al., 2015). Traditional cognitive training involves intensive in-person sessions that may not prove to be feasible and cost-eﬀective in the case of large-scale implementation. Citation: Manenti R, Gobbi E, Baglio F, Macis A, Ferrari C, Pagnoni I, Rossetto F, Di Tella S, Alemanno F, Cimino V, Binetti G, Iannaccone S, Bramanti P, Cappa SF and Cotelli M (2020) Effectiveness of an Innovative Cognitive Treatment and Telerehabilitation on Subjects With Mild Cognitive Impairment: A Multicenter, Randomized, Active-Controlled Study. Front. Aging Neurosci. 12:585988. doi: 10.3389/fnagi.2020.585988 Results: An improvement in memory, language and visuo-constructional abilities was observed after the end of face-to-face VRRS treatment compared to face-to-face treatment as usual. The application of home-based cognitive VRRS telerehabilitation November 2020 \| Volume 12 \| Article 585988 1 Frontiers in Aging Neuroscience \| www.frontiersin.org Manenti et al. Cognitive Treatment and Telerehabilitation MCI seems to induce more maintenance of the obtained gains than home-based unstructured stimulation. Discussion: The present study provides preliminary evidence in support of individualized VRRS treatment and telerehabilitation delivery for cognitive rehabilitation and should pave the way for future studies aiming at identifying optimal cognitive treatment protocols in subjects with MCI. Clinical Trial Registration: www.ClinicalTrials.gov, identiﬁer NCT03486704. Clinical Trial Registration: www.ClinicalTrials.gov, identiﬁer NCT03486704. Keywords: cognitive, telerehabilitation, dementia, mild cognitive impairment, home Keywords: cognitive, telerehabilitation, dementia, mild cognitive impairment, home Antonietti et al., 2016; Vermeij et al., 2016; Burton and O’Connell, 2018; Isernia et al., 2019). Frontiers in Aging Neuroscience \| www.frontiersin.org INTRODUCTION The average lifespan in the world almost doubled during the 20th century and has resulted in a large number of people living to old ages, causing an increased risk of developing age-related diseases, disability and dementia (Fratiglioni et al., 1999; Brown, 2015). In the coming years, the growing demand and the need to contain the costs of health care will dictate the need to reorganize the services dedicated to people at risk of developing cognitive impairment by taking advantage of technological developments (Bharucha et al., 2009; Astell, 2019; Moyle, 2019). Telerehabilitation via information and communication technologies (Brennan et al., 2011; Realdon et al., 2016; Pitt et al., 2019) represents an innovative approach to overcome the obstacles associated with face-to-face intervention. Telerehabilitation technologies allow to provide services remotely in patients’ homes or other environments, allowing access to health care to patients living in rural settings or with mobility diﬃculties (Brennan et al., 2002, 2009, 2011; Forducey et al., 2003; Mashima and Doarn, 2008; Zampolini et al., 2008; Hailey et al., 2011; Peretti et al., 2017). In addition, the telerehabilitation modality oﬀers the advantage of providing rehabilitation within the natural environment of the patient’s home, making the treatment more realistic and possibly more generalizable to the person’s daily life (McCue et al., 2010). The main aim of the current study was to evaluate the eﬃcacy of the cognitive face-to-face virtual reality rehabilitation system (VRRS) and to compare it to that of face-to-face cognitive treatment as usual for subjects with MCI. We hypothesized that the face-to-face VRRS system would ameliorate memory and attentional abilities more than treatment as usual in subjects with MCI. Recent studies have shown that the application of telerehabilitation methodology in individuals with physical impairments, post-stroke participants and patients with neurodegenerative diseases leads to clinical improvements that are generally equal to those induced by conventional face-to-face rehabilitation programs (Brennan et al., 2002; Rosen, 2004; Poon et al., 2005; Mashima and Doarn, 2008; Kairy et al., 2009; Cherney and van Vuuren, 2012; Jelcic et al., 2014; Moreover, we tested the hypothesis that the implementation of home-based treatment through the cognitive VRRS system could induce long-term beneﬁts, prolonging the beneﬁcial eﬀects of face-to-face. November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org 2 Cognitive Treatment and Telerehabilitation MCI Manenti et al. MATERIALS AND METHODS blinded to the type of treatment. Participants were randomized into three groups: (a) those who received face-to-face cognitive VRRS treatment followed by cognitive telerehabilitation (clinic-VRRS + Tele@H-VRRS), where subjects received face- to-face cognitive VRRS treatment (clinic-VRRS) followed by home-based VRRS treatment (Tele@H-VRRS); (b) those who received face-to-face cognitive VRRS treatment followed by at-home unstructured cognitive stimulation (clinic-VRRS + Tele@H-UCS), where subjects received clinic-VRRS treatment followed by at-home unstructured cognitive stimulation (Tele@H-UCS); and (c) those who Recruitment and treatment were conducted at the IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli of Brescia, the IRCCS Centro Neurolesi Bonino-Pulejo, Messina, and the IRCCS Fondazione Don Carlo Gnocchi Onlus of Milan, from April 2018 to February 2020 (see Figure 1). Study Design This was a multicenter rater-blinded, active-controlled and randomized study. The investigators and outcome assessors were IGURE 1 \| Flow chart showing study subject enrollment and sample processing. FIGURE 1 \| Flow chart showing study subject enrollment and sample processing. November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org 3 Cognitive Treatment and Telerehabilitation MCI Manenti et al. enrollment or during the entire duration of the present study (from baseline to the last follow-up assessment). received face-to-face cognitive treatment as usual (clinic- TAU), where participants received only face-to-face cognitive conventional rehabilitation. Participants Forty-nine older adults fulﬁlling the Petersen criteria for MCI (Petersen, 2011) were recruited. All participants were living independently in the community at the time of their baseline evaluation and were followed up annually during at least 2 years before recruitment in the present study. Assessment Procedures Evaluation Timeline The treatment group assigned to each patient was obtained by stratiﬁed randomization according to age and his/her performance in the Mini Mental State Examination (MMSE) (Folstein et al., 1975). Stratiﬁed randomization is achieved by generating a separate block for each combination of covariates, and participants are assigned to the appropriate block of covariates by a researcher blinded to the study aims. Details of the allocated group were given on cards contained in sequentially numbered, opaque and sealed envelopes. The evaluations were carried out for all groups at baseline (T0), at the end of face-to-face treatment (T1, 1 month from baseline), and after 4 months (T2) and 7 months (T3) from baseline. The T2 follow-up visit corresponded to the end of the at-home treatment for the clinic-VRRS + Tele@H-VRRS and clinic-VRRS + Tele@H-UCS groups. All the assessments were carried out by expert neuropsychologists blinded to the treatment allocation of the participants. Since the participants were aware of the type of intervention received, they were advised not to declare the type of treatment carried out during the post-treatment and follow-up evaluations, making the treatment conditions unknown to the clinical psychologist who conducted the evaluations. The study protocol was executed in its entirety with no signiﬁcant changes. This study was approved by the local ethics committees and was conducted in accordance with the Declaration of Helsinki and reported according to CONSORT guidelines (Boutron et al., 2008; Boutron et al., 2017). The trial was registered at clinicaltrials.gov (NCT number: NCT03486704). A comprehensive clinical, functional and neuropsychological evaluation was performed at all visits (T0, T1, T2, and T3). During baseline assessment, family history of dementia, record of medical events, current medication and complete neurologic examination results were recorded and the CDR scale (Morris, 1997) and the Cognitive Reserve Index questionnaire (CRIq) (Nucci et al., 2012) were completed. In addition, the participants in the clinic-VRRS + Tele@H-VRRS and clinic- VRRS + Tele@H-UCS groups underwent a computerized cognitive assessment and an assessment of system usability. All participants were made fully aware of the aims of the research, and written informed consent was obtained from all subjects from the local center. Clinical and Functional Assessment Since no previous studies have investigated cognitive VRRS treatment eﬀects on memory outcome, the sample size calculation was based on a prior study on patients with AD (Jelcic et al., 2014), with an eﬀect size of 0.85 (Cohen’s d) for the MMSE score (Folstein et al., 1975) improvement after telerehabilitation treatment, a signiﬁcance level (α) of 0.05 and a power (1-β) = 80 [two-tailed independent t-test)]. The estimated sample size was twelve participants for each group. The evaluation of subjective memory complaints was conducted using the 20-item version (range: 20–180) of the Everyday Memory Questionnaire (Sunderland et al., 1986; Calabria et al., 2011). Functional abilities were evaluated using basic (BADL) and instrumental activity of daily living (IADL) scales (Katz, 1983; Lawton and Brody, 1988). Depression was assessed with the 30- item version of the Geriatric Depression Scale (GDS) (Yesavage et al., 1982), whereas neuropsychiatric symptoms were recorded using the Neuropsychiatric Inventory (NPI) (Cummings et al., 1994; Binetti et al., 1998). Finally, quality of life was recorded using the Quality of Life in Alzheimer’s Disease (QOL-AD) scale (Bianchetti et al., 2017). All the participants had normal or corrected-to-normal vision and were characterized by the following: (a) memory complaints; (b) preservation of general cognitive functioning documented by MMSE scores from 24 to 30 (Folstein et al., 1975); (c) age over 65 years; (d) global Clinical Dementia Rating (CDR) score of 0.5 (Morris, 1997); (e) preservation of functional activities; (f) absence of criteria for a diagnosis of dementia according to DSM- V (American Psychiatric Association, 2014); and (g) absence of mood and anxiety disorders. Moreover, before inclusion in the cognitive training protocol, the availability and motivation of subjects to participate consecutively in the protocol for its entire duration and the presence of a caregiver for the completion of questionnaires at all time assessments were veriﬁed. Frontiers in Aging Neuroscience \| www.frontiersin.org Cognitive Rehabilitation Procedures The cognitive rehabilitation protocol was delivered to all participants according to the corresponding experimental group. 1http://khymeia.com/ Face-to-Face Cognitive VRRS Treatment (Clinic-VRRS) Participants assigned to the clinic-VRRS group received twelve 60-min sessions of individualized cognitive rehabilitation using VRRS1 in the clinic with a user-centered approach over TABLE 1 \| Face to face cognitive VRRS treatment (clinic-VRRS). Main domain Task and description Memory - Safe opening – forward: A s requested to memorize it. Wh the same order of presentatio - Visual memory: Pairs of geo and the subject is requested t requested to remember the p - Safe opening- backward: A is requested to memorize it. W in the reverse order to open a - Verbal memory: A list of wor memorize it. When words dis many other words. Attention and Executive functions - Complete the sequence of s the subject is requested to co - Change color: A geometric ﬁ select, from a series of ﬁgures - Rotation: An animal or an ar the sequence, selecting the c displayed direction of rotation - Complete the logical relation displayed on the screen and t cuisenaire rod that satisfy the Visuospatial abilities - Spatial orientation: A duck a four colored balls around the account the duck’s orientation - Road route: A road map is d has to pay attention to the ba - Find the symmetrical: An an asked to select, from a series - Recognize farm animals: A f series of farm animals. TABLE 1 \| Face to face cognitive VRRS treatment (clinic-VRRS). TABLE 1 \| Face to face cognitive VRRS treatment (clinic-VRRS). 1 \| Face to face cognitive VRRS treatment (clinic VRRS). omain Task and description Task duration y - Safe opening – forward: A sequence of numbers appears on the screen and the subject is requested to memorize it. When it disappears, the numbers must be typed on the screen in the same order of presentation to open a safe; 10 min - Visual memory: Pairs of geometric shapes or animals’ cards are displayed on the screen and the subject is requested to memorize them. When cards turn over, the subject is requested to remember the position of each pair of cards; 10 min - Safe opening- backward: A sequence of numbers appears on the screen and the subject is requested to memorize it. Assessment of System Usability Assessment of System Usability To record subjective assessments of the clinic-VRRS, we administered the System Usability Scale (SUS) (Brooke, 1996; Bangor et al., 2008, 2009; Peres et al., 2013) to all the subjects who received clinic-VRRS + Tele@H-VRRS and clinic-VRRS + Tele@H-UCS at T1. Moreover, we recorded the SUS scores at T2 in the clinic-VRRS + Tele@H-VRRS group to assess cognitive telerehabilitation usability (Tele@H- VRRS usability). The SUS is a 10-item, ﬁve-point Likert scale (1 = strongly disagree, 5 = strongly agree). The scoring instructions described by Brooke (35) were considered. The ﬁnal score ranges from 10 to 100. 1993) for visuo-constructional abilities; the Trail Making Test (TMT) part A and part B (Giovagnoli et al., 1996; Siciliano et al., 2019) for attention functions; and the Rey Auditory Verbal Learning Test (RAVLT), immediate and delayed recall (Carlesimo et al., 1996), the Free and Cued Selective Reminding Test (FCSRT) (Frasson et al., 2011) and the Rey– Osterrieth complex ﬁgure test-recall (Caﬀarra et al., 2002) for episodic memory. All the tests were administered and scored according to standard procedures (Lezak et al., 2012) (see Tables 3, 4 for details). Computerized Cognitive Tasks In the case of the participants who received face-to-face VRRS treatment (the clinic-VRRS + Tele@H-VRRS and clinic- VRRS + Tele@H-UCS groups), the performances achieved during VRRS treatment were further analyzed to assess memory, visuospatial abilities, attention and executive functions. In particular, we recorded the performance of each patient in the ﬁrst clinic-VRRS session (as the baseline score, T0), and then we registered the participants’ performances obtained during the last clinic-VRRS session as the post-treatment rating (T1). Moreover, participants underwent computerized cognitive assessment during follow-up assessments (T2, T3) to analyze long-term eﬀects. Face-to-Face Cognitive VRRS Treatment (Clinic-VRRS) When it disappears, the numbers must be typed on the screen in the reverse order to open a safe; 10 min - Verbal memory: A list of words appears on the screen and the subject is requested to memorize it. When words disappear, the subject is requested to identify them in a list of many other words. 10 min n and Executive functions - Complete the sequence of shapes: A sequence of shapes is displayed on the screen and the subject is requested to continue the sequence, selecting the correct elements; 10 min - Change color: A geometric ﬁgure appears on the screen and the subject is requested to select, from a series of ﬁgures, the one that differs from the target only for color; 10 min - Rotation: An animal or an arrow picture is shown and the subject is requested to complete the sequence, selecting the correct pictures from a series of elements, according to the displayed direction of rotation; 10 min - Complete the logical relationship: A cuisenaire rod and a comparison operator are displayed on the screen and the subject is asked to select, from a set of colored rods, the cuisenaire rod that satisfy the logical relationship. 10 min atial abilities - Spatial orientation: A duck appears on the screen and the subject is requested to place four colored balls around the duck, following the written spatial indications and taking into account the duck’s orientation; 10 min - Road route: A road map is displayed on the screen and a ball runs a route. The subject has to pay attention to the ball and to reproduce the route; 10 min - Find the symmetrical: An animal’s picture and a rotation axis are shown and the subject is asked to select, from a series of ﬁgures, the symmetrical one; 10 min - Recognize farm animals: A farm picture is displayed and the subject is requested to ﬁnd a series of farm animals. 10 min Task and description Memory Memory - Safe opening – forward: A sequence of numbers appears on the screen and the subject is requested to memorize it. When it disappears, the numbers must be typed on the screen in the same order of presentation to open a safe; - Safe opening – forward: A sequence of numbers appears on the screen and the subject is requested to memorize it. When it disappears, the numbers must be typed on the screen in the same order of presentation to open a safe; - Safe opening- backward: A sequence of numbers appears on the screen and the subject is requested to memorize it. When it disappears, the numbers must be typed on the screen in the reverse order to open a safe; Attention and Executive functions Attention and Executive functions - Complete the sequence of shapes: A sequence of shapes is displayed on the screen and the subject is requested to continue the sequence, selecting the correct elements; - Change color: A geometric ﬁgure appears on the screen and the subject is requested to select, from a series of ﬁgures, the one that differs from the target only for color; - Rotation: An animal or an arrow picture is shown and the subject is requested to complete the sequence, selecting the correct pictures from a series of elements, according to the displayed direction of rotation; - Rotation: An animal or an arrow picture is shown and the subject is requested to complete the sequence, selecting the correct pictures from a series of elements, according to the displayed direction of rotation; - Complete the logical relationship: A cuisenaire rod and a comparison operator are displayed on the screen and the subject is asked to select, from a set of colored rods, the cuisenaire rod that satisfy the logical relationship. - Spatial orientation: A duck appears on the screen and the subject is requested to place four colored balls around the duck, following the written spatial indications and taking into account the duck’s orientation; - Road route: A road map is displayed on the screen and a ball runs a route. Visuospatial abilities Neuropsychological Assessment In addition to clinical and functional assessments, all participants were tested at each visit with a standardized neuropsychological battery. Cognitive tests were applied to assess a broad range of cognitive abilities commonly aﬀected by MCI. The battery took approximately 90 min and included the MMSE (Folstein et al., 1975) for the assessment of global cognition; Raven’s Colored Progressive Matrices for non-verbal reasoning (Basso et al., 1987); verbal ﬂuency (phonemic, FPL; and semantic, FPC) (Novelli et al., 1986) and action and object naming subtests from the battery for the assessment of aphasic disorders (BADA, Miceli et al., 1994) for language production; the Rey–Osterrieth complex ﬁgure test-copy (Caﬀarra et al., 2002) and the Clock Drawing Test (CDT) (Shulman et al., The following stringent exclusion criteria were applied: (a) other prior or current neurological or major psychiatric disorders; (b) visual perception disorder and/or hearing loss; (c) history of traumatic brain injury, brain tumor or stroke; and (d) history of alcohol abuse. None of the participants had participated in cognitive training protocols within the year before November 2020 \| Volume 12 \| Article 585988 4 Cognitive Treatment and Telerehabilitation MCI Manenti et al. Attention and Executive functions Frontiers in Aging Neuroscience \| www.frontiersin.org Task and description The subject is requested to indicate whether the animal’s picture appears in front or behind the duck, considering the duck’s orientation; 10 mi - Indicates the rotation: A sequence of rotated animals or arrow pictures is shown and the subject has to indicate whether the direction of rotation of the elements in the sequence is clockwise or counterclockwise; - Puzzle: Individual pieces of the puzzle are shown on the screen and the subject is requested to arrange them in order to compose the whole puzzle; - Indicates the rotation: A sequence of rotated animals or arrow pictures is shown and the subject has to indicate whether the direction of rotation of the elements in the sequence is clockwise or counterclockwise; - Puzzle: Individual pieces of the puzzle are shown on the screen and the subject is requested to arrange them in order to compose the whole puzzle; - Connections of points: A series of circles with numbers or letters circles are randomly presented on the screen and the subject is invited to connect the numbered or letters circles in the correct sequence, following the numerical or alphabetical order. - Connections of points: A series of circles with numbers or letters circles are randomly presented on the screen and the subject is invited to connect the numbered or letters circles in the correct sequence, following the numerical or alphabetical order. 4 weeks. Subjects were seated in front of a computer screen in a quiet room. professionals, reality orientation therapy, reminiscence therapy, paper and pencil exercises and metacognitive training aiming to learn cognitive strategies and to use external aids were proposed. Face-to-face cognitive VRRS treatment included twelve exercises designed to enhance memory, visuospatial abilities, attention and executive functions (listed in Table 1). In each treatment session, the participant worked with six exercises, 10 min each, so that each exercise was completed six times over the twelve clinic-VRRS sessions. The subject was asked to continue to perform each task until the end of the set time. The therapist suggested to the participant some strategies aiming to improve performance in all the treatment sessions except for the ﬁrst and last sessions. At the end of each training session, subjects received feedback on their performances, and a detailed report of the results was made available to the therapist, allowing the monitoring of progress over time. Task and description The subject has to pay attention to the ball and to reproduce the route; - Find the symmetrical: An animal’s picture and a rotation axis are shown and the subject is asked to select, from a series of ﬁgures, the symmetrical one; - Find the symmetrical: An animal’s picture and a rotation axis are shown and the subject is asked to select, from a series of ﬁgures, the symmetrical one; - Recognize farm animals: A farm picture is displayed and the subject is requested to ﬁnd a series of farm animals. 10 min - Recognize farm animals: A farm picture is displayed and the subject is requested to ﬁnd a series of farm animals. November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org Cognitive Treatment and Telerehabilitation MCI Manenti et al. TABLE 2 \| At home-based VRRS treatment exercises (Tele@H-VRRS). Main domain Task and description Task duration Memory - Recognize banknotes and coins: A series of banknotes and coins is presented on the screen and the subject is requested to select the one that corresponds to the requested written quantity; 10 min - Collect money up to 10 euros: A group of banknotes and coins is displayed on the screen and the subject is invited to collect coins and banknotes needed to reach the required amount of money. The maximum amount of money is 10 euros; 10 min - Recognize banknotes and coins – back: The back of a series of banknotes and coins is presented on the screen and the subject is requested to select the one that corresponds to the requested written quantity; 10 min - Collect money up to 100 euros: Banknotes and coins is displayed on the screen and the subject is invited to collect coins and banknotes needed to reach the required amount of money. The maximum amount of money is 100 euros. Task and description Clinical VRRS treatment was tailored to the patient’s baseline characteristics through a pre-training session. The starting level and the number of trials were adjusted according to the subject’s performance level using an adaptive staircase procedure. Progress was continuously monitored, and the exercises adaptively progressed in diﬃculty. At the end of this face-to-face treatment, participants were requested not to perform any cognitive activity until the conclusion of the entire protocol. Home-Based Cognitive VRRS Treatment (Tele@H-VRRS) The participants assigned to the Tele@H-VRRS group received, after the end of face-to-face treatment, thirty-six 60-min sessions of home-based cognitive VRRS treatment (see “text footnote 1”), three sessions for week over 3 months. Twelve exercises designed to enhance memory, visuospatial abilities, attention and executive functions, diﬀerent from those used in face-to- face VRRS training, were selected (listed in Table 2). In each treatment session, a participant worked with six exercises, 10 min each, task diﬃculty adaptively progressed, and the performances were continuously monitored by the therapist. The subject was asked to continue to perform each task until the end of the set time. Attention and Executive functions Task and description 10 min Attention and Executive functions - Change of shape: A geometric ﬁgure appears on the screen and the subject is requested to select, from a series of ﬁgures, the one that differs from the target only for shape; 10 min - Find the missing cuisenaire rod: A cuisenaire rod is displayed on the screen and the subject is asked to select, from a set of colored rods, the one that logically completes the sequence; 10 min - Change all: A geometric ﬁgure appears on the screen and the subject is requested to select, from a series of ﬁgures, the one that differs from the target for color, shape and dimension as compared to the target; 10 min - Complete the sequence following the rule: A cuisenaire rod and a rule are displayed on the screen. The subject is asked to select, from a set of colored rods, the two that complete the sequence, taking into account the rule displayed (ascending or descending order). 10 min Visuospatial abilities - Spatial orientation-Front or rear: A duck on a spatial axis and an animal’s picture appear on the screen. The subject is requested to indicate whether the animal’s picture appears in front or behind the duck, considering the duck’s orientation; 10 min - Indicates the rotation: A sequence of rotated animals or arrow pictures is shown and the subject has to indicate whether the direction of rotation of the elements in the sequence is clockwise or counterclockwise; 10 min - Puzzle: Individual pieces of the puzzle are shown on the screen and the subject is requested to arrange them in order to compose the whole puzzle; 10 min - Connections of points: A series of circles with numbers or letters circles are randomly presented on the screen and the subject is invited to connect the numbered or letters circles in the correct sequence, following the numerical or alphabetical order. 10 min TABLE 2 \| At home-based VRRS treatment exercises (Tele@H-VRRS). Task and description - Spatial orientation-Front or rear: A duck on a spatial axis and an animal’s picture appear on the screen. Frontiers in Aging Neuroscience \| www.frontiersin.org Attention and Executive functions RESULTS A total of 79 subjects were evaluated for inclusion in this study. Ultimately, 30 subjects were excluded (27 subjects did not meet the inclusion criteria, and 3 subjects declined to participate), whereas 49 subjects were deemed eligible for participation. A total of 79 subjects were evaluated for inclusion in this study. Ultimately, 30 subjects were excluded (27 subjects did not meet the inclusion criteria, and 3 subjects declined to participate), whereas 49 subjects were deemed eligible for participation. Participants We enrolled 49 patients with MCI, 42 (86%) amnestic MCI and 7 (14%) non-amnestic MCI participants. In particular, the current sample included: (i) 20 amnestic single domain MCI (aMCI-s); (ii) 22 amnestic multiple domain MCI (aMCI-m); 10 of them showed an additional deﬁcit in visuo-constructional functions, 2 in attention, 3 in language, 4 in attention and visuo- constructional functions and 1 in attention and language; (iii) 6 non-amnestic single domain MCI (naMCI-s), presence of a disability in another cognitive area (1 of them showed deﬁcit in language, 3 in visuo-constructional functions and 2 in attention abilities), with normal memory; and (iv) 1 non-amnestic multiple domain MCI (naMCI-m), with disabilities in attention and visuo- constructional functions, with normal memory. Face-to-Face Cognitive Treatment as Usual (Clinic-TAU) The VRRS system has telerehabilitation functionalities, allowing the use of the same functionalities applied in the face-to-face treatment. For the at-home treatment, each participant received a home-based kit including a tablet that allowed access to a daily individualized training program, Participants assigned to the clinic-TAU group received twelve 60-min sessions of group cognitive stimulation in the clinic. During these group sessions, which were led by mental health November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org 6 Cognitive Treatment and Telerehabilitation MCI Manenti et al. a detailed VRRS tablet manual, an exercise instructions booklet and a participant diary. Before beginning the home- based treatment, the therapist downloaded all 36 sessions of the appropriate individualized cognitive training exercise on the patient’s tablet and assisted the participants and their caregivers at ﬁrst access to familiarize them with the technological device. During home-based treatment, the therapist provided continuous assistance for technical diﬃculties and individualized cognitive training exercises were adjusted by the therapist once a week. (time, group and time × group) were considered independent variables. Sequential Sidak correction was used for the evaluation of post hoc. Statistical signiﬁcance was set at p < 0.05. Statistical analyses were carried out by using SPSS 25.0. Graphs were generated using R software (R Core Team, 2013). Outcomes Primary outcome measures were the changes in two tasks of verbal episodic memory: (a) the RAVLT, immediate and delayed recall; and (b) the FCSRT. The secondary outcomes included quality of life measures, subjective memory complaints and neuropsychological measures evaluating memory, language, attention and visuo-constructional functions. The choice of outcomes was based on previous literature ﬁndings and on predeﬁned hypotheses of investigation. Inference on them was performed considering each outcome singularly (i.e., not simultaneous inference was done on all outcome, thus no multiple comparison was needed to compare outcomes results each other (see e.g., Proschan and Follmann, 1995; Bender and Lange, 2001). Home-Based Unstructured Cognitive Stimulation (Tele@H-UCS) Subjects assigned to the Tele@H-UCS group were requested to work on detailed activities 60 min a day, three times a week (36 sessions in total) over 12 weeks after the end of face-to-face treatment. They received, from the therapist, an instructions booklet and a participant diary. Conventional instruments, such as paper and pencil exercises, creative manual activities, reading newspapers and magazines, watching documentaries, crosswords and sudoku, were suggested. Each participant was requested to compile a detailed diary reporting the performed activities. These 49 subjects were randomized into the three experimental groups: 18 participants were allocated to the clinic-VRRS + Tele@H-VRRS group, 14 subjects to the clinic-VRRS + Tele@H-UCS group and 17 participants to the clinic-TAU group. All the participants completed the baseline (T0), post-treatment (T1) and follow-up (T2 and T3) evaluations (see Figure 1-Flowchart). Adherence to treatment was indexed by the number of sessions completed for each participant. Concerning the clinic-VRRS + Tele@H-VRRS group, all 18 participants completed the 12 sessions of face-to-face VRRS treatment, 6 participants completed all 36 sessions of at-home VRRS training, whereas all other subjects completed more than 70% of the telerehabilitation sessions. Moreover, all 14 participants in the clinic-VRRS + Tele@H-UCS group completed the 12 sessions of face-to-face VRRS treatment, 7 subjects completed the 36 sessions of at-home unstructured cognitive stimulation, and the other subjects completed more than 70% of the at-home unstructured cognitive stimulation sessions. Finally, 8 subjects in the clinic-TAU group completed the 12 sessions, whereas all the other subjects completed more than 70% of the usual treatment sessions. Computerized Cognitive Tasks p g Diﬀerent GLMMs were performed (Table 5), and it followed that the two groups were diﬀerent, across time, in the two tasks “Complete the logic relationship” and “Safe opening- forward” (interaction term p-values equal to 0.007 and 0.016, respectively). For the ﬁrst task, the performance of subjects was diﬀerent at all time points in the two groups (Figure 3). Post hoc analysis showed that there was a diﬀerent trend in the clinic-VRRS + Tele@H-VRRS and clinic-VRRS + Tele@H- UCS groups, with very similar scores at T1 (p = 0.825) and higher scores in the clinic-VRRS + Tele@H-VRRS group than in the clinic-VRRS + Tele@H-UCS group at both follow-up visits (T2: p = 0.033; T3: p = 0.042). For the safe opening-forward task, the score diﬀered signiﬁcantly over time between the two groups: only the clinic-VRRS + Tele@H-VRRS group showed an improvement from T0 and T1 (p = 0.017) and worsening from T1 and T2 (p = 0.044). All the resulting signiﬁcant outcomes are presented in Figure 2. In particular, the FCSRT IFR, CDT and TMT A scores improved only after clinic-VRRS (FCSRT IFR: p < 0.001; CDT: p < 0.001; TMT A: p = 0.002). Although in TMT A and CDT scores a signiﬁcant diﬀerence was observed between the two groups at baseline assessment, an improvement in these tests could be recorded exclusively in clinic-VRRS group. Regarding the FPC, the clinic-VRRS group showed an increase from T0 to T1, whereas the clinic-TAU group showed worse FPC performance, inducing a signiﬁcant time × group interaction (although the changes within groups did not reach signiﬁcance). Thus, by combining the information provided by the models and ﬁgures, we showed that the clinic-VRRS was more eﬃcient than clinic-TAU, improving memory (FCSRT IFR), language (FPC), attention (TMT A) and visuo-constructional abilities (CDT). Home-Based VRRS Rehabilitation (Tele@H-VRRS) vs. Home-Based Unstructured Cognitive Stimulation (Tele@H-UCS) vs. No Additional Treatment Statistical Analysis Summary statistics are expressed as means and standard deviations. Comparisons of socio-demographic features between groups were evaluated by parametric (t-tests) or corresponding non-parametric (Mann–Whitney) tests. Variable distribution was inspected through histograms and the use of the Kolmogorov– Smirnov and Shapiro–Wilk tests. Consistent with the type of variable distribution (Gaussian, Poisson or Gamma), generalized linear mixed models (GLMMs) were performed to evaluate score diﬀerences across time points and between groups. In detail, the ﬁrst series of GLMMs was applied to compare two groups (clinic-TAU vs. clinic-VRRS + Tele@H-VRRS together with clinic-VRRS + Tele@H-UCS) across two time points (baseline and post-treatment). Subsequently, GLMMs were performed to compare the three subject groups across four time points (baseline, post-treatment, 4-month follow- up, and 7-month follow-up). Diﬀerent test scores were used as dependent variables (one for each model), and the eﬀects The mean age of the participants was 76.5 years (SD = 4.2), the mean number of years of education was 10.7 years (SD = 4.4), and 24 participants (49.0%) were male. The clinic-VRRS + Tele@H- VRRS group included 13 male and 5 female participants (age: mean, 75.3 years, SD: 3.3; education: 11.8 years, SD 4.8); the clinic-VRRS + Tele@H-UCS group included 4 male and 10 November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org 7 Cognitive Treatment and Telerehabilitation MCI Manenti et al. female participants (age: mean, 76.3 years, SD: 4.9; education: 10.5 years, SD 4.8); and the clinic-TAU group included 7 male and 10 female participants (age: mean, 78.1 years, SD: 4.1; education: 9.8 years, SD 3.7). provided, while those who received usual care did not receive any additional treatment during the following months. Our second aim was to assess whether the telerehabilitation VRRS system could extend the beneﬁcial eﬀects of face-to- face VRRS treatment. The three groups did not diﬀer regarding age (p = 0.142) or education (p = 0.405), but there was a signiﬁcant diﬀerence in sex (p = 0.036). Descriptive statistics of all clinical features of the three groups of patients, measured at the four time points, are reported in Table 3. To verify this hypothesis, diﬀerent GLMMs were performed (one for each outcome) using time (four time points), group (three groups) and their interaction as independent variables. The results of the models are shown in Table 4 (last columns). Face-to-Face Cognitive Virtual Reality Rehabilitation System vs. Face-to-Face Cognitive Treatment as Usual Regarding the neuropsychological assessment, the outcome that behaved in a signiﬁcantly diﬀerent way in the three groups over time was the FCSRT IFR. As reported earlier, the FCSRT IFR showed an improvement from T0 to T1 in the clinic-VRRS group. By splitting the clinic-VRRS group into two subgroups, this improvement signiﬁcantly decreased after the end of face-to-face treatment in the clinic- VRRS + Tele@H-UCS group (T1 vs. T2 p = 0.025; T1 vs. T3 p = 0.003), whereas no strong evidence of changes in the clinic-VRRS + Tele@H-VRRS group was recorded (T1 vs. T2 p = 0.055; T1 vs. T3 p = 0.084). Although the mean changes from T1 to T2 were similar in clinic- VRRS + Tele@H-UCS and in clinic-VRRS + Tele@H-VRRS groups, diﬀerent statistical signiﬁcance were mainly driven by diﬀerence in variability (standard deviations) recorded in the two experimental groups. As stated above, the clinic-TAU group did not show any signiﬁcant modiﬁcation across all time points (see Table 4). Our ﬁrst aim was to evaluate the eﬃcacy of the face-to-face VRRS (clinic-VRRS) and to compare it to that of cognitive treatment as usual (clinic-TAU). For this purpose, we considered two groups of subjects: those who received VRRS (i.e., the clinic-VRRS + Tele@H-VRRS and clinic-VRRS + Tele@H-UCS groups) and those who received treatment as usual (i.e., the clinic- TAU group), and we compared their scores at the ﬁrst two time points (baseline, T0; and after face-to-face treatment, T1). We performed diﬀerent GLMMs, one for each outcome, with time (two time points), group (two treatments) and their interaction as independent variables, to verify whether the two treatments had diﬀerent eﬀects on the outcomes. The results are shown in Table 4 (ﬁrst columns). The performances of the two groups at the two time points were signiﬁcantly diﬀerent for the FCSRT IFR, FPC and CDT (p-value of the interaction term equal to 0.010, 0.024, and 0.010, respectively). Moreover, for the TMT A, there was a trend toward signiﬁcance (p = 0.058). Cut-o >33 28.5 >4.68 19.5 assessment. y Face to Face VRRS followed by Home-based unstructured cognitive stimulation (n = 14) Face to Face cognitive treatment as usual (n = 17) onths ow-up 7 Months Follow-up Baseline Post- treatment 4 Months Follow-up 7 Months Follow-up Baseline Post- treatment 4 Months Follow-up 7 Months Follow-up Cut- an (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) 5 (27.9) 61.0 (24.1) 76.6 (30.9) 76.0 (30.6) 76.2 (30.0) 72.4 (26.8) 66.2 (27.1) 61.9 (26.3) 62.0 (26.5) 71.1 (25.2) 9 (4.0) 32.6 (8.2) 33.0 (5.9) 33.4 (5.7) 32.1 (6.9) 32.3 (7.3) 34.9 (4.4) 35.1 (3.4) 34.7 (4.2) 32.7 (4.9) >3 9 (8.6) 26.1 (7.6) 31.0 (7.0) 32.1 (6.7) 30.2 (8.1) 33.4 (8.4) 30.2 (7.7) 30.1 (6.5) 30.9 (6.6) 31.5 (7.8) >28 9 (3.9) 3.6 (3.3) 4.6 (2.3) 4.3 (3.4) 5.1 (3.6) 6.0 (4.2) 4.5 (3.2) 4.2 (3.0) 4.2 (2.6) 4.8 (3.9) >4. System Usability Scale Interestingly, the SUS, administered at T1 to the two groups who received clinic-VRRS, showed good usability performance of the clinic-VRRS system (67.8, SD 11.6), and the SUS scores obtained at T2 in the clinic-VRRS + Tele@H-VRRS group, which received home-based telerehabilitation from T1 and T2, highlighted good usability performance from the VRRS telerehabilitation system (69.6, SD 8.8) (Bangor et al., 2008). After the ﬁrst month, the participants who received clinic- VRRS were divided into two subgroups in which cognitive telerehabilitation and unstructured cognitive stimulation were November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org 8 2 (8.5) 17.8 (8.0) 20.4 (6.1) 24.4 (6.5) 22.1 (6.3) 21.4 (6.2) 19.7 (7.9) 19.9 (9.0) 21.4 (8.4) 21.7 (10.2) >19 2 (6.5) 32.0 (4.4) 33.4 (4.9) 33.6 (3.8) 33.9 (3.2) 34.2 (3.1) 33.4 (3.2) 32.5 (4.9) 33.1 (3.9) 33.5 (3.6) ≥3 7 (3.6) 6.6 (4.1) 6.7 (2.8) 8.3 (2.6) 8.6 (3.2) 8.1 (3.2) 6.4 (4.3) 7.0 (3.8) 6.6 (4.0) 7.2 (4.1) >6.3 4 (1.8) 10.4 (2.0) 10.9 (1.9) 11.1 (1.6) 11.3 (1.5) 11.4 (1.2) 10.8 (2.1) 10.9 (1.9) 10.8 (1.5) 11.2 (1.2) ≥1 8 (0.2) 0.8 (0.2) 0.9 (0.2) 0.8 (0.2) 0.9 (0.1) 0.9 (0.1) 0.9 (0.1) 0.8 (0.2) 0.9 (0.2) 0.9 (0.1) ≥0 2 (6.6) 30.1 (7.6) 29.8 (6.7) 33.4 (8.4) 30.6 (8.7) 29.8 (9.5) 28.9 (8.4) 31.7 (8.8) 31.2 (11.5) 30.4 (7.9) >1 1 (5.5) 29.1 (6.4) 29.6 (5.9) 29.2 (6.8) 28.5 (3.9) 27.8 (4.6) 30.9 (6.3) 29.4 (6.0) 29.1 (7.8) 29.2 (6.1) >2 7 (2.4) 26.9 (2.5) 26.4 (1.7) 27.0 (2.5) 26.4 (2.6) 27.2 (1.7) 26.9 (2.3) 27.1 (1.8) 27.2 (1.8) 27.8 (2.2) 9 (2.5) 24.9 (2.8) 24.1 (2.4) 24.6 (3.0) 24.4 (3.0) 25.0 (2.8) 24.4 (2.9) 25.6 (2.4) 25.1 (2.3) 25.5 (2.6) (24.7) 55.3 (24.1) 59.5 (21.2) 53.9 (17.7) 65.9 (26.1) 64.5 (27.2) 46.8 (16.0) 47.1 (16.2) 52.4 (21.4) 60.0 (37.9) <9 (139.4) 245.3 (126.4) 237.6 (160.0) 238.3 (119.7) 241.4 (142.2) 276.7 (118.5) 206.9 (123.8) 231.2 (184.7) 219.8 (164.3) 268.2 (201.5) <28 7 (0.9) 1.7 (0.8) 2.9 (1.0) 2.2 (1.1) 2.6 (1.2) 2.6 (0.9) 1.9 (0.7) 1.8 (0.7) 2.0 (1.1) 1.8 (0.7) ≤2 ackets. Cut-off scores according to Italian normative data are reported. msec: milliseconds; VRRS: virtual reality rehabilitation system. Cognitive Treatment and Telerehabilitation MCI Manenti et al. TABLE 4 \| Generalized Linear Mixed Models results for neuropsychological test. clinic-VRRS (clinic-VRRS + Tele@H-VRRS and clinic-VRRS + Tele@H-UCS) vs. clinic-TAU longitudinal evaluation at two time points clinic-VRRS + Tele@H-VRRS vs. clinic-VRRS + Tele@H-UCS vs. clinic-TAU longitudinal evaluation at four time points p_Time p_Time p_Time × Group p_Time p_Group p_Time × Group Clinical and Functional Assessment Everyday Memory Questionnaire (EMQ) 0.005 0.450 0.713 0.072 0.412 0.288 Quality of Life in Alzheimer’s Disease (QOL-AD) QOL-AD – composite score 0.701 0.333 0.999 0.184 0.687 0.472 Memory Rey Auditory Verbal Learning Test (RAVLT), immediate recall 0.796 0.962 0.699 0.767 0.269 0.088 Rey Auditory Verbal Learning Test (RAVLT), delayed recall 0.779 0.933 0.629 0.601 0.636 0.086 Free and Cued Selective Reminding Test (FCSRT) FCSRT – Immediate free recall (IFR) 0.004 0.675 0.010 <0.001 0.275 0.003 FCSRT – Immediate total recall (ITR) 0.222 0.773 0.253 0.293 0.431 0.335 FCSRT – delayed free recall (DFR) 0.012 0.932 0.471 0.003 0.256 0.085 FCSRT – delayed total recall (DTR) 0.545 0.661 0.942 0.103 0.486 0.702 FCSRT – index of sensitivity of cueing (ISC) 0.137 0.347 0.222 0.031 0.219 0.829 Language Verbal ﬂuency, phonemic (FPL) <0.001 0.767 0.904 0.006 0.955 0.783 Verbal ﬂuency, semantic (FPC) 0.967 0.640 0.024 0.536 0.880 0.085 Battery for Analysis of Aphasic Deﬁcits (B.A.D.A.) B.A.D.A. – Objects naming 0.239 0.828 0.671 0.129 0.747 0.677 B.A.D.A. – Actions naming 0.005 0.630 0.289 0.024 0.778 0.797 Attentional functions Trail Making Test (TMT) TMT, part A (msec) 0.098 0.104 0.058 0.002 0.411 0.150 TMT, part B (msec) 0.836 0.686 0.176 0.315 0.900 0.549 Visuo-constructional functions Clock Drawing Test (CDT) 0.001 0.211 0.010 0.006 0.005 0.276 msec: milliseconds; VRRS: virtual reality rehabilitation system; bold font indicates statistical signiﬁcance. TABLE 4 \| Generalized Linear Mixed Models results for neuropsychological test Frontiers in Aging Neuroscience \| www.frontiersin.org DISCUSSION Diﬀerent strategies, including stimulation restorative cognitive training, compensatory cognitive training and multicomponent training, have been used to reduce cognitive diﬃculties in subjects with MCI when assessing diﬀerent outcomes (for review see Li et al., 2011; Hong et al., 2015; Sherman et al., 2017). In recent years, the potential usefulness of cognitive training in normal aging and MCI has received increased attention (Brown, 2015; Vannini et al., 2017). Recent meta-analyses and reviews have reported that non-pharmacological cognitive interventions are eﬀective in maintaining cognitive function in high-risk older adults (Kidd, 2008; Li et al., 2011, 2014, 2017; Hong et al., 2015; Janoutova et al., 2015; Sherman et al., 2017; Yao et al., 2020). The main purpose of this study was to investigate the eﬃcacy of the face-to-face cognitive VRRS and to compare it to that of face-to-face cognitive treatment as usual for subjects with MCI. Frontiers in Aging Neuroscience \| www.frontiersin.org November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org 10 Cognitive Treatment and Telerehabilitation MCI Manenti et al. FIGURE 2 \| Effects of Face to Face cognitive VRRS (clinic-VRRS) vs. Face to Face cognitive treatment as Usual (clinic-TAU) on neuropsychological assessment. Asterisks indicate statistical signiﬁcance. FIGURE 2 \| Effects of Face to Face cognitive VRRS (clinic-VRRS) vs. Face to Face cognitive treatment as Usual (clinic-TAU) on neuropsychological assessment. Asterisks indicate statistical signiﬁcance FIGURE 2 \| Effects of Face to Face cognitive VRRS (clinic-VRRS) vs. Face to Face cognitive treatment as Usual (clinic-TAU) on neuropsychological assessment. Asterisks indicate statistical signiﬁcance. TABLE 5 \| Descriptive statistics and Generalized Linear Mixed Models results for Computerized Cognitive Tasks. DISCUSSION Face to Face VRRS followed by home-based VRRS (n = 18) Face to Face VRRS followed by home-based unstructured cognitive stimulation (n = 14) Baseline Post- treatment 4 Months Follow-up 7 Months Follow-up Baseline Post- treatment 4 Months Follow-up 7 Months Follow-up p_Time p_Group p_Time × Group Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Memory Safe opening – forward Mean score (%) 25.6 (13.2) 32.7 (9.4) 26.7 (11.6) 31.7 (11.3) 30.8 (15.5) 31.3 (12.1) 36.5 (18.0) 31.0 (14.5) 0.047 0.418 0.016 Visual memory Mean score (%) 80.0 (4.7) 80.0 (5.5) 80.1 (7.1) 78.2 (6.2) 79.7 (4.8) 81.3 (3.7) 79.8 (3.3) 81.5 (3.4) 0.827 0.41 0.168 Safe opening – backward Mean score (%) 29.6 (11.2) 33.7 (9.5) 32.3 (13.8) 37.0 (21.1) 30.6 (14.9) 36.3 (9.8) 36.9 (8.1) 37.1 (12.8) 0.021 0.547 0.690 Verbal memory Mean score (%) 82.6 (6.5) 84.4 (6.4) 84.9 (6.0) 84.4 (5.1) 81.5 (5.6) 85.5 (3.9) 85.8 (4.9) 84.4 (4.9) 0.010 0.993 0.484 Attention and Executive functions Complete the sequence of shapes Mean score (%) 93.1 (7.8) 97.3 (2.8) 97.6 (2.3) 97.2 (3.3) 91.9 (9.1) 96.7 (3.7) 97.6 (2.0) 97.5 (2.7) 0.004 0.751 0.914 Change color Mean score (%) 99.6 (0.3) 99.9 (0.2) 99.9 (0.2) 99.9 (0.1) 99.4 (0.3) 99.8 (0.2) 99.8 (0.2) 99.7 (0.4) <0.001 0.021 0.205 Rotation Mean score (%) 90.6 (4.4) 94.4 (4.9) 90.3 (16.9) 89.0 (19.7) 85.7 (5.9) 89.0 (8.6) 87.2 (9.5) 88.7 (8.5) <0.001 0.224 0.665 Complete the logical relationship Mean score (%) 98.7 (1.4) 99.4 (0.8) 99.3 (1.0) 99.2 (0.9) 97.6 (2.3) 99.4 (0.8) 98.5 (1.0) 98.5 (1.6) <0.001 0.019 0.007 Visuospatial abilities Spatial orientation Mean score (%) 98.4 (1.9) 99.2 (0.8) 98.8 (1.4) 99.2 (1.2) 97.0 (3.2) 99.1 (0.7) 98.4 (1.4) 98.4 (1.6) 0.009 0.101 0.403 Road route Mean score (%) 84.8 (7.6) 90.4 (4.3) 86.2 (4.3) 88.0 (6.8) 85.2 (4.4) 91.0 (4.9) 86.0 (5.8) 85.4 (6.2) <0.001 0.679 0.610 Find the symmetrical Mean score (%) 95.3 (1.7) 95.7 (1.2) 96.2 (0.9) 95.6 (1.3) 94.0 (1.2) 94.6 (1.0) 94.6 (1.1) 94.7 (0.8) 0.037 <0.001 0.470 Recognize farm animals Mean score (%) 86.5 (10.3) 92.4 (3.6) 91.7 (4.3) 88.1 (5.7) 82.7 (7.4) 92.4 (5.2) 89.9 (6.8) 86.9 (7.6) <0.001 0.288 0.370 *data available for 16 patients of this group. Standard deviation between brackets. msec: milliseconds; VRRS: virtual reality rehabilitation system; bold font indicates statistical signiﬁcance. DISCUSSION Overall, high rates of participant agreement, recruitment and treatment adherence supported the feasibility of both face-to- face and telerehabilitation interventions. Moreover, the analyses on system usability evidenced good usability of clinic-VRRS and Tele@H-VRRS. The available evidence is insuﬃcient to draw ﬁrm conclusions on the eﬀects of diﬀerent interventions on functional activities or quality of life. Further, well-designed studies investigating the eﬃcacy of cognitive telerehabilitation are necessary. The current ﬁndings show a signiﬁcant improvement in memory, language and visuo-constructional abilities after the end of face-to-face cognitive VRRS treatment compared to face- to-face cognitive treatment as usual. Our ﬁndings are in line with previous studies that described the usefulness of face-to- face virtual reality rehabilitation protocols aimed at improving memory and executive functioning in older participants with mild cognitive diﬃculties (Liao et al., 2019; Moreno et al., 2019; Tuena et al., 2020) as well as previous studies of eﬀective cognitive treatments to prevent and slow the progression of MCI (Belleville, 2008). We acknowledge that our study has some limitations. First, given our relatively small sample size, the recorded ﬁndings should be conﬁrmed in larger samples to reach a ﬁrm conclusion. A larger sample size would make it possible to take into account in the analyses individual variables such as age, sex and gender. Furthermore, the adoption of longer follow-up visits in future studies would better highlight the duration of the long- term eﬀects induced by the applied treatments. Finally, in the present study some intergroup variabilities in cognitive scores are recorded and the use of a crossover design could be used in future studies in order to avoid problems of comparability of the experimental groups with regard to confounding characteristics. Regarding at-home treatment, we found that cognitive VRRS telerehabilitation has comparable eﬀects to conventional rehabilitation in improving cognitive abilities in patients with neurodegenerative diseases. We evidenced positive eﬀects of VRRS telerehabilitation interventions, generally comparable with those cognitive unstructured home-based intervention. This comparable eﬀect may be related to the modalities in which the VRRS intervention was delivered: a computerized cognitive training modality without a real interaction and feedback between patient and therapist. Frontiers in Aging Neuroscience \| www.frontiersin.org DISCUSSION ABLE 5 \| Descriptive statistics and Generalized Linear Mixed Models results for Computerized Cognitive Tasks. November 2020 \| Volume 12 \| Article 585988 11 Frontiers in Aging Neuroscience \| www.frontiersin.org Cognitive Treatment and Telerehabilitation MCI Manenti et al. FIGURE 3 \| Effects of home-based cognitive telerehabilitation (Tele@H-VRRS) vs. home-based unstructured cognitive stimulation (Tele@H-UCS) vs. no treatment on computerized cognitive tasks. Asterisks indicate statistical signiﬁcance. FIGURE 3 \| Effects of home-based cognitive telerehabilitation (Tele@H-VRRS) vs. home-based unstructured cognitive stimulation (Tele@H-UCS) vs. no treatment on computerized cognitive tasks. Asterisks indicate statistical signiﬁcance. Speciﬁcally, we hypothesized that face-to-face cognitive VRRS treatment may lead to an improvement in cognitive functions, that is, memory and attentional abilities, compared with face-to- face cognitive treatment as usual. asynchronous (in which patient and therapist do not interact in real time) interactions, and the type of monitoring feedback, i.e., on line (during the intervention) or oﬀline (delayed), are key components that may inﬂuence the eﬃcacy of the telerehabilitation and have thus been considered (Di Tella et al., 2019) in future studies. Importantly, clinic-VRRS + Tele@H- VRRS group showed greater maintenance of treatment gains in episodic memory (i.e., FCSRT). Moreover, participants in the clinic-VRRS + Tele@H-VRRS group exhibited signiﬁcant intervention-related improvements in executive functions as assessed by computerized cognitive tasks, with maintenance of gains at the 7-month follow-up. Our results might be related to patient’s engagement and adherence in a telerehabilitation design involving asynchronous therapist-patient interactions (Matamala-Gomez et al., 2020). Moreover, we assessed whether an innovative cognitive telerehabilitation program could induce long-term cognitive beneﬁts. To address these questions, we compared the eﬀects of face- to-face cognitive VRRS followed by cognitive telerehabilitation (clinic-VRRS + Tele@H-VRRS), face-to-face cognitive VRRS followed by at-home unstructured cognitive stimulation (clinic- VRRS + Tele@H-UCS), and face-to-face cognitive rehabilitation program as usual (clinic-TAU) on cognition in patients with MCI. Overall, high rates of participant agreement, recruitment and treatment adherence supported the feasibility of both face-to- face and telerehabilitation interventions. Moreover, the analyses on system usability evidenced good usability of clinic-VRRS and Tele@H VRRS To address these questions, we compared the eﬀects of face- to-face cognitive VRRS followed by cognitive telerehabilitation (clinic-VRRS + Tele@H-VRRS), face-to-face cognitive VRRS followed by at-home unstructured cognitive stimulation (clinic- VRRS + Tele@H-UCS), and face-to-face cognitive rehabilitation program as usual (clinic-TAU) on cognition in patients with MCI. November 2020 \| Volume 12 \| Article 585988 REFERENCES nonpharmacologic treatment: explanation and elaboration. Ann. Intern. Med. 148, 295–309. doi: 10.7326/0003-4819-148-4-200802190-00008 American Psychiatric Association (2014). DSM-5: Manuale Diagnostico e Statistico dei Disturbi Mentali. Milan: Raﬀaello Cortina editore. Brennan, D., Georgeadis, A., and Baron, C. (2002). Telerehabilitation tools for the provision of remote speech-language treatment. Top. 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Disord. 47, 129–130. doi: 10.1159/000497799 Brooke, J. (1996). SUS-A quick and dirty usability scale. Usability Eval. Ind. 189, 4–7. Bangor, A., Kortum, P., and Miller, J. (2009). Determining what individual SUS scores mean: adding an adjective rating scale. J. Usability Stud. 4, 114–123. Brown, G. C. (2015). Living too long: the current focus of medical research on increasing the quantity, rather than the quality, of life is damaging our health and harming the economy. EMBO Rep. 16, 137–141. doi: 10.15252/embr. 201439518 Bangor, A., Kortum, P. T., and Miller, J. T. (2008). An empirical evaluation of the system usability scale. Int. J. Hum. Comput. Interact. 24, 574–594. doi: 10.1080/10447310802205776 Basso, A., Capitani, E., and Laiacona, M. (1987). Raven’s coloured progressive matrices: normative values on 305 adult normal controls. Funct. Neurol. 2, 189–194. Burdea, G., Polistico, K., Krishnamoorthy, A., House, G., Rethage, D., Hundal, J., et al. (2015). Feasibility study of the BrightBrainerTM integrative cognitive rehabilitation system for elderly with dementia. Disabil. Rehabil. Assist. Technol. 10, 421–432. doi: 10.3109/17483107.2014.900575 Belleville, S. (2008). Cognitive training for persons with mild cognitive impairment. Int. Psychogeriatr. 20, 57–66. doi: 10.1017/s104161020700631x Burton, R. L., and O’Connell, M. E. (2018). Telehealth rehabilitation for cognitive impairment: randomized controlled feasibility trial. JMIR Res. Protoc. 7:e43. doi: 10.2196/resprot.9420 Bender, R., and Lange, S. (2001). Adjusting for multiple testing—when and how? J. Clin. Epidemiol. 54, 343–349. AUTHOR CONTRIBUTIONS or a lack of local services. The recent COVID-19 emergency has clearly indicated the importance of remote delivery of cognitive rehabilitation to support ongoing rehabilitation services and guarantee continuity of care to subjects with cognitive impairment. RM, FB, FA, SI, PB, SC, and MC: conception and methodology. AM and CF: formal analysis. RM, EG, FB, IP, FR, SD, FA, VC, GB, SI, PB, SC, and MC: investigation. RM, EG, FB, AM, CF, IP, FR, SD, FA, VC, GB, and MC: data curation. RM, EG, AM, CF, IP, and MC: writing – original draft preparation. All authors contributed to writing – review and editing and have read and agreed to the published version of the manuscript. ETHICS STATEMENT This work was supported by the Italian Ministry of Health (Ricerca Corrente and Rete IRCCS delle Neuroscienze e della Neuroriabilitazione – Teleneuroriabilitazione). AM and CF were supported by the Italian Ministry of Health (Ricerca Corrente, Rete IRCCS delle Neuroscienze e della Neuroriabilitazione – Teleneuroriabilitazione and Cinque per Mille). This work was supported by the Italian Ministry of Health (Ricerca Corrente and Rete IRCCS delle Neuroscienze e della Neuroriabilitazione – Teleneuroriabilitazione). AM and CF were supported by the Italian Ministry of Health (Ricerca Corrente, Rete IRCCS delle Neuroscienze e della Neuroriabilitazione – Teleneuroriabilitazione and Cinque per Mille). The studies involving human participants were reviewed and approved by Comitato Etico IRCCS Istituto Centro San Giovanni di Dio–Fatebenefratelli; 25125 BRESCIA–Via Pilastroni, 4. The patients/participants provided their written informed consent to participate in this study. DISCUSSION The synchronous (in which patient and therapist perform exercises in real time) or Notwithstanding this, our study provides preliminary evidence in support of individualized VRRS treatment, and telerehabilitation delivery for cognitive rehabilitation is quite encouraging and should pave the way for future studies aiming at identifying optimal treatment protocols in subjects with MCI. This research supports the feasibility and beneﬁts of cognitive rehabilitation provided by telerehabilitation systems. This may be particularly important for subjects with limited access to therapy due to geographical distance, transport diﬃculties November 2020 \| Volume 12 \| Article 585988 12 Cognitive Treatment and Telerehabilitation MCI Manenti et al. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. nonpharmacologic treatment: explanation and elaboration. Ann. Intern. Med. 148, 295–309. doi: 10.7326/0003-4819-148-4-200802190-00008 REFERENCES doi: 10.1016/s0895-4356(00)00314-0 Caﬀarra, P., Vezzadini, G., Dieci, F., Zonato, F., and Venneri, A. (2002). Rey- Osterrieth complex ﬁgure: normative values in an Italian population sample. Neurol. Sci. 22, 443–447. doi: 10.1007/s100720200003 Bharucha, A. J., Anand, V., Forlizzi, J., Dew, M. A., Reynolds, C. F. III, Stevens, S., et al. (2009). 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Rev. 27, 440–484. doi: 10.1007/s11065-017-9363-3 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. Shulman, K. I., Pushkar Gold, D., Cohen, C. A., and Zucchero, C. A. (1993). Clock- drawing and dementia in the community: a longitudinal study. Int. J. Geriatr. Psychiatry 8, 487–496. November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org REFERENCES doi: 10.1002/gps.930080606 Copyright © 2020 Manenti, Gobbi, Baglio, Macis, Ferrari, Pagnoni, Rossetto, Di Tella, Alemanno, Cimino, Binetti, Iannaccone, Bramanti, Cappa and Cotelli. 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. Copyright © 2020 Manenti, Gobbi, Baglio, Macis, Ferrari, Pagnoni, Rossetto, Di Tella, Alemanno, Cimino, Binetti, Iannaccone, Bramanti, Cappa and Cotelli. 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. Siciliano, M., Chiorri, C., Battini, V., Sant’elia, V., Altieri, M., Trojano, L., et al. (2019). Regression-based normative data and equivalent scores for Trail Making Test (TMT): an updated Italian normative study. Neurol. Sci. 40, 469–477. doi: 10.1007/s10072-018-3673-y Solana, J., Cáceres, C., García-Molina, A., Opisso, E., Roig, T., Tormos, J. M., et al. (2015). Improving brain injury cognitive rehabilitation by personalized November 2020 \| Volume 12 \| Article 585988 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org 15
https://openalex.org/W2597245724	https://www.frontiersin.org/articles/10.3389/fcimb.2017.00079/pdf	English	null	A Co-infection Model System and the Use of Chimeric Proteins to Study Chlamydia Inclusion Proteins Interaction	Frontiers in cellular and infection microbiology	2,017	cc-by	8,346	Edited by: Anders Omsland, Washington State University, USA Reviewed by: Jason A. Carlyon, Virginia Commonwealth University School of Medicine, USA Laszlo Kari, National Institute of Allergy and Infectious Diseases (NIH), USA Keywords: Chlamydia, inclusion membrane protein, IncD, IncE, chimeric Inc protein, homo- and heterotypic interaction Correspondence: Isabelle Derré id8m@virginia.edu Correspondence: Isabelle Derré id8m@virginia.edu Correspondence: Isabelle Derré id8m@virginia.edu ORIGINAL RESEARCH published: 14 March 2017 doi: 10.3389/fcimb.2017.00079 Ying Han 1 and Isabelle Derré 2 1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA, 2 Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA Chlamydia trachomatis is an obligate intracellular bacterium associated with trachoma and sexually transmitted diseases. During its intracellular developmental cycle, Chlamydia resides in a membrane bound compartment called the inclusion. A subset of Type III secreted effectors, the inclusion membrane proteins (Inc), are inserted into the inclusion membrane. Inc proteins are strategically positioned to promote inclusion interaction with host factors and organelles, a process required for bacterial replication, but little is known about Inc proteins function or host interacting partners. Moreover, it is unclear whether each Inc protein has a distinct function or if a subset of Inc proteins interacts with one another to perform their function. Here, we used IncD as a model to investigate Inc/Inc interaction in the context of Inc protein expression in C. trachomatis. We developed a co-infection model system to display different tagged Inc proteins on the surface of the same inclusion. We also designed chimeric Inc proteins to delineate domains important for interaction. We showed that IncD can self-interact and that the full-length protein is required for dimerization and/or oligomerization. Altogether our approach can be generalized to any Inc protein and will help to characterize the molecular mechanisms by which Chlamydia Inc proteins interact with themselves and/or host factors, eventually leading to a better understanding of C. trachomatis interaction with the mammalian host. Keywords: Chlamydia, inclusion membrane protein, IncD, IncE, chimeric Inc protein, homo- and heterotypic interaction INTRODUCTION Received: 22 November 2016 Accepted: 27 February 2017 Published: 14 March 2017 Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for the most common preventable blindness from infectious origin and is the leading cause of sexually transmitted infection of bacterial origin (Schachter, 1999). The ocular and genital tract epithelia are the primary sites of infection. After entry, C. trachomatis resides in a membrane-bound compartment, called the inclusion (Moulder, 1991). Within the lumen of the inclusion, the bacteria undergo a complex developmental cycle alternating between infectious and replicative forms. During co-evolution with the mammalian host, the C. trachomatis genome was reduced to about 900 open reading frames (ORF) (Stephens et al., 1998) and C. trachomatis has evolved sophisticated mechanisms to hijack cellular organelles and manipulate cellular pathways to acquire essential Citation: Han Y and Derré I (2017) A Co-infection Model System and the Use of Chimeric Proteins to Study Chlamydia Inclusion Proteins Interaction. Front Cell Infect Microbiol 7 79 Front. Cell. Infect. Microbiol. 7:79. doi: 10.3389/fcimb.2017.00079 March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Chlamydia Inclusion Membrane Proteins Interaction Han and Derré nutrients (Elwell et al., 2016). Central to these processes is a bacterial Type III secretion system (T3SS), which allows for the translocation of bacterial eﬀectors from the bacterial cytosol into the host cell (Mueller et al., 2014). One family of C. trachomatis T3SS eﬀectors, are inserted into the inclusion membrane and referred to as the inclusion membrane proteins (Inc) (Dehoux et al., 2011; Lutter et al., 2012; Moore and Ouellette, 2014). a bacterial two-hybrid system (Gauliard et al., 2015). Inc222 and Inc850 interaction was observed using this experimental set up, and the data also suggested that IncD interacted with itself. In addition, IncA, IncG, IncF, CT229 (CTL0481), CT058 (CTL0314), and CT222 (CTL0475) were identiﬁed as potential interacting partners of IncD. While the CT058- and CT222-IncD interactions were reciprocal, the IncA-, IncG-, IncF-, and CT229- IncD interactions were unidirectional with this subset of Inc being able to interact with IncD, but IncD did not interact with these proteins. Chlamydia inclusion membrane proteins are characterized by a large bilobed hydrophobic domain containing at least 50 amino acids (Bannantine et al., 2000), and amino- and carboxy- terminal tails that are presumably exposed on the cytosolic surface of the inclusion membrane. Based on the presence of the bilobed hydrophobic domain, the C. trachomatis genome is predicted to encode up to 60 Inc proteins. The inclusion membrane localization of a large number of predicted C. trachomatis Inc proteins has been conﬁrmed using primary antibodies (Li et al., 2008) or expression of tagged proteins from the bacteria (Weber et al., 2015). These Type III secreted eﬀectors are unique to Chlamydia and are strategically positioned to mediate the interaction of the inclusion with cellular factors and organelles. Here we have developed a system to test Inc/Inc homo- and heterotypic interactions in the context of C. trachomatis infection and to identify domains that support these interactions. Our system relies on the homotypic fusion properties of C. trachomatis inclusions, on the co-infection with C. Citation: trachomatis strains expressing Inc proteins fused to diﬀerent tags and on the expression of chimeric Inc proteins. Cell Lines and Bacterial Strains HeLa cells were obtained from ATCC (CCL-2) and cultured at 37◦C with 5% CO2 in DMEM high glucose (Invitrogen) supplemented with 10% heat inactivated FBS (Invitrogen). C. trachomatis Lymphogranuloma venereum, Type II were obtained from ATCC (L2/434/Bu VR-902B). Chlamydia propagation and infection were performed as previously described (Derre et al., 2007). Plasmid Construction Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA). PCR was performed using Herculase DNA polymerase (Stratagene). PCR primers were obtained from Integrated DNA Technologies. All the plasmids used in this study are derivatives of p2TK2-SW2 mCh(Gro) (Agaisse and Derre, 2014). They express mCherry under the control of the groESL operon promoter and terminator, the TetR repressor and the indicated Inc under the control of the tetA gene promoter and incDEFG operon terminator. The p2TK2-SW2 mCh(Gro) Tet IncD-, IncE-, and IncG-3xFLAG plasmids were described previously (Agaisse and Derre, 2014; Mirrashidi et al., 2015). p2TK2-SW2 mCh(Gro) Tet IncD-Myc, CTL0314-3xFLAG, CTL0475-3xFLAG, or IncD/IncE-3xFLAG chimera were constructed similarly using the primers listed in Supplementary Table S1. It is unclear whether each Inc protein has a distinct function or if a subset of Inc proteins can act in concert, potentially through direct interaction. Some Inc proteins are evenly distributed on the surface of the inclusion membrane, while others are concentrated in microdomains. This is best illustrated with IncB, Inc101, Inc222, and Inc850, which co-localize to discrete punctae of the inclusion membrane (Mital et al., 2010). Inc222 and Inc850 were shown to interact, suggesting that Inc proteins could form stable complexes with one another. The homo- or heterotypic interaction of Inc proteins was independently investigated using Ethics Statement g Only a few C. trachomatis Inc proteins have been assigned a host interacting partner and/or a function. IncA is involved in homotypic fusion of inclusions (Hackstadt et al., 1999). IncG interacts with 14-3-3ß (Scidmore and Hackstadt, 2001) and CT229 with Rab4 (Rzomp et al., 2006). IncD interacts with and recruits the ceramide transfer protein, CERT, to ER- inclusion membrane contact sites (Derre et al., 2011; Agaisse and Derre, 2014). CT228 interacts with the myosin phosphatase target subunit 1 MYPT1 and regulates the mechanisms by which C. trachomatis egresses from the host cell (Lutter et al., 2013). CT850 interacts with the dynein light chain DYNLT1 (Mital et al., 2015). InaC (CT813/CTL0184) mediates the recruitment of 14-3-3ß, 14-3-3ǫ, and ARF1 to the inclusion membrane and is involved in actin assembly and Golgi positioning around the inclusion (Kokes et al., 2015). IncE binds to the sorting nexins SNX5 and SNX6, and recruits these retromer complex components to the inclusion, resulting in inclusion membrane tubulation (Aeberhard et al., 2015; Mirrashidi et al., 2015). Mirrashidi et al. have also identiﬁed putative mammalian interacting partners for nearly 40 C. trachomatis Inc proteins (Mirrashidi et al., 2015). The study was performed with the Inc proteins overexpressed in mammalian cells, so these interactions remain to be validated during infection, but the study detected IncD/CERT and CT228/MYPT1 interactions, suggesting that this human/Inc interactome is a solid foundation to further investigate the function of C. trachomatis Inc proteins. All genetic manipulations and containment work were approved by the UVA Biosafety Committee and are in compliance with the section III-D-1-a of the National Institutes of Health guidelines for research involving recombinant DNA molecules. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Antibodies The following primary antibodies were used: Mouse monoclonal anti-FLAG [1:1,000 (IF), 1:20,000 (WB), Sigma], mouse monoclonal anti-Myc [1:1,000 (IF), 1:1,000 (WB), Cell Signaling], rabbit polyclonal anti-Actin (1:1,000, Sigma), rabbit polyclonal anti-tRFP (1:2,000, Evrogen), and rabbit polyclonal anti-IncA (1:200, kind gift from T. Hackstadt, Rocky Mountain Laboratories). The following secondary antibodies were used: Peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Jackson ImmunoResearch), peroxidase-conjugated goat anti-mouse IgG (1:10,000, Jackson ImmunoResearch), goat anti-mouse AlexaFluor 488 or 514 (IF: 1:1,000, Molecular Probes), and goat anti-rabbit Paciﬁc Blue (IF: 1:1,000, Molecular Probes). To verify the expression of the constructs, HeLa cells were infected with the above listed C. trachomatis strains in the absence or in the presence of aTc and the corresponding cell lysates were analyzed by western-blot (Figure 1A). Detection of actin and mCherry respectively conﬁrmed equal cell and bacterial number in the absence (-aTc) or presence of aTc (+aTc). Anti-FLAG antibodies were used to detect the respective 3xFLAG tagged Inc proteins and anti-Myc antibodies were used to detect IncD- Myc. As expected, in the absence of inducer the proteins were not expressed (Figure 1A, -aTc), however a signal corresponding to the expected molecular weight of the respective proteins was observed in the presence of aTc (Figure 1A, +aTc). C. trachomatis Transformation Our calcium-based transformation protocol was adapted from Wang et al. (2011) and is described in Agaisse and Derre (2013). March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 2 Chlamydia Inclusion Membrane Proteins Interaction Han and Derré Reproducibility h Each experiment was performed at least three times. Representative results are shown. Co-infection and Immunoﬂuorescence Analysis of Co-infected Cells y Each strain was used at an MOI of 5 to ensure that each eukaryotic cell would receive at least one of each bacterium leading to a mixed bacterial population in each inclusion and the cellular localization of the 3xFLAG- and Myc-tagged Inc proteins was analyzed by immunoﬂuorescence. In our original experimental set up, the samples were co-stained with a rabbit anti-FLAG and a mouse anti-Myc. However, the rabbit anti- FLAG led to uneven staining of the inclusions (even at higher concentration). Duplicate coverslips from the same co-infection were therefore stained with mouse anti-FLAG or mouse anti- Myc antibodies. Although not ideal, the MOI were carefully optimized so that, for each co-immunoprecipitation experiment, 100% of the inclusions were positive for both FLAG and Myc. The samples were not analyzed if it was not the case. In addition, the inclusion membrane localization of the above listed 3xFLAG- or Myc-tagged Inc proteins was analyzed by immuno-ﬂuorescence. For this purpose, HeLa cells infected with the respective Chlamydia strains in the absence or in the presence of aTc were ﬁxed 24 h post-infection and stained with antibodies against the FLAG or Myc tag. The respective Inc proteins were not detected in the absence of inducer (not shown). However, in the presence of aTc, 100% of the inclusions were positive for the respective constructs and the pattern indicated that, as previously shown for IncD-, IncE-, and IncG-3xFLAG (Agaisse and Derre, 2014; Mirrashidi et al., 2015), all constructs localized to the inclusion membrane (Figure 1B). The inclusion localization of CTL0314-3xFLAG and CTL0475-3xFLAG was further conﬁrmed by co-staining of the inclusions with an anti- IncA antibody (Supplementary Figure 1). RESULTS To investigate IncD interaction with a subset of Inc proteins in the context of C. trachomatis infection, we have generated C. trachomatis strains co-expressing mCherry under a constitutive promoter and IncD-3xFLAG, IncD-Myc, IncE-3xFLAG, IncG- 3xFLAG, CTL0314-3xFLAG (CT058), or CTL0475-3xFLAG (CT222) under the control of the anhydrotetracycline (aTc) inducible promoter. IncG, CTL0314 and CTL0475 were chosen based on their potential ability to interact with IncD (Gauliard et al., 2015) and IncE was included as a negative control. Immunoblotting lysates were centrifuge at 13,000 rpm for 10 min. An aliquot of the clariﬁed lysate was collected (Lysate). The clariﬁed lysates were incubated for 2 h in the presence of 10 µl of anti-FLAG M2 agarose beads (Sigma). The beads were washed three times (20 mM Tris pH7.5, 150 mM NaCl, 2 mM EDTA, 1%Triton X-100) and the bound proteins were eluted with 15 µl of elution buﬀer [20 mM Tris pH7.5, 150 mM NaCl, 2 mM EDTA, 100 µg/ml 3XFLAG peptide (Sigma)]. Ten microliters of the eluted fraction was collected (IP). All steps were conducted at 4◦C. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 1xPBS containing 0.05% Tween and 5% Fat-free milk. Primary and HRP-conjugated secondary antibobies were diluted in 1xPBS containing 0.05% Tween and 5% Fat-free milk and respectively incubated over-night at 4◦C and 1 h at room temperature. Proteins were detected using the Amersham ECL western blotting detection reagent as per manufacturer recommendation and a Biorad ChemiDoc imaging system. Immunoﬂuorescence and Microscopy At the indicated times, HeLa cells seeded onto glass coverslips were ﬁxed for 30 min in PBS containing 4% paraformaldehyde. Immunostainings were performed at room temperature. Antibodies were diluted in PBS containing 0.1% BSA and 0.1% Triton X-100. Samples were washed with PBS and examined under an epiﬂuorescence or spinning disc confocal microscope. Co-immunoprecipitation 8.105 HeLa cells plated in 6-well tissue culture dishes and infected with the indicated C. trachomatis strains for 24 h were washed once with 1x PBS and lysed for 20 min in 300 µl of lysis buﬀer [20 mM Tris pH7.5, 150 mM NaCl, 2 mM EDTA, 1%Triton X- 100, 1 mM PMSF and protease inhibitor cocktail (Roche)]. The To test the potential interaction between IncD and a subset of Inc proteins during C. trachomatis intracellular developmental cycle, we took advantage of the homotypic fusion properties of the C. trachomatis inclusions (Ridderhof and Barnes, 1989; Agaisse and Derre, 2013). HeLa cells were co-infected with two March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3 Chlamydia Inclusion Membrane Proteins Interaction Han and Derré FIGURE 1 \| Expression and inclusion localization of the subset of inclusion membrane proteins used in this study. (A) Immuno-blot of cell lysates from HeLa cells infected with strains of C. trachomatis expressing mCherry constitutively and IncD-, IncE-, IncG-, CTL0314-, CTL0475-3xFLAG (3F), or IncD-Myc under the control of the aTc inducible promoter. The cells were infected for 24 h in the absence (-aTc) or in the presence (+aTc) of 2 ng/ml aTc and immuno-blot of the corresponding lysates were probed using antibodies against FLAG or Myc, mCherry, and Actin. (B,C) Confocal micrographs of inclusions of the C. trachomatis strains listed in (A). The cells were infected in the presence of 2 ng/ml aTc, ﬁxed 24 h post-infection, immunostained with anti-FLAG or anti-Myc antibodies and imaged using a confocal microscope. A single plane crossing the middle of the inclusion is shown. The left panels correspond to the bacteria (Chlamydia, red) and the middle panels to the 3xFLAG or Myc signal of the Inc constructs (Inc, green). The merge is shown on the right. Scale bar: 10 µm. FIGURE 1 \| Expression and inclusion localization of the subset of inclusion membrane proteins used in this study. (A) Immuno-blot of cell lysates from HeLa cells infected with strains of C. trachomatis expressing mCherry constitutively and IncD-, IncE-, IncG-, CTL0314-, CTL0475-3xFLAG (3F), or IncD-Myc under the control of the aTc inducible promoter. The cells were infected for 24 h in the absence (-aTc) or in the presence (+aTc) of 2 ng/ml aTc and immuno-blot of the corresponding lysates were probed using antibodies against FLAG or Myc, mCherry, and Actin. Co-immunoprecipitation (B,C) Confocal micrographs of inclusions of the C. trachomatis strains listed in (A). The cells were infected in the presence of 2 ng/ml aTc, ﬁxed 24 h post-infection, immunostained with anti-FLAG or anti-Myc antibodies and imaged using a confocal microscope. A single plane crossing the middle of the inclusion is shown. The left panels correspond to the bacteria (Chlamydia, red) and the middle panels to the 3xFLAG or Myc signal of the Inc constructs (Inc, green). The merge is shown on the right. Scale bar: 10 µm. C. trachomatis strains expressing either IncE-3xFLAG or IncD- Myc. Each strain was used at an MOI of 5 to ensure that each eukaryotic cell would receive at least one of each bacterium leading to a mixed bacterial population in each inclusion. The cellular localization of the IncE-3xFLAG and IncD-Myc proteins was analyzed by immunoﬂuorescence (Figure 2). Close to 100% of the inclusions were positive for FLAG (Figure 2, Top Panels) or Myc (Figure 2, Bottom Panels) when the samples were immuno-labeled with one or the other antibody. Altogether, this result conﬁrmed that the co-infection method allows for the insertion of diﬀerent Inc proteins into the same inclusion membrane when the proteins are produced by diﬀerent strains. co-immuno-precipitate with IncD-3xFLAG (Figure 3, last lane), showing that IncD interacts with itself. C. trachomatis strains expressing either IncE-3xFLAG or IncD- Myc. Each strain was used at an MOI of 5 to ensure that each eukaryotic cell would receive at least one of each bacterium leading to a mixed bacterial population in each inclusion. The cellular localization of the IncE-3xFLAG and IncD-Myc proteins was analyzed by immunoﬂuorescence (Figure 2). Close to 100% of the inclusions were positive for FLAG (Figure 2, Top Panels) or Myc (Figure 2, Bottom Panels) when the samples were immuno-labeled with one or the other antibody. Altogether, this result conﬁrmed that the co-infection method allows for the insertion of diﬀerent Inc proteins into the same inclusion membrane when the proteins are produced by diﬀerent strains. We next investigated whether the IncD/IncD interaction was occurring when the proteins were inserted into the inclusion membrane or “in vitro” after the cell lysates were prepared and processed for immuno-precipitation. To address this question, we compared IncD self-interaction as described above, or after collecting and co-incubating cell lysates from cells that were singly infected with either the IncD-3xFLAG strain or the IncD- Myc strain. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Co-immunoprecipitation The molecular weight ladder is shown on the right. FIGURE 3 \| IncD interacts with itself in the context of C. trachomatis infection. Lysates from HeLa cells co-infected in the presence of 2 ng/ml aTc with a strain of C. trachomatis expressing mCherry constitutively and IncD-Myc under the control of the aTc inducible promoter [CtL2mCh(Gro)TetIncD-Myc] and a strain of C. trachomatis expressing mCherry constitutively and IncE-, IncG-, CTL0314-, CTL0475-, or IncD-3xFLAG (3F) under the control of the aTc inducible promoter [CtL2mCh(Gro)Tet] were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). The molecular weight ladder is shown on the right. FIGURE 3 \| IncD interacts with itself in the context of C. trachomatis infection. Lysates from HeLa cells co-infected in the presence of 2 ng/ml aTc with a strain of C. trachomatis expressing mCherry constitutively and IncD-Myc under the control of the aTc inducible promoter [CtL2mCh(Gro)TetIncD-Myc] and a strain of C. trachomatis expressing mCherry constitutively and IncE-, IncG-, CTL0314-, CTL0475-, or IncD-3xFLAG (3F) under the control of the aTc inducible promoter [CtL2mCh(Gro)Tet] were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). The molecular weight ladder is shown on the right. that is characteristic of Inc proteins, is most likely required for their insertion into the inclusion membrane, which would make its deletion incompatible with insertion of the corresponding Inc protein variant into the inclusion membrane. comparable to the one of IncD- or IncE-3xFLAG (Figure 1A) and the inclusion localization of the constructs was also conﬁrmed by immunoﬂuorescence (Figure 5C). p To address these issues, we generated chimeric proteins between IncD and IncE. IncE was chosen because it does not interact with IncD (Figure 3) and IncD and IncE have similar molecular weight and display similar hydropathy proﬁles (Figure 5A). Based on IncD and IncE respective hydropathy proﬁles, each protein was divided into three domains: The N- terminal domain (IncD: aa 1–39, IncE: aa 1–38), the hydrophobic domain (IncD: aa 40–91, IncE: aa 39–90), and the C-terminal domain (IncD: aa 92–146, IncE: aa 91–132). Co-immunoprecipitation IncD self-interaction was observed upon co-infection (Figure 4, IP lane 3), however the IncD/IncD interaction was not detected when lysates from singly infected cells were combined prior immuno-precipitation (Figure 4, IP lane 4). To investigate if IncD could interact with a subset of Inc proteins, including itself, HeLa cells were co-infected with the IncD-Myc expressing strain and a strain that expressed IncE-, IncG-, CTL0314-, CTL0475-, or IncD-3xFLAG, using the experimental set up described in Figure 2. For each condition, we conﬁrmed that nearly 100% of the inclusions were positive for both markers (not shown). The samples were subjected to co-immuno-precipitation and the results were analyzed by western-blot (Figure 3). IncE-, IncG-, CTL0314- , CTL0475-, and IncD-3xFLAG were expressed and eﬃciently immuno-precipitated with the anti-FLAG conjugated beads. IncD-Myc did not co-immuno-precipitate with IncE-, IncG- , CTL0314-, or CTL0475-3xFLAG, suggesting that IncD does not interact with these Inc proteins. However, IncD-Myc did Altogether, these results indicate that, under our experimental set up, when C. trachomatis inclusion membrane proteins are inserted into the inclusion membrane, IncD interacts with itself but not with IncE, IncG, CTL0314, or CTL0475. Moreover, the IncD self-interaction was only observed when the IncD molecules were inserted into the same inclusion membrane. We next sought to determine the IncD domain(s) mediating the IncD/IncD interaction. One possible approach would be to generate various internal, N- and C-terminal truncated variant of IncD and assay for their self-interaction. However, Inc proteins secretion through the Chlamydia type III secretion system requires a N-terminal secretion signal, that, if truncated, would prevent secretion. Moreover, the central hydrophobic domain, March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 4 Chlamydia Inclusion Membrane Proteins Interaction Han and Derré FIGURE 3 \| IncD interacts with itself in the context of C. trachomatis infection. Lysates from HeLa cells co-infected in the presence of 2 ng/ml aTc with a strain of C. trachomatis expressing mCherry constitutively and IncD-Myc under the control of the aTc inducible promoter [CtL2mCh(Gro)TetIncD-Myc] and a strain of C. trachomatis expressing mCherry constitutively and IncE-, IncG-, CTL0314-, CTL0475-, or IncD-3xFLAG (3F) under the control of the aTc inducible promoter [CtL2mCh(Gro)Tet] were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). Co-immunoprecipitation The molecular weight ladder is shown on the right. FIGURE 2 \| Inclusion membrane localization of Inc proteins expressed by different C. trachomatis strains in a co-infection model system. Epiﬂuorescence micrographs of HeLa cells co-infected with strains of C. trachomatis expressing mCherry constitutively and IncE-3xFLAG or IncD-Myc under the control of the aTc inducible promoter. The cells were infected in the presence of 2 ng/ml aTc, ﬁxed 24 h post-infection, immunostained with mouse anti-Myc antibodies (top panels) or mouse anti- FLAG antibodies (bottom panels) and imaged using an epiﬂuorescence microscope. The left panels correspond to the bacteria (Chlamydia, red) and the middle panels to the Myc or FLAG signal of the Inc constructs (Inc, green). The merge is shown on the right. Scale bar: 50 µm. FIGURE 2 \| Inclusion membrane localization of Inc proteins expressed by different C. trachomatis strains in a co-infection model system. Epiﬂuorescence micrographs of HeLa cells co-infected with strains of C. trachomatis expressing mCherry constitutively and IncE-3xFLAG or IncD-Myc under the control of the aTc inducible promoter. The cells were infected in the presence of 2 ng/ml aTc, ﬁxed 24 h post-infection, immunostained with mouse anti-Myc antibodies (top panels) or mouse anti- FLAG antibodies (bottom panels) and imaged using an epiﬂuorescence microscope. The left panels correspond to the bacteria (Chlamydia, red) and the middle panels to the Myc or FLAG signal of the Inc constructs (Inc, green). The merge is shown on the right. Scale bar: 50 µm. FIGURE 3 \| IncD interacts with itself in the context of C. trachomatis FIGURE 3 \| IncD interacts with itself in the context of C. trachomatis infection Lysates from HeLa cells co infected in the presence of 2 ng/ml aTc FIGURE 3 \| IncD interacts with itself in the context of C. trachomatis infection. Lysates from HeLa cells co-infected in the presence of 2 ng/ml aTc with a strain of C. trachomatis expressing mCherry constitutively and IncD-Myc under the control of the aTc inducible promoter [CtL2mCh(Gro)TetIncD-Myc] and a strain of C. trachomatis expressing mCherry constitutively and IncE-, IncG-, CTL0314-, CTL0475-, or IncD-3xFLAG (3F) under the control of the aTc inducible promoter [CtL2mCh(Gro)Tet] were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). Co-immunoprecipitation FIGURE 4 \| IncD-IncD interaction occurs on C.trachomatis inclusion membrane. HeLa cells were singly infected in the presence of 2 ng/ml with a strain of C. trachomatis expressing mCherry constitutively and IncD-3xFLAG (3F) or IncD-Myc under the control of the aTc inducible promoter or co-infected with these two strains (IncD-3F+IncDMyc CoInf). Lysates from singly and co-infected cells were collected and a fourth sample was prepared by mixing equal part of lysates from the singly infected cells (IncD-3F+IncDMyc Mix). The lysates were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). We were able to detect IncD/IncD interaction indicating that IncD may act as a dimer or an oligomer. Our data are also consistent with IncD self-interaction occurring only when the protein is inserted into the inclusion membrane. In the light of our previous data, showing that IncD interacts with the PH domain of CERT (Derre et al., 2011; Agaisse and Derre, 2014), one could envision that IncD dimerization or oligomerization may enhance the eﬃcacy of CERT recruitment to the inclusion. inclusion membrane and could be used to dissect the molecular mechanisms driving Inc/Inc protein interaction. Our investigation of IncD self-interaction revealed that although IncD/IncE chimeric proteins containing the N-terminus and hydrophobic domain (DDE) or the hydrophobic domain and the C-terminus (EDD) of IncD were able to interact with IncD, it appears that the full length IncD protein is required for eﬃcient self-interaction. In our model system, we did not detect the IncD/CTL0314 and IncD/CTL0475 interactions previously observed in a bacterial two-hybrid system. There are two possible explanations to this discrepancy: (1) we failed to detect these interactions under our experimental set up or (2) the interactions observed using the bacterial two-hybrid system were not physiologically relevant. These results emphasize the need of validating any Inc/Inc interaction by diﬀerent approaches, preferably in the context of infected cells. Co-immunoprecipitation The following chimeric constructs were generated: IncDED, IncEED, IncDEE, IncEDE, IncDDE, and IncEDD where the ﬁrst, second and third letter respectively correspond to the N-terminal, hydrophobic, or C-terminal domain of the respective Inc protein. The hydropathy proﬁles of the IncD/IncE chimeric proteins are shown in Figure 5A, where the IncD domains are indicated by the red circles. Each chimera was fused to a 3xFLAG tag and expressed under the control of the aTc inducible promoter into p2TK2-SW2mCh(Gro) and the corresponding plasmids were introduced into C. trachomatis. When HeLa cells were infected with the resulting C. trachomatis strains, mCherry was constitutively expressed and the IncD/IncE- 3xFLAG chimeric proteins were only detected in the presence of inducer (Figure 5B). The levels of expression of the chimera were If the N-terminal, the hydrophobic, or the C-terminal domain of IncD is suﬃcient to mediate IncD self-interaction, we rationalized that this domain would promote IncD/IncD- E chimera interaction in our co-infection experimental set up. To test our hypothesis, HeLa cells were co-infected with C. trachomatis strains respectively expressing IncD-Myc and IncD-3xFLAG or IncD-Myc and one of the six IncD/IncE- 3xFLAG chimeric proteins. For each co-infection combination, we conﬁrmed that nearly 100% of the inclusions were positive for each construct by immuno-ﬂuorescence (data not shown). The lysates were subjected to immuno-precipitation using anti- FLAG antibodies and co-immuno-precipitation of IncD-Myc was assayed by western-blot (Figure 6). The IncD/IncE-3xFLAG chimeric proteins were immuno-precipitated as eﬃciently as IncD-3xFLAG. As observed before (Figures 3, 4) IncD-Myc co- immuno-precipitated with IncD-3xFLAG. In addition, IncD- Myc co-immuno-precipitated with two of the IncD/IncE chimeric constructs, IncDDE and IncEDD, but the IncD/IncDDE and IncD/IncEDD interactions were not as robust as the one observed with IncD/IncD. Altogether these results conﬁrmed that chimeric Inc proteins could be engineered and successfully inserted into C. trachomatis March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 5 Chlamydia Inclusion Membrane Proteins Interaction Han and Derré FIGURE 4 \| IncD-IncD interaction occurs on C.trachomatis inclusion membrane. HeLa cells were singly infected in the presence of 2 ng/ml with a strain of C. trachomatis expressing mCherry constitutively and IncD-3xFLAG (3F) or IncD-Myc under the control of the aTc inducible promoter or co-infected with these two strains (IncD-3F+IncDMyc CoInf). Co-immunoprecipitation Lysates from singly and co-infected cells were collected and a fourth sample was prepared by mixing equal part of lysates from the singly infected cells (IncD-3F+IncDMyc Mix). The lysates were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). membrane, potentially leading to interaction artifacts due to the absence of lipids and/or bacterial and host proteins that would normally constitute the natural environment surrounding the Inc proteins. With the development of Chlamydia genetic tools (Wang et al., 2011; Sixt and Valdivia, 2016), it is now possible to express Inc proteins of interest from the bacteria and to study their roles in the context of the inclusion membrane (Agaisse and Derre, 2014; Bauler and Hackstadt, 2014; Kokes et al., 2015; Weber et al., 2015, 2016). We have previously used this method to further investigate IncD/CERT interaction in the context of the infection and to conﬁrm the direct role of IncD in CERT recruitment to the inclusion membrane (Derre et al., 2011; Agaisse and Derre, 2014). In the current study, we sought to follow up on a bacterial two-hybrid study suggesting that IncD may interact with itself and with a subset of Inc proteins (Gauliard et al., 2015). To probe for Inc/Inc interaction in the context of C. trachomatis infection, we took advantage of the fact that nascent inclusions originating from bacteria expressing two diﬀerent Inc protein variants will eventually undergo fusion resulting in inclusions that display both Inc proteins on the surface of their membranes. FIGURE 4 \| IncD-IncD interaction occurs on C.trachomatis inclusion membrane. HeLa cells were singly infected in the presence of 2 ng/ml with a strain of C. trachomatis expressing mCherry constitutively and IncD-3xFLAG (3F) or IncD-Myc under the control of the aTc inducible promoter or co-infected with these two strains (IncD-3F+IncDMyc CoInf). Lysates from singly and co-infected cells were collected and a fourth sample was prepared by mixing equal part of lysates from the singly infected cells (IncD-3F+IncDMyc Mix). The lysates were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org DISCUSSION (B) Immuno-blot of cell lysates from HeLa cells infected with strains of C. trachomatis expressing mCherry constitutively and the indicated IncD/IncE-3xFLAG (3F) chimeric proteins under the control of the aTc inducible promoter. The cells were infected for 24 h in the absence (-aTc) or in the presence (+aTc) of 2 ng/ml aTc and immuno-blot of the corresponding lysates were probed using antibodies against FLAG, mCherry, and Actin. (C) Confocal micrographs of inclusions of the C. trachomatis strains listed in (B). The cells were infected in the presence of 2 ng/ml aTc, ﬁxed 24 h post-infection, immunostained with anti-FLAG antibodies and imaged using a confocal microscope. A single plane crossing the middle of the inclusion is shown. The left panels correspond to the bacteria (Chlamydia, red) and the middle panels to the FLAG signal of the IncD/IncE chimera (Inc, green). The merge is shown on the right. Scale bar: 10 µm. FIGURE 5 \| IncD/IncE chimeric proteins localize to C. trachomatis inclusion membrane. (A) Hydropathy plots of IncD, IncE, and the IncD/IncE chimeric proteins used in this study. The red circles indicate domains of IncD. (B) Immuno-blot of cell lysates from HeLa cells infected with strains of C. trachomatis expressing mCherry constitutively and the indicated IncD/IncE-3xFLAG (3F) chimeric proteins under the control of the aTc inducible promoter. The cells were infected for 24 h in the absence (-aTc) or in the presence (+aTc) of 2 ng/ml aTc and immuno-blot of the corresponding lysates were probed using antibodies against FLAG, mCherry, and Actin. (C) Confocal micrographs of inclusions of the C. trachomatis strains listed in (B). The cells were infected in the presence of 2 ng/ml aTc, ﬁxed 24 h post-infection, immunostained with anti-FLAG antibodies and imaged using a confocal microscope. A single plane crossing the middle of the inclusion is shown. The left panels correspond to the bacteria (Chlamydia, red) and the middle panels to the FLAG signal of the IncD/IncE chimera (Inc, green). The merge is shown on the right. Scale bar: 10 µm. We therefore turned to chimeric Inc proteins that contained various permutations of the N-, C-terminal, and hydrophobic domains of IncD and IncE, another small Inc protein that did not interact with IncD. Only two chimeras were able to interact with IncD. DISCUSSION Inclusion membrane proteins are speciﬁc to Chlamydia and are strategically positioned to mediate inclusion interaction with host factors and organelles. However, little is known about Inc proteins interacting partners and/or function. Inc proteins are diﬃcult to study because of their inherent biochemical properties. Their large hydrophobic domain complicates protein puriﬁcation in a native and soluble state and limits subsequent in vitro studies to soluble domains (Ronzone and Paumet, 2013; Ronzone et al., 2014). Up until recently, in the absence of genetic tools to manipulate Chlamydia, studies have focused on over-expressing Inc proteins in mammalian cells (Rzomp et al., 2006; Derre et al., 2011; Mital et al., 2013; Mirrashidi et al., 2015) or using two-hybrid system in yeast (Scidmore and Hackstadt, 2001; Rzomp et al., 2006; Lutter et al., 2013; Mital et al., 2015) or bacteria (Gauliard et al., 2015). While these studies have led to the identiﬁcation of Inc proteins interacting partners and shed light on the putative function of some Inc proteins, the big caveat of these studies is that the Inc proteins were not studied in the natural context of the inclusion If IncD self-interacts, what are the domains driving this interaction? IncD is a 15 kDa protein that can be separated into three major domains: A N-terminal domain (aa1– 39) that contains the Type III secretion signal, a central hydrophobic domain (aa40–91) responsible for inclusion membrane anchoring and a C-terminal domain (aa92–146). Both the N- and C-terminal domains are predicted to face the cytosol. To study IncD variants, that are potentially defective for IncD/IncD interaction, in the context of the inclusion membrane, these variants should retain the Type III secretion signal and the hydrophobic domain for inclusion membrane localization. Given that IncD is a fairly small protein, the type of truncated variants that can be generated and studied is therefore limited. We did generate a variant of IncD that lacked the C-terminal domain, but although the corresponding protein was expressed in C. trachomatis, it failed to display strong inclusion localization (data not shown). March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 6 Chlamydia Inclusion Membrane Proteins Interaction Han and Derré FIGURE 5 \| IncD/IncE chimeric proteins localize to C. trachomatis inclusion membrane. (A) Hydropathy plots of IncD, IncE, and the IncD/IncE chimeric proteins used in this study. The red circles indicate domains of IncD. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org REFERENCES Derre, I., Pypaert, M., Dautry-Varsat, A., and Agaisse, H. (2007). RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection. PLoS Pathog. 3:e030155. doi: 10.1371/journal.ppat.0030155 Aeberhard, L., Banhart, S., Fischer, M., Jehmlich, N., Rose, L., Koch, S., et al. (2015). The proteome of the isolated Chlamydia trachomatis containing vacuole reveals a complex traﬃcking platform enriched for retromer components. PLoS Pathog. 11:e1004883. doi: 10.1371/journal.ppat.1004883 Derre, I., Swiss, R., and Agaisse, H. (2011). The lipid transfer protein CERT interacts with the Chlamydia inclusion protein IncD and participates to ER-Chlamydia inclusion membrane contact sites. PLoS Pathog. 7:e1002092. doi: 10.1371/journal.ppat.1002092 Agaisse, H., and Derre, I. (2013). A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing ﬂuorescent proteins under the control of a C. trachomatis promoter. PLoS ONE 8:e57090. doi: 10.1371/journal.pone.0057090 Elwell, C., Mirrashidi, K., and Engel, J. (2016). Chlamydia cell biology and pathogenesis. Nat. Rev. Microbiol. 14, 385–400. doi: 10.1038/nrmicro.2016.30 Gauliard, E., Ouellette, S. P., Rueden, K. J., and Ladant, D. (2015). Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis. Front. Cell. Infect. Microbiol. 5:13. doi: 10.3389/fcimb.2015.00013 Agaisse, H., and Derre, I. (2014). Expression of the eﬀector protein IncD in Chlamydia trachomatis mediates recruitment of the lipid transfer protein CERT and the endoplasmic reticulum-resident protein VAPB to the inclusion membrane. Infect. Immun. 82, 2037–2047.doi: 10.1128/IAI.01530-14 Hackstadt, T., Scidmore-Carlson, M. A., Shaw, E. I., and Fischer, E. R. (1999). The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion. Cell. Microbiol. 1, 119–130. doi: 10.1046/j.1462-5822.1999.00012.x Bannantine, J. P., Griﬃths, R. S., Viratyosin, W., Brown, W. J., and Rockey, D. D. (2000). A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane. Cell. Microbiol. 2, 35–47. doi: 10.1046/j.1462-5822.2000.00029.x Kokes, M., Dunn, J. D., Granek, J. A., Nguyen, B. D., Barker, J. R., Valdivia, R. H., et al. (2015). Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia. Cell Host Microbe 17, 716–725. doi: 10.1016/j.chom.2015.03.014 Bauler, L. D., and Hackstadt, T. (2014). Expression and targeting of secreted proteins from Chlamydia trachomatis. J. Bacteriol. 196, 1325–1334. doi: 10.1128/JB.01290-13 Li, Z., Chen, C., Chen, D., Wu, Y., Zhong, Y., and Zhong, G. (2008). Characterization of ﬁfty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome. Infect. Immun. 76, 2746–2757. doi: 10.1128/IAI.00010-08 Dehoux, P., Flores, R., Dauga, C., Zhong, G., and Subtil, A. (2011). DISCUSSION Lysates from HeLa cells co-infected in the presence of 2 ng/ml aTc with a strain of C. trachomatis expressing mCherry constitutively and IncD-Myc under the control of the aTc inducible promoter [CtL2mCh(Gro)TetIncD-Myc] and strain of C. trachomatis expressing mCherry constitutively and the indicated IncD/IncE-3xFLAG (3F) chimera under the control of the aTc inducible promoter [CtL2mCh(Gro)Tet] were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). ultimately leading to a better understanding of Chlamydia life cycle and interaction with the mammalian host. FUNDING NIH NIAID grant R01AI101441 to ID. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fcimb. 2017.00079/full#supplementary-material Supplementary Figure 1 \| Inclusion localization of CTL0314- and CTL0475-3xFLAG. Confocal micrographs of inclusions of C. trachomatis strains expressing mCherry constitutively and CTL0314-3xFLAG (Top panels) or CTL0475-3xFLAG (Bottom panels) under the control of the aTc inducible promoter. The cells were infected in the presence of 2 ng/ml aTc, ﬁxed 24 h post-infection, immunostained with anti-FLAG (Yellow), and anti-IncA (Blue) antibodies and imaged using a confocal microscope. A single plane crossing the middle of the inclusion is shown. The left panels correspond to the bacteria (Chlamydia, red). The merge is shown on the right. Scale bar: 10 µm. membrane proteins in the context of an infected cell. The co-infection system described here and the use of chimeric Inc proteins, together with the generation of C. trachomatis strains lacking Inc proteins of interest will be powerful tools to assay not only Inc protein functions, but also the molecular mechanisms by which Inc proteins interact with themselves or with host factors, Supplementary Table 1 \| Primers used in this study. ACKNOWLEDGMENTS FIGURE 6 \| IncD-IncD self-interaction requires full length IncD. Lysates from HeLa cells co-infected in the presence of 2 ng/ml aTc with a strain of C. trachomatis expressing mCherry constitutively and IncD-Myc under the control of the aTc inducible promoter [CtL2mCh(Gro)TetIncD-Myc] and strain of C. trachomatis expressing mCherry constitutively and the indicated IncD/IncE-3xFLAG (3F) chimera under the control of the aTc inducible promoter [CtL2mCh(Gro)Tet] were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). We would like to thank Hervé Agaisse for constructive discussion and critical reading of the manuscript. AUTHOR CONTRIBUTIONS YH: Performed the experiments, Analyzed the data. ID: Designed the experiments, Performed the experiments, Analyzed the data, Wrote the manuscript. DISCUSSION The combination of the N-terminal and hydrophobic domains of IncD (DDE) led to weak interaction and the hydrophobic domain combined to the C-terminal domain (EDD) led to a slightly more eﬃcient binding. The eﬃcacy of binding of these two chimeric proteins was however weaker than the one observed with the full- length protein. Our data suggest that the full-length IncD protein is required for dimerization and/or oligomerization. Alternatively, the hydrophobic domain may be suﬃcient to mediate IncD self-interaction but optimal self-interaction may require additional amino acids that were not included in our constructs, to accommodate optimal self-interaction. Finally, it is possible that, although the hydrophobic domain is suﬃcient to mediate IncD self-interaction, oligomers formed less eﬃciently between un-identical IncD units. Testing the self-interaction of the IncDDE and IncEDD chimera could address this question. Our data suggest that, if not the full-length protein, a large portion of IncD is required for self-interaction and potentially for function. This would be in contrast with IncE, which has been proposed to be a monomer (Gauliard et al., 2015) and for which the C-terminal domain is suﬃcient for binding the PX domain of SNX5 and SNX6 (Mirrashidi et al., 2015). Altogether, one could envision that some Inc proteins, such as IncE, act as monomers and have distinct domains dedicated to interaction with host factors, while others, like IncD, require oligomerization of the full-length protein to eﬃciently recruit their host target to the inclusion membrane. With the recent advances in Chlamydia genetics, it is now possible to investigate the role of C. trachomatis inclusion March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 7 Han and Derré Chlamydia Inclusion Membrane Proteins Interaction FIGURE 6 \| IncD-IncD self-interaction requires full length IncD. Lysates from HeLa cells co-infected in the presence of 2 ng/ml aTc with a strain of C. trachomatis expressing mCherry constitutively and IncD-Myc under the control of the aTc inducible promoter [CtL2mCh(Gro)TetIncD-Myc] and strain of C. trachomatis expressing mCherry constitutively and the indicated IncD/IncE-3xFLAG (3F) chimera under the control of the aTc inducible promoter [CtL2mCh(Gro)Tet] were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, Lysate) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies against Myc (Top Panels) and FLAG (Bottom Panels). FIGURE 6 \| IncD-IncD self-interaction requires full length IncD. REFERENCES Multi-genome identiﬁcation and characterization of chlamydiae-speciﬁc type III secretion substrates: the Inc proteins. BMC Genomics 12:109. doi: 10.1186/1471-2164-12-109 Lutter, E. I., Barger, A. C., Nair, V., and Hackstadt, T. (2013). 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Molecular genetic analysis of Chlamydia species. Annu. Rev. Microbiol. 70, 179–198. doi: 10.1146/annurev-micro-102215-095539 Mital, J., Miller, N. J., Dorward, D. W., Dooley, C. A., and Hackstadt, T. (2013). Role for chlamydial inclusion membrane proteins in inclusion membrane structure and biogenesis. PLoS ONE 8:e63426. doi: 10.1371/journal.pone.0063426 Stephens, R. S., Kalman, S., Lammel, C., Fan, J., Marathe, R., Aravind, L., et al. (1998). Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754–759. doi: 10.1126/science.282.5389.754 Mital, J., Miller, N. J., Fischer, E. R., and Hackstadt, T. (2010). Speciﬁc chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network. Cell. Microbiol. 12, 1235–1249. REFERENCES doi: 10.1111/j.1462-5822.2010.01465.x Wang, Y., Kahane, S., Cutcliﬀe, L. T., Skilton, R. J., Lambden, P. R., and Clarke, I. N. (2011). 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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. Copyright © 2017 Han and Derré. 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. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Copyright © 2017 Han and Derré. 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. REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Ronzone, E., Wesolowski, J., Bauler, L. D., Bhardwaj, A., Hackstadt, T., and Paumet, F. (2014). An alpha-helical core encodes the dual functions of the chlamydial protein IncA. J. Biol. Chem. 289, 33469–33480. doi: 10.1074/jbc.M114.592063 March 2017 \| Volume 7 \| Article 79 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 9
https://openalex.org/W1947362175	https://europepmc.org/articles/pmc4603698?pdf=render	English	null	Spheroidal carbonaceous particles are a defining stratigraphic marker for the Anthropocene	Scientific reports	2,015	cc-by	3,869	Spheroidal carbonaceous particles are a defining stratigraphic marker for the Anthropocene received: 23 January 2015 accepted: 07 April 2015 Published: 28 May 2015 Graeme T. Swindles1, Elizabeth Watson1, T. Edward Turner1, Jennifer M. Galloway2, Thomas Hadlari2, Jane Wheeler1 & Karen L. Bacon1 There has been recent debate over stratigraphic markers used to demarcate the Anthropocene from the Holocene Epoch. However, many of the proposed markers are found only in limited areas of the world or do not reflect human impacts on the environment. Here we show that spheroidal carbonaceous particles (SCPs), a distinct form of black carbon produced from burning fossil fuels in energy production and heavy industry, provide unambiguous stratigraphic markers of the human activities that have rapidly changed planet Earth over the last century. SCPs are found in terrestrial and marine sediments or ice cores in every continent, including remote areas such as the high Arctic and Antarctica. The rapid increase in SCPs mostly occurs in the mid-twentieth century and is contemporaneous with the ‘Great Acceleration’. It therefore reflects the intensification of fossil fuel usage and can be traced across the globe. We integrate global records of SCPs and propose that the global rapid increase in SCPs in sedimentary records can be used to inform a Global Standard Stratigraphic Age for the Anthropocene. A high-resolution SCP sequence from a lake or peatland may provide the much-needed ‘Golden Spike’ (Global Boundary Stratotype Section and Point). The Anthropocene has become a term widely adopted by both the scientific community and the media1,2. It reflects the current time of the Earth’s history when human activities have become one of the dominant forces shaping the planet implying that a new geological time division may be required. There has been much debate over the timing of the Anthropocene; some authors have used archaeological evidence to suggest that the rise of human impacts began in the early to mid-Holocene3, ~2 millennia ago4, or from the time of the industrial revolution1. However, mounting evidence suggests that human impacts on the planet at these times were diachronous and highly spatially variable5. There is rising support for the base of the Anthropocene to be placed at ca. AD 1950 that approximates the ‘Great Acceleration’, a time of rapidly increasing and globally-widespread anthropogenic impacts on planet Earth. This includes unprecedented burning of fossil fuels leading to a rapid rise in global atmospheric CO2, deployment of nuclear weapons, and pollution from industrial, agricultural and domestic processes5,6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1School of Geography, University of Leeds, Leeds, LS2 9JT. 2Geological Survey of Canada, Calgary, Alberta, T2L 2A7. Correspondence and requests for materials should be addressed to G.T.S. (email: g.t.swindles@leeds.ac.uk) Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 Table 1. The global occurrence of spheroidal carbonaceous particles. The reported ages of the first occurrence and rapid increase of SCP concentration are provided with key references. Spheroidal carbonaceous particles are a defining stratigraphic marker for the Anthropocene There is a need for a stratigraphic marker that reflects the significant global impact of humans on Earth and defines the Anthropocene. This stratigraphic marker must represent the onset of the Anthropocene in marine and terrestrial sediments and ice, be present across the globe, and be related to the types of human impacts that characterize the Anthropocene. Anthropogenic soils4, chemical tracers7, and radionuclides5 have been proposed as Anthropocene markers. However, many of these are diachronous, regionally variable, occur at the wrong time, or require a complex analytical procedure to decipher. The Tambora 1815 volcanic event has also been proposed as a possible marker for dating the onset of the Anthropocene8. This volcanic event is registered in chemical profiles from Greenland and Antartica ice core records; however, tephra from this eruption is only found in Asia9. More importantly, it does not derive from human impacts on the environment that defines the Anthropocene. Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 1 Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 www.nature.com/scientificreports/ www.nature.com/scientificreports/ tificreports/ Figure 1. Kernel density plot of the decade of rapid increase in global SCPs (data are from Table 1). The plot illustrates that the rapid increase mainly occurs in the mid-twentieth century across the globe. The earliest decade was used in the case of the event spanning two decades (e.g. 1950s–1960s). Figure 1. Kernel density plot of the decade of rapid increase in global SCPs (data are from Table 1). The plot illustrates that the rapid increase mainly occurs in the mid-twentieth century across the globe. The earliest decade was used in the case of the event spanning two decades (e.g. 1950s–1960s). An unambiguous ‘index fossil’ of the human activities that have changed the face of planet Earth in recent centuries is therefore needed. Spheroidal carbonaceous particles (SCPs) are a distinct component of black carbon only produced from the high-temperature (> 1000 °C) combustion of fossil fuels (coal and oil) (Supplementary file). SCPs are produced as a by-product of energy production as well as heavy industry and have no natural sources in the Quaternary10. SCPs are highly abundant in areas close to pol- lution sources11,13, and are also found across the continents of planet Earth (Table 1). Importantly, they have also been recorded in remote areas distal from industrial sources including Greenland14, Svalbard15, Arctic Canada16,17 and Antarctica18. Several studies have shown that SCPs are correlated with other types of industrial pollution including sulphur and polycyclic aromatic hydrocarbons (PAHs)13,19. In addition, SCPs are also well-preserved in lake and marine sediments, peats and glacial ice as they are chemically inert, owing to their composition of elemental carbon20,21. g p SCPs are suitable indicators for the Anthropocene for the following reasons: 1. They are present across the globe (Table 1);h h 2. They are an unambiguous marker of anthropogenic fossil fuel combustion that composition of our atmosphere and driven recent climate change22;h h 2. They are an unambiguous marker of anthropogenic fossil fuel combustion that has changed the composition of our atmosphere and driven recent climate change22; p p g 3. They record unprecedented impacts of human activity on the environment;h h y p p y 4. They are documented in ice cores, marine and terrestrial sediments;hi y p p They are documented in ice cores, marine and terrehi h 5. They are easily extracted and identified by researchers. Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. The global occurrence of spheroidal carbonaceous particles. The reported ages of the first occurrence and rapid increase of SCP concentration are provided with key references. Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 2 www.nature.com/scientificreports/ Spheroidal carbonaceous particle record from Malham Tarn Moss, a peat bog in the Yorkshire Dales, Northern England. The first occurrence of SCPs in the mid-19th century reflects the onset of industrial combustion of coal at high temperature. The rapid increase in the 1950s reflects the increase in total energy production after the Second World War. Human impacts on Malham Tarn Moss become unprecedented at this time, including atmospheric deposition of Pb and soil erosion from intensive agricultural practices (reflected in the loss-on-ignition and Fe data from the peat bog) and a rapid increase in SCP deposition. The top of core represents the year of sampling (2009). The Medieval Warm Period (MWP) and Little Ice Age (LIA), marked by drier and wetter bog surface wetness respectively, are shown and the proposed Holocene–Anthropocene boundary of AD 1950 is illustrated by the grey line. Figure 2. Spheroidal carbonaceous particle record from Malham Tarn Moss, a peat bog in the Yorkshire D l N th E l d Th fi t f SCP i th id 19th t fl t th t f Figure 2. Spheroidal carbonaceous particle record from Malham Tarn Moss, a peat bog in the Yorkshire Dales, Northern England. The first occurrence of SCPs in the mid-19th century reflects the onset of industrial combustion of coal at high temperature. The rapid increase in the 1950s reflects the increase in total energy production after the Second World War. Human impacts on Malham Tarn Moss become unprecedented at this time, including atmospheric deposition of Pb and soil erosion from intensive agricultural practices (reflected in the loss-on-ignition and Fe data from the peat bog) and a rapid increase in SCP deposition. The top of core represents the year of sampling (2009). The Medieval Warm Period (MWP) and Little Ice Age (LIA), marked by drier and wetter bog surface wetness respectively, are shown and the proposed Holocene–Anthropocene boundary of AD 1950 is illustrated by the grey line. record would need to be calibrated to a specific year using a marker horizon, such as an ash bed as close as possible to the rapid increase in SCPs. www.nature.com/scientificreports/ Variation in the timing and extent of coal and oil usage are reflected in temporal differences of the first occurrence (First Occurrence Datum) of SCPs in different regions (Table 1). The peak (acme) in SCP con- centration is also variable spatially, reflecting proximity to pollution sources. However, the rapid increase in SCPs reflects the rise to dominance of oil as the major fossil fuel source on Earth22 and mostly occurs in the mid-twentieth century across the globe (Fig. 1) – contemporaneous with the ‘Great Acceleration’3 and rapid increase in global population. The rapid increase in SCPs is thus a key chronostratigraphic marker for the Anthropocene because it is a global signature. We know of only two pre-Holocene occur- rences of SCPs in the sedimentary record due to non-anthropogenic phenomena and they both corre- late to significant geological timescale boundaries and mass extinctions. SCPs were derived from (I) the combustion of coal by flood basalts at the latest Permian extinction23; and (II) the combustion of fossil organic matter from bolide impact at the Cretaceous-Palaeogene boundary24. Furthermore, it has been suggested that the Cretaceous–Palaeogene examples are easily distinguished from modern SCPs due to a lower burn temperature resulting in lighter colouration of the particles25. We suggest that the appearance of SCPs in Anthropocene sediments will appear geologically instantaneous in the far future. p pp g g y We propose that lake and/or peatland sequences with detailed SCP records should be used to inform either a Global Standard Stratigraphic Age (GSSA), or used as the Global Boundary Stratotype Section and Point (GSSP) for the Anthropocene. Many lakes and peatlands have continuous sedimentation/ accumulation over this time period and deposited an adequate thickness of sediment to capture SCPs. In Britain for example, SCP records commonly begin between AD 1830–186025, somewhat later than the earliest industrialisation. However, the rapid increase in SCPs (AD 1950–1960s) reflects the increased intensity of fossil fuel use in industry and power generation after the second world war that: (1) left an unambiguous expression in sedimentary records; and (II) reflects impact of global significance. The rapid increase in SCPs provides a marker of the point in time when human activities became globally unprecedented, rather than reflecting first intense industrialisation. An exceptional lake or peatland SCP Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 3 www.nature.com/scientificreports/ Figure 2. www.nature.com/scientificreports/ The deposition of the Hekla 1947 ash in Irish peatlands imme- diately prior to the rapid increase in SCPs may provide such a calibration26.h y p p y p There are numerous secondary markers that could be used to mark the onset of the Anthropocene, including chemical signatures of anthropogenic pollution (e.g. PAHs, Pb, Hg), land degradation (e.g. dust from soil erosion), or changes to biodiversity such as extinctions of native biota or introductions of non-native species. However, these signals are not consistent globally. Biostratigraphic evidence of recent climate or human impacts in lakes and peatlands (e.g. microfossils such as chironomids, diatoms, testate amoebae, and non-native pollen) may also be used in some localities. Recent peats and sediments have been dated using radio-isotopes such as 210Pb, 137Cs and high-resolution 14C techniques, permitting precise dating of the rapid increase in SCPs. p g p To support our argument we provide a reference example from Malham Tarn Moss, a raised bog in the Yorkshire Dales, N. England (Fig. 2). A high-resolution SCP sequence combined with records of lead pollution and soil erosion (Fe, loss-on-ignition) are illustrated. These reflect increased land-use inten- sity, direct human impacts on the peatland and peatland response to climate change (water table depth reconstruction based on testate amoebae microfossils). This record illustrates the unprecedented human impacts on the environment in N. England after c. AD1950 which occurs alongside the rapid increase in SCPs. Our example clearly demonstrates the utility of SCPs as a defining stratigraphic marker for the Anthropocene. References Late Holocene ecohydrological and carbon dynamics of a UK raised bog: impac of human activity and climate change. Quaternary Sci Rev 84, 65–85, doi:10.1016/j.quascirev.2013.10.030 (2014). Methods We carried out a detailed analysis of published literature to assess the occurrence of spheroidal carbona- ceous particles (SCPs) in sediment and peat profiles and ice cores across the world (Table 1). All sources were compiled by country and continent and the established dates of the first occurrence and the onset of rapid increase of SCPs were noted (based on independent dating methods including 210Pb determi- nations and tephra). A Kernal density function was used to estimate the probability density function of the date of rapid increase in SCPs. Two adjacent cores from Malham Tarn Moss Yorkshire Dales, Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 4 www.nature.com/scientificreports/ Northern England (54.0975946°, − 2.1730828°) were taken using a Russian-type D section corer. One core was analysed for 21 chemical elements using a Cox Analytical Systems ITRAX X-ray fluorescence core scanner at 500 μ m intervals to semi-quantitatively determine Pb and Fe content. The other core was divided into 1-cm contiguous sections for SCP analysis. SCP concentrations were analysed under high-powered microscopy following acid digestion and presented as n per g dry peat27. Calendar ages for the first occurrence, rapid increase, and peak concentration of SCP were assigned to the record20,25. Loss-on-ignition was determined using standard methods28. Water table depth reconstruction was car- ried out on subfossil testate amoebae using a transfer function based on a local training set29. Northern England (54.0975946°, − 2.1730828°) were taken using a Russian-type D section corer. One core was analysed for 21 chemical elements using a Cox Analytical Systems ITRAX X-ray fluorescence core scanner at 500 μ m intervals to semi-quantitatively determine Pb and Fe content. The other core was divided into 1-cm contiguous sections for SCP analysis. SCP concentrations were analysed under high-powered microscopy following acid digestion and presented as n per g dry peat27. Calendar ages for the first occurrence rapid increase and peak concentration of SCP were assigned to the record20,25 i g Loss-on-ignition was determined using standard methods28. Water table depth reconstruction was car- ried out on subfossil testate amoebae using a transfer function based on a local training set29. References & Battarbee, R. W. Dating of recent catchment peats using spheroidal carbonaceous particle (SCP) t ti fil ith ti l f t L h S tl d H l 11 593 597 d i 10 1191/095968301680223549 , pp y, g pp g y p p y Kingdom. J Paleolimnol 34, 349–361, doi:10.1007/s10933-005-4925-4 (2005). 21. Yang, H. D., Rose, N. L. & Battarbee, R. W. Dating of recent catchment peats using spheroidal carbonaceous particle (SCP) concentration profiles with particular reference to Lochnagar, Scotland. Holocene 11, 593–597, doi:10.1191/095968301680223549 (2001). 21. Yang, H. D., Rose, N. L. & Battarbee, R. W. Dating of recent catchment peats using spheroidal carbonaceous particle ( concentration profiles with particular reference to Lochnagar, Scotland. Holocene 11, 593–597, doi:10.1191/09596830168022 (2001).ilh 22. Oldfield, F. Can the magnetic signatures from inorganic fly ash be used to mark the onset of the Anthropocene? The Anthropocene Review, 1–11, doi:10.1177/2053019614534402 (2014).l 3. Grasby, S. E., Sanei, H. & Beauchamp, B. Catastrophic dispersion of coal fly ash into oceans during the latest Permian extinction Nat Geosci 4, 104–107, doi:10.1038/Ngeo1069 (2011). 24. Harvey, M. C., Brassell, S. C., Belcher, C. M. & Montanari, A. Combustion of fossil organic matter at the Cretaceous-Paleo (K-P) boundary. Geology 36, 355–358, doi:10.1130/G24646a.1 (2008). 25. Rose, N. L., Harlock, S., Appleby, P. G. & Battarbee, R. W. Dating of recent lake-sediments in the United-Kingdom and Ire using spheroidal carbonaceous particle (SCP) concentration profiles. Holocene 5, 328–335, doi:10.1177/09596836950050 (1995). 26. Swindles, G. T. & Roe, H. M. Constraining the age of spheroidal carbonaceous particle (SCP) stratigraphies in peats u tephrochronology. Quaternary Newsletter 110, 2–9 (2006).i p gy Q y , ( ) 27. Swindles, G. T. Dating recent peat profiles using spheroidal carbonaceous particles (SCPs). Mires and Peat 7, 1–10 (2010). i 28. Chambers, F. M., Beilman, D. W. & Yu, Z. Methods for determining peat humification and for quantifying peat bulk de organic matter and carbon content for palaeostudies of climate and peatland carbon dynamics. Mires and Peat 7, 1–10 (20 g p p y 29. Turner, T. E., Swindles, G. T. & Roucoux, K. H. Late Holocene ecohydrological and carbon dynamics of a UK raised bog: impact of human activity and climate change. Quaternary Sci Rev 84, 65–85, doi:10.1016/j.quascirev.2013.10.030 (2014). g y 9. Turner, T. E., Swindles, G. T. & Roucoux, K. H. References 1. Crutzen, P. J. Geology of mankind. Nature 415, 23–23, doi:10.1038/415023a (2002).h gy 2. Crutzen, P. J. & Stoermer, E. F. The ‘Anthropocene’. Global Change Newsletter 41, 17–18 (2000).h 2. Crutzen, P. J. & Stoermer, E. F. The ‘Anthropocene’. Global Change Newsletter 41, 17–18 (2000)h 2. Crutzen, P. J. & Stoermer, E. F. The ‘Anthropocene’. Global Ch h 3. Ruddiman, W. F. The anthropogenic greenhouse era began thousands of years ago. Climatic Change 61, 261–293 doi:10.1023/B:Clim.0000004577.17928.Fa (2003). 4. Certini, G. & Scalenghe, R. Anthropogenic soils are the golden spikes for the Anthropocene. Holocene 21, 1269–1274, doi:10.1177/0959683611408454 (2011).i 5. Zalasiewicz, J., Williams, M. & Waters, C. N. Can an Anthropocene Series be defined and recognized? Geol Soc Spec Publ 395 39–53, doi:10.1144/Sp395.16 (2014).fh p 6. Steffen, W. et al. The Anthropocene: From global change to planetary stewardship. Ambio 40, 739–761, doi:10.1007/s13280-011 0185-x (2011).h 7. Dean, J. R., Leng, M. J. & Mackay, A. W. Is there an isotopic signature of the Anthropocene? The Anthropocene Review, 1–12, doi:10.1177/2053019614541631 (2014). 8. Smith, V. C. Volcanic markers for dating the onset of the Anthropocene. A Stratigraphical Basis for the Anthropocene, Geologica Society of London Special Publications 395, 283–299 (2013).h Society of London Special Publications 395, 283–299 (2013). y f p , ( ) 9. Kandlbauer, J., Carey, S. 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J Paleolimnol 33, 393–431, doi:10.1007/s10933-005-2516-z (2005).l J , , ( ) 18. Rose, N. L. et al. Long-range transport of pollutants to the Falkland Islands and Antarctica: Evidence from lake Sediment fly ash particle records. Environ Sci Technol 46, 9881–9889, doi:10.1021/Es3023013 (2012).h 19. Broman, D., Naf, C., Wik, M. & Renberg, I. The importance of spheroidal carbonaceous particles (SCPs) for the distribution of Particulate Polycyclic Aromatic-Hydrocarbons (PAHs) in an estuarine-like urban coastal water area. Chemosphere 21, 69–77, doi:10.1016/0045-6535(90)90379-8 (1990). ( ) ( ) 20. Rose, N. L. & Appleby, P. G. Regional applications of lake sediment dating by spheroidal carbonaceous particle analysis I: U Kingdom. J Paleolimnol 34, 349–361, doi:10.1007/s10933-005-4925-4 (2005). 20. Rose, N. L. & Appleby, P. G. Regional applications of lake sediment dating by spheroidal carbonaceous particle analysis I: United Kingdom. J Paleolimnol 34, 349–361, doi:10.1007/s10933-005-4925-4 (2005). 21. Yang, H. D., Rose, N. L. Acknowledgements g We acknowledge NERC Training Grants NE/G52398X/1 to Elizabeth Watson and NE/G52398X/1 to Ed Turner. Special thanks go to Henry Lamb of Aberystwyth University for valuable advice on the XRF analysis. We thank Phil Gibbard for constructive comments on an earlier version of the manuscript. Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 5 www.nature.com/scientificreports/ Author Contributions GTS conceived the idea, carried out data analysis and wrote the first draft of the paper; EW carried out data analysis; TET provided the dataset from Malham; JMG, TH and KLB contributed expertise and text on geological aspects; JW contributed expertise and text on historic context. All authors contributed to and reviewed the final manuscript. Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 Additional Informationi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Swindles, G. T. et al. Spheroidal carbonaceous particles are a defining stratigraphic marker for the Anthropocene. Sci. Rep. 5, 10264; doi: 10.1038/srep10264 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 5:10264 \| DOI: 10.1038/srep10264 6
https://openalex.org/W2898728009	https://europepmc.org/articles/pmc6315647?pdf=render	English	null	Preliminary Scale of Reference Values for Evaluating Reactive Strength Index-Modified in Male and Female NCAA Division I Athletes	Sports	2,018	cc-by	5,938	Received: 29 September 2018; Accepted: 19 October 2018; Published: 29 October 2018 Abstract: The purpose of this analysis was to construct a preliminary scale of reference values for reactive strength index-modiﬁed (RSImod). Countermovement jump data from 151 National Collegiate Athletic Association (NCAA) Division I collegiate athletes (male n = 76; female n = 75) were analyzed. Using percentiles, scales for both male and female samples were constructed. For further analysis, athletes were separated into four performance groups based on RSImod and comparisons of jump height (JH), and time to takeoff (TTT) were performed. RSImod values ranged from 0.208 to 0.704 and 0.135 to 0.553 in males and females, respectively. Males had greater RSImod (p < 0.001, d = 1.15) and JH (p < 0.001, d = 1.41) as compared to females. No statistically signiﬁcant difference was observed for TTT between males and females (p = 0.909, d = 0.02). Only JH was found to be statistically different between all performance groups. For TTT no statistical differences were observed when comparing the top two and middle two groups for males and top two, bottom two, and middle two groups for females. Similarities in TTT between sexes and across performance groups suggests JH is a primary factor contributing to differences in RSImod. The results of this analysis provide practitioners with additional insight as well as a scale of reference values for evaluating RSImod scores in collegiate athletes. Keywords: countermovement jump; jump height; time to takeoff; force platform; athlete monitoring Christopher J. Sole 1,, Timothy J. Suchomel 2 and Michael H. Stone 3 1 Department of Health and Human Performance, The Citadel-The Military College of South Carolina, Charleston, SC 29409, USA 2 Department of Human Movement Sciences, Carroll University, Waukesha, WI 53186, USA; tsuchome@carrollu.edu 2 Department of Human Movement Sciences, Carroll University, Waukesha, WI 53186, USA; tsuchome@carrollu edu 3 Center of Excellence for Sport Science and Coach Education, Department of Exercise and Sport Science, East Tennessee State University, Johnson City, TN 37614, USA; stonem@etsu.edu C d l @ i d l d T l 1 843 953 6386 * Correspondence: csole@citadel.edu; Tel.: +1-843-953-6386 * Correspondence: csole@citadel.edu; Tel.: +1-843-953-6386 Sports 2018, 6, 133; doi:10.3390/sports6040133 Preliminary Scale of Reference Values for Evalu Reactive Strength Index-Modiﬁed in Male and Female NCAA Division I Athletes Christopher J. Sole 1,*, Timothy J. Suchomel 2 and Michael H. Stone 3 sports sports sports sports Brief Report 1. Introduction When assessing athlete testing and training data for the purpose of performance monitoring, coaches and sport scientists are often faced with questions regarding the worth of the data they have collected. Questions such as, “Is that score good?” or “How does that score compare to peer and aspirant performers?” are common as athlete performance data are reviewed. Unfortunately, answers to questions such as these are not always immediately apparent. In order to ﬁnd answers, or determine the worth of data, coaches and sport scientists must engage in the process of evaluation. Although the evaluation of data can be achieved through several means, one common approach is through a norm-referenced perspective. In this form of evaluation, an individual athlete’s score is compared to other scores considered representative of a population, and are commonly presented using percentiles. Overall, norm-referenced evaluation provides coaches and practitioners with a general idea of how an individual compares to a group or population. Normative data for various ﬁtness and performance tests have been previously published for a variety of populations [1]. Depending on www.mdpi.com/journal/sports Sports 2018, 6, 133 2 of 10 the purpose of the testing, this method of evaluation can be useful, such as in talent identiﬁcation, or grouping individuals based on their abilities. However, if data for a speciﬁc measure or population do not exist, this process is not possible. With the growing interest in vertical jump testing in athlete performance monitoring [2], as well as the increased accessibility of technology such as portable force platforms [3–7], many new variables derived from a single vertical jump have arisen from the literature. Although the addition of new variables used to characterize jump performance is not necessarily negative, it does however present interpretation and evaluation challenges for practitioners. One such variable is reactive strength index-modiﬁed (RSImod) [8]. Reactive strength index-modiﬁed can be calculated from a standing countermovement jump (CMJ) and represents the ratio of jump height (JH) to movement time, referred to as time to takeoff (TTT). Since it was ﬁrst introduced [8], RSImod has been reported to be both a reliable [9] and valid [10,11] indication of the athlete’s lower-body impulsive or “explosive” ability. Furthermore, considering RSImod takes into consideration an outcome variable (i.e., JH) and a process variable (i.e., TTT), it appears to be a simple and effective means for evaluating jumping strategy and ultimately neuromuscular functional state (i.e., adaptation or fatigue) [12]. 2.1. Participants This analysis was completed retrospectively using archived data that were collected as part of an ongoing athlete performance monitoring program [17]. Additionally, all athletes provided written informed consent at the time of data collection. The methods and scope of this analysis were reviewed and approved by the University’s institutional review board. Countermovement jump data from a total of 151 collegiate athletes (male n = 76; female n = 75) were included in this analysis. All athletes were NCAA Division I, representing a variety of sports including baseball (n = 29; height = 182.1 ± 6.2 cm, body mass 88.0 ± 9.0 kg), men’s tennis (n = 7; height = 176.9 ± 9.0 cm, body mass = 74.7 ± 9.5 kg), men’s soccer (n = 25; height = 179.5 ± 6.8 cm, body mass = 78.5 ± 9.2 kg), men’s track and ﬁeld (n = 15; height = 183.1 ± 6.3 cm; body mass = 94.4 ± 29.0 kg), women’s tennis (n = 11; height = 167.6 ± 5.6 cm, body mass = 68.6 ± 12.7 kg), women’s soccer (n = 22; height = 166.1 ± 6.2 cm, body mass = 63.9 ± 8.1 kg), women’s track and ﬁeld (n = 13; height = 166.5 ± 7.2 cm, body mass = 67.1 ± 20.5 kg), softball (n = 14; height = 168.5 ± 6.7 cm, body mass = 70.4 ± 10.1 kg), and women’s volleyball (n = 15; height = 175.3 ± 7.5 cm, body mass = 70.7 ± 7.7 kg). Athletes ranged in age from 18–23 years. 1. Introduction Although a variety of normative data have been published related to CMJ’s criterion variable of jump height [13–16], normative data have yet to be published on many speciﬁc CMJ variables such as RSImod. If RSImod is to be effectively used in athlete performance testing and monitoring, more information is needed related to the interpretation and evaluation of this variable. The purpose of this report is twofold (1) to present a preliminary scale of reference values for RSImod, and (2) to examine differences in the constituents of RSImod (JH and TTT) across the scale in an effort to provide additional context to aid in the evaluation of this variable. 2.3. Data Analysis Following data collection, voltage data obtained from the force platform were converted to the vertical component of the ground reaction force using laboratory calibrations. Force-time curves were then constructed. All data were collected and analyzed using custom programs (LabVIEW version 15, National Instruments, Austin, TX, USA). To reduce noise in the signal, a digital low-pass Butterworth ﬁlter with a cutoff frequency of 10 Hz was used. From the force-time data, RSImod was then calculated using procedures outlined by previous authors [8,9,19]. Equation (1) RSImod = CMJ height (m) time to takeoff (s) (1) (1) The CMJ height (JH) was calculated from the vertical displacement of the jumper’s center of mass estimated from ﬂight time [20] and TTT was deﬁned as the time interval between the initiation of the countermovement and the instant when the jumper left the force platform. A threshold of 10 N was used to identify the beginning and end of this period. 2.2. Data Collection In order to avoid any possible inﬂuence of fatigue from training or competition, all CMJ data included in this analysis were selected from the athlete’s preseason testing sessions. All athletes were injury-free at the time of data collection. All data were collected under the same standardized testing protocol. Speciﬁcally, participants completed a general warm-up consisting of 25 jumping-jacks, one set of ﬁve repetitions of mid-thigh clean pulls with a 20 kg barbell, and three sets of ﬁve repetitions of mid-thigh clean pulls with barbell totaling 40 kg for females and 60 kg for males. Immediately following the general warm-up, participants began jump testing where they completed a speciﬁc warm-up consisting of two submaximal CMJs performed at 50% and 75% of their perceived maximum effort. Following the speciﬁc warm-up, participants performed two maximal CMJs with approximately 3 of 10 Sports 2018, 6, 133 60 seconds of rest between each jump. Brieﬂy, each participant stood motionless on the force platform and then received a countdown of “3, 2, 1, jump!” After the jump command, participants performed a rapid countermovement to a self-selected depth before propelling themselves upward with the intent of jumping as high as possible. To control for arm swing, all jumps were performed while holding a near-weightless (≤0.5 kg) plastic bar across the shoulders, approximately between the seventh cervical and third thoracic vertebra [9,18]. All jumps were performed on a force platform (0.91 m × 0.91 m, RoughDeck HP, Rice Lake Weighing Systems, Rice Lake, WI, USA) sampling at 1000 Hz. 2.4. Statistical Analyses Relative and absolute reliability of RSImod and its constituent variables were assessed using a two-way mixed-effect model intraclass correlation coefﬁcient (ICC) and typical error expressed as a coefﬁcient of variation (CV%) performed between the two CMJ trials. Reliability was found to be acceptable with high test-retest correlation and low CV% for all variables for both males (RSImod: ICC = 0.963, CV% 7.6%; JH: ICC = 0.978, CV% = 4.7%; TTT: ICC 0.899, CV% = 5.4%) and females (RSImod: ICC = 0.967, CV% 7.9%, JH: ICC = 0.978, CV% = 4.1%; TTT: ICC 0.892, CV% = 6.0%), therefore the mean of the CMJ trials was used for all analyses [21,22]. Once averaged, RSImod data were aggregated by sex to form male (n = 76) and female (n = 75) groups. RSImod scores for both males and females were assessed and found to be normally distributed (males: Kolmogorov-Smirnov (D (76) = 0.090, p > 0.200; Skewness: 0.170, SE = 0.276; Kurtosis: −0.154, SE = 0.545; females: Kolmogorov-Smirnov (D (75) = 0.064, p > 0.200; Skewness: 0.221, SE = 0.277; Kurtosis: −0.272, SE = 0.548). Additionally, no outliers were identiﬁed [23,24] for either group. Male and female scales were then constructed using percentile rank. In order to allow for further analysis of the scales, male and female participants were then ranked based on RSImod scores and then divided into four performance groups using quartiles as cut points (Figures 1 and 2). Independent samples t-tests were used to compare differences in RSImod, JH, and TTT between males and females. To compare differences between each performance group, a series of one-way analysis of variances (ANOVAs) were used to examine the four performance groups with Bonferroni post hoc analysis used when appropriate. Levene’s test was used to assess equality of variance for all group comparisons. To provide an indication of the practical signiﬁcance of any observed differences, Cohen’s d effect sizes were calculated and interpreted in accordance with the scale developed by Hopkins [25]. The critical alpha was set at p < 0.05 for all analyses. All statistical analyses were performed using SPSS version 23 (IBM, Armonk, NY, USA) and Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA). 4 of 10 4 of 10 Sports 2018, 6, 133 Sports 2018, 6, x FO Figure 1. Ranked reactive strength index-modified (RSImod) scores and performance groups for male athletes. 2.4. Statistical Analyses Note: U = upper performance group; UM = upper-middle performance group; LM = lower- middle performance group; L = lower performance group. Figure 1. Ranked reactive strength index-modiﬁed (RSImod) scores and performance groups for male athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower-middle performance group; L = lower performance group. Figure 1. Ranked reactive strength index-modified (RSImod) scores and performance groups for male athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower- middle performance group; L = lower performance group. Figure 1. Ranked reactive strength index-modified (RSImod) scores and performance groups for male Figure 1. Ranked reactive strength index-modified (RSImod) scores and performance groups for male athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower- middle performance group; L = lower performance group Figure 1. Ranked reactive strength index-modiﬁed (RSImod) scores and performance groups for male athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower-middle performance group; L = lower performance group. athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower- middle performance group; L = lower performance group. p g p p g p Figure 2. Ranked reactive strength index-modified scores and performance groups for female athletes. Note: U = upper performance group; UM = upper middle performance group; LM = lower middle Figure 2. Ranked reactive strength index-modified scores and performance groups for female athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower-middle performance group; L = lower performance group. Figure 2. Ranked reactive strength index-modiﬁed scores and performance groups for female athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower-middle performance group; L = lower performance group. Figure 2. Ranked reactive strength index-modified scores and performance groups for female athletes. N t U f UM iddl f LM l iddl Figure 2. Ranked reactive strength index-modified scores and performance groups for female athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower-middle performance group; L = lower performance group. Figure 2. Ranked reactive strength index-modiﬁed scores and performance groups for female athletes. Note: U = upper performance group; UM = upper-middle performance group; LM = lower-middle performance group; L = lower performance group. Note: perfor 3 Results 3. Results p g p p g p 3. Results Descriptive statistics for RSImod and its constituents for male and female athletes are displayed in Table 1. The preliminary reference value scales for RSImod for both male and female athletes are 3. Results Descriptive statistics for RSImod and its constituents for male and female athletes are displayed in Table 1. The preliminary reference value scales for RSImod for both male and female athletes are displayed in Tables 2 and 3. Descriptive statistics for male and female athletes stratified by performance group are displayed in Table 4 Descriptive statistics for RSImod and its constituents for male and female athletes are displayed in Table 1. The preliminary reference value scales for RSImod for both male and female athletes are displayed in Tables 2 and 3. Descriptive statistics for male and female athletes stratiﬁed by performance group are displayed in Table 4. 5 of 10 Sports 2018, 6, 133 Table 1. Descriptive statistics for reactive strength index-modiﬁed for male and female athletes (n = 151, mean ± standard deviation. Male (n = 76) Female (n = 75) RSImod (m/s) 0.424 ± 0.102 0.314 ± 0.089 JH (m) 0.36 ± 0.07 0.27 ± 0.06 TTT (s) 0.868 ± 0.105 0.870 ± 0.114 Note: RSImod = reactive strength index-modiﬁed; JH = jump height; TTT = time to takeoff Table 1. Descriptive statistics for reactive strength index-modiﬁed for male and female athletes (n = 151, mean ± standard deviation. Male (n = 76) Female (n = 75) RSImod (m/s) 0.424 ± 0.102 0.314 ± 0.089 JH (m) 0.36 ± 0.07 0.27 ± 0.06 TTT (s) 0.868 ± 0.105 0.870 ± 0.114 Note: RSImod = reactive strength index-modiﬁed; JH = jump height; TTT = time to takeoff Table 2. Reference value scale for reactive strength index-modiﬁed for male collegiate athletes (n = 76). Percentile RSImod (m/s) 97 0.630 95 0.604 90 0.547 85 0.523 80 0.508 75 0.492 70 0.487 65 0.476 60 0.461 55 0.448 50 0.419 45 0.398 40 0.376 35 0.369 30 0.366 25 0.352 20 0.331 15 0.316 10 0.308 5 0.257 3 0.216 Table 2. Reference value scale for reactive strength index-modiﬁed for male collegiate athletes (n = 76). Table 3. Reference value scale for reactive strength index-modified for female collegiate athletes (n = 75). Note: perfor 3 Results 3. Results Percentile RSImod (m/s) 97 0.497 95 0.461 90 0.434 85 0.413 80 0.391 75 0.379 70 0.366 65 0.351 60 0.333 55 0.315 50 0.308 45 0.293 40 0.279 35 0.273 30 0.266 25 0.248 20 0.241 15 0.214 10 0.202 5 0.173 3 0.139 Table 3. Reference value scale for reactive strength index-modified for female collegiate athletes (n = 75). 6 of 10 Sports 2018, 6, 133 Table 4. Descriptive statistics for reactive strength index-modiﬁed between performance groups (mean ± standard deviation). Table 4. Descriptive statistics for reactive strength index-modiﬁed between performance groups (mean ± standard deviation). Male Performance Group n RSImod (m/s) JH (m) TTT (s) Female Perfomance Group n RSImod (m/s) JH (m) TTT (s) U 20 0.549 ± 0.057 0.43 ± 0.05 0.795 ± 0.087 U 19 0.429 ± 0.045 0.34 ± 0.03 0.792 ± 0.077 UM 18 0.464 ± 0.021 0.39 ± 0.04 0.850 ± 0.067 UM 18 0.343 ± 0.023 0.29 ± 0.04 0.848 ± 0.117 LM 20 0.379 ± 0.019 0.33 ± 0.04 0.871 ± 0.100 LM 20 0.277 ± 0.017 0.24 ± 0.03 0.880 ± 0.092 L 18 0.296 ± 0.044 0.28 ± 0.05 0.964 ± 0.090 L 18 0.203 ± 0.036 0.19 ± 0.03 0.964 ± 0.100 Note: RSImod = reactive strength index-modiﬁed; JH = jump height; TTT = time to takeoff, U = upper performance group; UM = upper-middle performance group; LM = lower-middle performance group, L = lower performance group. Statistically signiﬁcant differences were observed when comparing male and female athletes with regard to RSImod (t (149) = 7.09, p < 0.001, d = 1.15) and JH (t (149) = 8.64, p < 0.001, d = 1.41). No statistically signiﬁcant difference was observed for TTT between male and female athletes (t (149) = 0.11, p = 0.909, d = 0.02). Statistically signiﬁcant differences were observed for JH and TTT between individual performance groups for both male (JH: (F (3,72) = 43.68, p < 0.001; TTT: (F (3,72) = 12.17, p < 0.001) and female athletes (JH: (F (3,71) = 63.90, p < 0.001; TTT: (F (3,71) = 10.11, p < 0.001). Speciﬁcally, post hoc analyses for the males revealed that JH was statistically different (p < 0.05) between all four performance groups with moderate to very large effect sizes observed ranging from d = 0.86 to 2.96. Note: perfor 3 Results 3. Results For TTT all comparisons were found to be statistically signiﬁcant with effect sizes ranging from moderate to large (d = 0.81 to 1.44), with the exception of the comparisons of upper and upper-middle groups and upper-middle and lower-middle groups that were found not to be statistically different exhibiting moderate (d = 0.71) and small (d = 0.25) effect sizes, respectively. Similarly, for females, statistically signiﬁcant (p < 0.05) differences were identiﬁed between all four performance groups for JH with large to very large effect sizes observed ranging from d = 1.26 to 4.50. For TTT, all groups were found to be statistically different (p < 0.05) with moderate to large effect sizes observed ranging from d = 1.05 to 1.93 with the exception of comparisons of upper and upper middle (d = 0.57), upper-middle and lower-middle (d = 0.31), and lower-middle and lower (d = 0.87) groups, who were determined not to be statistically different, exhibiting small to moderate effect sizes. 4. Discussion The purpose of this analysis was to construct a preliminary scale of reference values for evaluating RSImod. Additionally, this report included an analysis of the variables used to calculate RSImod in effort to improve the interpretation of this variable. RSImod values ranged from 0.208 to 0.704 and 0.135 to 0.553 in males and females, respectively. When comparing male and female athletes overall, males exhibited statistically greater RSImod values. This ﬁnding is in agreement with previous research that examined RSImod differences between male and female athletes [9]. On average, male athletes were found to have RSImod values 29.8% greater than their female counterparts. Interestingly, when comparing TTT values males and females were found to be quite similar, exhibiting only a 0.2% difference on average. This similarity in TTT between males and females is in agreement with previous investigations examining the temporal structure of the CMJ [26,27]. The observed similarities in TTT values and statistically different RSImod values, indicates that the primary factor inﬂuencing the sex differences observed in RSImod may be attributed to jump height. In fact, in the present analysis, there was an approximate 30% difference in JH between males and females. The strong inﬂuence of JH on RSImod over TTT can be further illustrated by examining the relationships between these variables. The relationship between RSImod and TTT was r = −0.60 for females and r = −0.40 for males, whereas the relationship between RSImod and JH was r = 0.87 and r = 0.89 for males and females, respectively. Although not explicitly examined in the present study, there are several reasons why differences in JH may have existed between males and females. Two reasons may be due to differences in muscular strength or in jump strategy. Previous research has indicated that RSImod 7 of 10 Sports 2018, 6, 133 displayed strong relationships with maximal isometric strength [28]; however, it should be noted that additional research has indicated that RSImod differences existed between males and females despite controlling for strength level and using it as a covariate [29]. Furthermore, McMahon et al. [30], as well as Sole et al. [26], have indicated that male participants applied a larger concentric impulse and achieved a greater velocity throughout the concentric phase, which ultimately leads to a greater jump height. 4. Discussion It is clear that although differences in RSImod and JH may exist between males and females, further research is needed to determine the factors that produce these differences. p A secondary analysis of the present data scale involved a comparison of four performance groups representing the upper, upper-middle, lower-middle, and lower male and female athletes, as determined by RSImod. When examining both JH and TTT between the performance groups, only JH was found to be statistically different between all groups. In contrast, no statistical differences and only small to moderate effect sizes were observed when comparing the top two (upper and upper-middle) and middle two (upper-middle and lower-middle) groups for males in TTT values. Similarly for females, no statistical differences and only small to moderate effect sizes were observed between the top two (upper and upper-middle), bottom two (lower and lower-middle), and middle two (upper- and lower-middle) groups. These results indicate that TTT alone was not enough to differentiate between groups, as was the case with JH. As mentioned above, the current ﬁndings may be partially explained by muscular strength differences between each group. An abundance of research supports the idea that stronger individuals produce superior jump performances compared to weaker individuals [31]. Another possible explanation for the current ﬁndings may be due to differences in musculotendinous stiffness characteristics. Kipp et al. [32] indicated that vertical stiffness was strongly correlated to the traditional measure of reactive strength index (drop jump height/ground contact time). Given that the RSImod and reactive strength index variants are strongly correlated [33], it is possible that vertical stiffness may also play a role in JH performance during a CMJ. To the authors’ knowledge, no study has yet to investigate the inﬂuence of stiffness on RSImod. The reference values presented in the current study were constructed using athletes from various sporting disciplines. While the use of multiple sports within a single scale may be viewed as a limitation, it should be noted that no other study has used a sample size as large as the one used within the current study. As displayed in Figures 1 and 2, the ranking and grouping of athletes by RSImod resulted in a disproportionate sport representation within groups. Although this lack of homogeneity within performance groups may be viewed as problematic, it also may simply highlight sport speciﬁc differences in jumping strategies as noted by previous reports [34]. 4. Discussion Moreover, it should be noted that previous research indicated that within-team differences based on player position may exist when examining RSImod [35]. Thus, while the RSImod reference values produced in this study may serve as an initial step in RSImod comparisons between athletes, it is important for future research to continue to collect normative RSImod data for different sports and levels of sports so that additional scales may be developed. Furthermore, additional data will allow for the comparison of athletes within a single sport or between different levels of the same sport [1]. Although the scales and analyses provide insight for evaluating RSImod and its constituent parts, practitioners should be cognizant of some potential limitations of this data. The present scales and analyses used athlete data collected from a single university, and although all athletes were competitive at the NCAA Division I level, this sample may not be representative of all collegiate athletes. Thus, as noted above, it is important that if practitioners see value in using RSImod as a monitoring tool, additional data for male and female athletes is needed. A second limitation of the present study may be related to the speciﬁc data analysis procedures used to calculate RSImod. Although all data were collected using force plates sampling at an optimal sampling frequency [36] and analyzed using identical procedures, the present study used arbitrary thresholds to identify the initiation of the countermovement (unweighting phase) as well as takeoff and landing events. Furthermore, the jump height values used to calculate RSImod were estimated using time in air. Recently researchers have identiﬁed this as potentially problematic as it relates to RSImod as any errors in identifying TTT 8 of 10 Sports 2018, 6, 133 or JH will ultimately inﬂuence RSImod [12]. Future analyses should consider using more robust methods [22,37] for identifying these key time points during the jump, in effort to reduce potential error. Although this limitation is valid, within this analysis it may be partially obviated by the fact that all CMJ analyses were completed using identical procedures. 5. Conclusions The results of the present analysis provide practitioners with preliminary scales of reference values for interpreting RSImod scores in collegiate athletes. A primary ﬁnding of this report was that the difference in RSImod between male and female Division I athletes is largely attributed to differences in jump height. Interestingly, when compared as a whole, there were no discernable differences in TTT between male and female athletes. Analysis of the RSImod scales indicate that there are clear performance differences between upper performers and lower male and female performers, in both RSImod and its constituent parts. Given the importance of comparing performances between individuals, these reference values may provide a valuable resource for practitioners seeking to evaluate their athlete’s performance. Author Contributions: C.J.S. and T.J.S. conceived and designed the experiments; C.J.S. and T.J.S. performed the experiments; C.J.S. analyzed the data; C.J.S. and M.H.S. contributed materials and analysis tools; C.J.S, T.J.S., and M.H.S wrote the paper. Funding: No ﬁnancial support was provided for the completion of this project. Funding: No ﬁnancial support was provided for the completion of this project. Funding: No ﬁnancial support was provided for the completion of this project. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Vertical jump and leg power norms for young adults. 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https://openalex.org/W2103243654	https://europepmc.org/articles/pmc3991020?pdf=render	English	null	Role of Glycogen Synthase Kinase-3β in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons	Pharmaceuticals	2,010	cc-by	6,139	Pharmaceuticals 2010, 3, 42-58; doi:10.3390/ph3010042 Pharmaceuticals 2010, 3, 42-58; doi:10.3390/ph3010042 Role of Glycogen Synthase Kinase-3β in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons Cristina Ploia 1, Alessandra Sclip 1, Alessio Colombo 1, Mariaelena Repici 2, Fabrizio Gardoni 3, Monica Di Luca 3, Gianluigi Forloni 1, Xanthi Antoniou 1 and Tiziana Borsello 1,* 1 Istituto di ricerche Farmacologiche "Mario Negri", Via La Masa 19, 20156 Milano, Italy; E-Mails: cristina.ploia@marionegri.it (C.P.); alessandra.sclip@marionegri.it (A.S.); alessiovittorio.colombo@marionegri.it (A.C.); gianluigi.forloni@marionegri.it (G.F.); xanthi.antoniou@marionegri.it (X.A.) 1 Istituto di ricerche Farmacologiche "Mario Negri", Via La Masa 19, 20156 Milano, Italy; E-Mails: cristina.ploia@marionegri.it (C.P.); alessandra.sclip@marionegri.it (A.S.); alessiovittorio.colombo@marionegri.it (A.C.); gianluigi.forloni@marionegri.it (G.F.); xanthi.antoniou@marionegri.it (X.A.) 2 UMR 7102 Neurobiologie des Processus Adaptatifs, Universite P. et M. Curie, 9 quai St Bernard, 75005, Paris, France; E-Mail: mr200@le.ac.uk (M.R.) 2 UMR 7102 Neurobiologie des Processus Adaptatifs, Universite P. et M. Curie, 9 quai St Bernard, 75005, Paris, France; E-Mail: mr200@le.ac.uk (M.R.) 3 Dipartimento Scienze Farmacologiche, Università degli Studi di Milano, Via Balzaretti, 9, 20133 Milano, Italy; E-Mail: fabrizio.gardoni@unimi.it (F.G.); monica.diluca@unimi.it (M.D.L.) * Author to whom correspondence should be addressed; E-Mail: tiziana.borsello@marionegri.it; Tel.: +39-02-39014469/39014592; Fax: +39-02-3546277. Received: 23 October 2009; in revised form: 3 December 2009 / Accepted: 5 January 2010 / Published: 7 January 2010 Abstract: The phosphorylation of Amyloid Precursor Protein (APP) at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (Cdk5) can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 μM) for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3β activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3β reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3β signaling pathway. Pharmaceuticals 2010, 3 43 Keywords: APP; NMDA; GSK-3β; JNK; Cdk5 Keywords: APP; NMDA; GSK-3β; JNK; Cdk5 1. Introduction Alzheimer’s disease (AD) is the most common form of dementia in the elderly. One of the pathological hallmarks of AD is the abnormal accumulation of amyloid-β (Aβ) produced by proteolytic cleavage of the Amyloid Precursor Protein (APP). APP processing is influenced by post- translational modifications, N- and O-glycosylations, and phosphorylations that can induce a preferential cleavage of APP in the amyloidogenic pathway eventually leading to Aβ production [1,2]. In particular the hyperphosphorylation of APP at Threonine 668 (Thr668) in the cytoplasmic domain plays a pivotal role in APP processing and is highly relevant to AD [3]. The candidate kinases, responsible for Thr668 phosphorylation, are three: Cyclin-dependent kinase 5 (Cdk5), glycogen synthase kinase-3β (GSK-3β) and c-jun-N-terminal kinase (JNK) [4-7], but the conditions and the precise role of these kinases in APP metabolism have still to be elucidated [8]. These three kinases are also associated with neurotoxicity [9,10] and are implicated in Alzheimer’s disease [5,6,11,12]. Amongst them, JNK is a key pathway in excitotoxicity [13–15], Aβ toxicity [16] and AD pathology [3] and modulates APP phosphorylation in differentiated neurons [7,17]. Cdk5 is another kinase with an important role in excitotoxicity [18] as well as AD pathogenesis [19]. Cdk5 and its activator p25 accumulate in neurons during oxidative stress and treatment with Aβ [9,10,20], while altered Cdk5/p25 levels have been reported in AD brains [21]. Finally, GSK-3β is of particular relevance to neurological disorders since it phosphorylates APP at Thr668, as well as Tau and Presenilin1 [22–24], inducing amyloid plaques and neurofibrillary tangles formation [25]. An abnormal increase of GSK-3β activity is associated with AD pathogenesis [26] and inhibition of GSK-3β hyperactivation induced by exposure of neurons to Aβ peptide, prevents neurodegeneration [27]. Moreover GSK-3β inhibition reduces neuronal death in models of oxygen- glucose deprivation as well as glutamate excitotoxicity in vitro [28]. Because glutamate plays a key role in AD pathology and NMDA stimulation for prolonged periods leads to increased production and secretion of Aβ fragments in primary neuronal cultures [29] we investigated APP phosphorylation at Thr668 in cortical neurons stimulated with a high dose of NMDA (100 μM). This dose of NMDA correlated with an increase in APP phosphorylation and amyloidogenic processing (β-APPs) and led to activation of all three kinases without causing neuronal death in this short temporal window. 2.1. NMDA treatment of cortical neurons Cortical neurons were exposed to N-methyl-D-aspartate (NMDA) 100 μM for 30’-45’. As reported previously by Borsello et al., this dose of NMDA induces 90% neuronal death after 24 h [15]. Instead, in the temporal window used in our experiments, NMDA administration did not induce neuronal death Pharmaceuticals 2010, 3 2.2. NMDA for 30’-45’ induces APP hyperphosphorylation at Thr668 Application of 100 μM NMDA in cortical neurons induced hyperphosphorylation of APP at Thr668. P-APP was normalized against the total APP using the 22C11 antibody that recognizes the APP full length (Figure 2, a). Quantification of western blots revealed that, compared to control conditions, NMDA application increased APP phosphorylation at Thr668. In particular, at 30’ the P-APP/APP ratio reached 1.7 fold increase (p = 0.02) and at 45’ rose further (2.1-fold increase, p = 0.003) (Figure 2b). Total APP protein levels were not affected by NMDA treatment (Figure 2, d). The ratio APP/tubulin did not change, while the ratio P-APP/tubulin increased confirming the augmentation of P-APP (Figure 2, c). Notably, NMDA resulted in an increase of the secreted APPs fragments in the corresponding neuronal media (Figure 2, e). More specifically, by 45’ we observed a 1.7-fold increase (p = 0.003), (Figure 2, f). Moreover NMDA treatment induced a significant increase in the amyloidogenic processing of APP as demonstrated by the increase of βAPPs/APP ratio after 45’ (p = 0.02) and reduction of αAPPs/APP ratio at 30’-45’ (p = 0.037 and p = 0.039) (see Figure 3, a-b-c). These results suggest that NMDA stimulation, not only induces an increase in APP phosphorylation, but promotes the amyloidogenic processing by increasing βAPPs in the media. Pharmaceuticals 2010, 3 45 The first clear signs of neuronal death became apparent after 1 h of NMDA stimulation, with (a) a significant increase in LDH release in comparison to control and NMDA treated cultures for 30’-45’ (b) nuclear fragmentation (Figure 1b, D arrows) and (c) the presence of “ghosts” neurons (denucleated neurons with an unstained shadowy center where the nucleus used to be) (Figure 1b, D arrowheads) further underlying that at this point NMDA becomes toxic. Pharmaceuticals 2010, 3 44 as demonstrated by LDH analysis and Hoechst staining (Figure 1, a-b). Following 30’-45’ NMDA stimulation, some neurons presented a swelling appearance with enlarged nuclei, a hallmark of neuronal death (Figure 1b, B-C arrows). Figure 1. Neuronal death is not induced after 30’-45’ NMDA application. (a) Neurons were exposed to NMDA (30’-45’-1 h) and neuronal viability was assessed by LDH assay. NMDA-1h significantly increased neuronal death. Quantification is from five independent experiments (±S.E.M.), (* p < 0.05). (b) Hoechst staining of neurons exposed to NMDA. At 30’-45’, NMDA induces some neuronal swelling (arrows fig. B-C, magnification 20 × and higher magnification (40 ×) in the box B). After 1 h some neurons undergo nuclear fragmentation (arrows fig. D) while we can also note the presence of the first “ghost” neuron (arrowheads fig. D; higher magnification (40 ×) in the box). 2.3. NMDA stimulation induces JNK, Cdk5 and GSK-3β Because JNK, Cdk5 and GSK-3β kinases can all contribute to APP phosphorylation at Thr668 we studied their activation following NMDA stimulation. we (1) JNK. In agreement with our previous reports [14,15], we could show that NMDA induces JNK activation both at 30’ and 45’ (1.3- and 1.75-fold, p = 0.03) as shown by quantification of the P- JNK/JNK ratio (Figure 4, a-b). (1) JNK. In agreement with our previous reports [14,15], we could show that NMDA induces JNK activation both at 30’ and 45’ (1.3- and 1.75-fold, p = 0.03) as shown by quantification of the P- JNK/JNK ratio (Figure 4, a-b). (2) Cdk5. The effect of NMDA on Cdk5 activity was examined with Cdk5 immunoprecipitation kinase assays using histone H1 as protein kinase substrate. NMDA application for 45’ induced a significant increase (1.8-fold, p = 0.05) in Cdk5 activity (Figure 4, c-d). (3) GSK-3β. Concomitantly, NMDA application led to a powerful activation of GSK-3β (here expressed as a decrease of the inhibitory phosphorylation in Ser-9). GSK-3β phosphorylation decreased to 0.5 (p = 0.002) by 30’, to reach a 0.7 value (p = 0.0002) by 45’ (Figure 4, e-f). These results demonstrate that NMDA treatment leads to the activation of all three kinases, indicating the involvement of complex signaling mechanisms. Pharmaceuticals 2010, 3 46 Figure 2. NMDA application induces APP hyperphosphorylation. (a) Neurons were exposed to NMDA (30’-45’), cell lysates were immunoblotted for P-APP and total-APP. Loading control: Tubulin. (b) Quantification showed that NMDA increased P-APP level (P-APP/APP ratio), at 30’ (70%) and 45’ (110%). (c) NMDA increased P-APP/Tub ratio after 45’ (60%). (d) NMDA did not affect the total-APP protein level (APP/Tub). (e) Proteins from culture media were blotted for total-APP secreted (APPs). Loading control: Total medium proteins identified with Coomassie Blue. (f) NMDA-45’ treatment increased APPs level (70%). Quantifications are from six independent experiments (±S.E.M.), * p < 0.05, ** p < 0.01. Figure 2. NMDA application induces APP hyperphosphorylation. (a) Neurons were exposed to NMDA (30’-45’), cell lysates were immunoblotted for P-APP and total-APP. Loading control: Tubulin. (b) Quantification showed that NMDA increased P-APP level (P-APP/APP ratio), at 30’ (70%) and 45’ (110%). (c) NMDA increased P-APP/Tub ratio after 45’ (60%). (d) NMDA did not affect the total-APP protein level (APP/Tub). (e) Proteins from culture media were blotted for total-APP secreted (APPs). Loading control: Total medium proteins identified with Coomassie Blue. 2.3. NMDA stimulation induces JNK, Cdk5 and GSK-3β (f) NMDA-45’ treatment increased APPs level (70%). Quantifications are from six independent experiments (±S.E.M.), * p < 0.05, ** p < 0.01. 47 Pharmaceuticals 2010, 3 Figure 3. Inhibition of GSK-3β and APP amyloidogenic processing. (a) NMDA induces an increase in the APP amyloidogenic processing as shown by the increase of βAPPs and the decrease of αAPPs fragments. (b) Quantification showed a significant increase of βAPPs/APP ratio during NMDA-45’ treatment (p = 0.02) in comparison to untreated controls. L803-mts/NMDA-45’ treatment reduced βAPPs levels, this reduction is not significant compared to NMDA-45’ treatment. (c) NMDA reduced the αAPPs/APP ratio at 30’-45’ (p = 0.037, p = 0.039) compared to untreated controls. Instead, L803-mts induced an increase of the αAPPs/APP ratio, although it was not able to restore control levels. Loading control: Tubulin. Quantifications are from three independent experiments (±S.E.M.), * p < 0.05. Figure 3. Inhibition of GSK-3β and APP amyloidogenic processing. (a) NMDA induces an increase in the APP amyloidogenic processing as shown by the increase of βAPPs and the decrease of αAPPs fragments. (b) Quantification showed a significant increase of βAPPs/APP ratio during NMDA-45’ treatment (p = 0.02) in comparison to untreated controls. L803-mts/NMDA-45’ treatment reduced βAPPs levels, this reduction is not significant compared to NMDA-45’ treatment. (c) NMDA reduced the αAPPs/APP ratio at 30’-45’ (p = 0.037, p = 0.039) compared to untreated controls. Instead, L803-mts induced an increase of the αAPPs/APP ratio, although it was not able to restore control levels. Loading control: Tubulin. Quantifications are from three independent experiments (±S.E.M.), * p < 0.05. Pharmaceuticals 2010, 3 48 Figure 4. JNK, Cdk5 and GSK-3β are activated following NMDA application. (a) NMDA increased JNK activity as revealed by the increment of P-JNK. (b) Quantification showed a 30% and 75% increase in JNK activity at 30’-45'. (c) Cdk5 activity assay showed an increase in enzyme activity after NMDA-45’. (d) Quantification confirmed an 80% induction of Cdk5 activity after NMDA-45’. (e) Neurons were exposed to NMDA and blotted for P-GSK-3β and total GSK-3β. (f) NMDA induced an increase of GSK-3β activity at 30’ (50%) and 45’ (70%). Loading control: Tubulin. Quantifications are from six independent experiments (±S.E.M.), * p < 0.05, ** p < 0.01. Figure 4. JNK, Cdk5 and GSK-3β are activated following NMDA application. (a) NMDA increased JNK activity as revealed by the increment of P-JNK. 2.3. NMDA stimulation induces JNK, Cdk5 and GSK-3β (b) Quantification showed a 30% and 75% increase in JNK activity at 30’-45'. (c) Cdk5 activity assay showed an increase in enzyme activity after NMDA-45’. (d) Quantification confirmed an 80% induction of Cdk5 activity after NMDA-45’. (e) Neurons were exposed to NMDA and blotted for P-GSK-3β and total GSK-3β. (f) NMDA induced an increase of GSK-3β activity at 30’ (50%) and 45’ (70%). Loading control: Tubulin. Quantifications are from six independent experiments (±S.E.M.), * p < 0.05, ** p < 0.01. Pharmaceuticals 2010, 3 NMDA for 1 h significantly increased neuronal death while treatment with D-JNKI protected neurons. Quantification is from five independent experiments (±SEM), (∗ p < 0.05). Neurons were pre-treated with Roscovitine (10 μM) for 30’ before NMDA administration and the P-APP/APP ratio was compared to untreated neurons. Roscovitine effectively blocked NMDA induced activation of Cdk5 but did not reduce APP phosphorylation levels. Quantification of P-APP/APP ratio confirmed that there was no significant reduction in the Roscovitine/NMDA samples compared to NMDA alone (Figure 5, c-d). ( g ) Thus Cdk5 does not play a pivotal role in NMDA mediated hyperphosphorylation of APP. Pharmaceuticals 2010, 3 49 Pharmaceuticals 2010, 3 2.4. Neither JNK nor Cdk5 are responsible for NMDA induced APP hyperphosphorylation JNK nor Cdk5 are responsible for NMDA induced APP hyperphosphorylation As previously shown, in control conditions, D-JNKI1 treatment for 24h prevented APP phosphorylation on Thr668 in cortical neurons [17]. To investigate the role of JNK in APP phosphorylation following NMDA application we used the same D-JNKI1 inhibitor. Neurons were pre-treated with D-JNKI1 (4 μM) 30’ before NMDA stimulation and the P-APP/APP ratio was compared to untreated neurons (Figure 5, a). D-JNKI1 did not prevent NMDA-induced APP phosphorylation as shown by quantification of Western blots (Figure 5, b). On the contrary, D- JNKI1/NMDA co-treatment induced an increase of P-APP compared to NMDA alone. Application of D-JNKI alone or with NMDA did not lead to neuronal death (see Figure 6). n conclude that NMDA-induced hyperphosphorylation of APP is not mediated by JNK. We then investigated the role of Cdk5 in our model. To prevent Cdk5 action we used the ATP competitive inhibitor Roscovitine, a well-characterized inhibitor of cdc-2 like kinases and the most common inhibitor to block Cdk5 activity [30]. Figure 5. JNK and Cdk5 inhibition do not reverse NMDA induced APP hyperphosphorylation. (a) Neurons were pre-treated with D-JNKI1 and then exposed to NMDA (30’-45’). (b) Western blot analysis and quantification showed that co-treatment with D-JNKI1/NMDA did not reduce P-APP increment induced by NMDA alone. On the contrary, D-JNKI1 pre-treatment increased P-APP/APP level. (NM = NMDA, DJ = D-JNKI1) (c) Neurons were pre-treated with Roscovitine and then exposed to NMDA. (d) Roscovitine did not reduce P-APP following NMDA application. Roscovitine/NMDA co-treatment further increased P-APP/APP levels compared to controls. (Ros = Roscovitine) Loading control: Tubulin. Quantifications are from six independent experiments (±S.E.M.), * p < 0.05, ** p < 0.01. . . 50 Pharmaceuticals 2010, 3 Figure 6. Neurons were exposed to NMDA (30’-45’-1 h) and neuronal viability was assessed by LDH assay. Application of D-JNKI alone or in combination with NMDA (30’-45’) did not affect neuronal survival. NMDA for 1 h significantly increased neuronal death while treatment with D-JNKI protected neurons. Quantification is from five independent experiments (±SEM), (∗ p < 0.05). Figure 6. Neurons were exposed to NMDA (30’-45’-1 h) and neuronal viability was assessed by LDH assay. Application of D-JNKI alone or in combination with NMDA (30’-45’) did not affect neuronal survival. Pharmaceuticals 2010, 3 51 Pharmaceuticals 2010, 3 Figure 7. L803-mts action on APP phosphorylation. (a) Neurons were pre-treated with L803- mts and then exposed to NMDA (30’-45’). Western blot analysis shows that L803- mts/NMDA co-treatment inverts the increase of P-APP/APP ratio following NMDA administration. (b) Quantification revealed a significant reduction (65%) of P-APP in L803- mts/NMDA-45’ neurons compared to NMDA-45’(#=p < 0.05). Loading control: Tubulin. Quantifications are from six independent experiments (±S.E.M.), * p < 0.05, ** p < 0.01. 2.6. GSK-3β regulates APP amyloidogenic processing induced by NMDA stimulation K-3β regulates APP amyloidogenic processing induced by NMDA stimulatio 2.6. GSK-3β regulates APP amyloidogenic processing induced by NMDA stimulation 2.6. GSK-3β regulates APP amyloidogenic processing induced by NMDA stimulation To detect the effect of L803-mts during NMDA treatment on APP amyloidogenic processing we analysed the level of βAPPs (amyloidogenic pathway) and αAPPs fragments (non-amyloidogenic pathway) (Figure 3, a). As described above, NMDA treatment induced a significant increase of βAPPs and a decrease of αAPPs fragments. Following treatment of NMDA stimulated neurons with L803- mts, the βAPPs/APP ratio returned to almost control levels (Figure 3, b), while the αAPPs/APP ratio showed a clear tendency to increase (Figure 3, c). These results indicate that L803-mts modulates the APP amyloidogenic processing induced by NMDA. 2.5. GSK-3β regulates NMDA induced APP hyperphosphorylation To examine if GSK-3β plays a role in NMDA induced hyperphosphorylation we used L803-mts, a small cell permeable peptide, which competes for the substrate binding site of GSK-3β [31]. Application of L803-mts (10 μM) 30’ before NMDA stimulation led to a reduction of APP phosphorylation during NMDA stimulation at 45’ compared to NMDA alone (p = 0.0006). Notably at 45’, P-APP levels in L803-mts/NMDA treated neurons were comparable to those of non-treated control neurons (Figure 7, a-b) underlying the fundamental role of GSK-3β in this process. We conclude that GSK-3β is the kinase responsible for NMDA mediated hyperphosphorylation of APP. 2.7. Discussion Amyloid Precursor Protein metabolism has a fundamental role in AD pathogenesis. Very important in the processing of APP is the role of the phosphorylation at Thr668, in the C-terminal cytoplasmic domain of APP (AICD). Such phosphorylation facilitates β-secretase cleavage and can lead to an Pharmaceuticals 2010, 3 Pharmaceuticals 2010, 3 52 increase in Aβ production [3]. It is thus important to elucidate the signaling pathways involved in this phosphorylation and how do they relate to different stressful stimuli. We investigated the impact of an excitotoxic stimulus such as NMDA on the phosphorylation of APP and on the activation of the three kinases that are involved in this phosphorylation, namely: JNK [6,17], Cdk5 [5,7] and GSK-3β [4]. The neurotoxic effect of extracellular aggregates of Aβ peptide, derived from APP processing, is mediated by excitotoxic events such as dysregulation of Ca2+ homeostasis, oxidative stress and NMDA responses [32–35] and recent studies have demonstrated the close interaction between Aβ, Ca2+ homeostasis and oxidative stress through the NMDA receptors (NMDA-R) [36,37]. Concomitantly, the NMDA-R has important functions in synaptic transmission, synaptic plasticity and excitotoxicity [38] and deregulation of glutamatergic neurotransmission may contribute to the cognitive deficits present in AD. In fact, memantine, an NMDA receptor antagonist, is a drug used in the treatment of AD and can improve memory in AD patients [39,40]. In order to investigate the direct effect of NMDA on APP, we stimulated 12 DIV fully differentiated cortical neurons with NMDA 100 μM for 30’-45’, a combination of concentration and duration that is not toxic for our neuronal in vitro model. Stimulation of cortical neuronal cultures with NMDA for 30’-45’ induces APP hyperphosphorylation at Thr668. Similar data were described by Hoey et al., [48]. Additionally, increment in P-APP correlated with an enhanced APP processing as shown by APPs release in neuronal media. Moreover in these conditions we observed an increase of βAPPs and a decrease of αAPPs fragments in neuronal lysates, confirming the important role of APP phosphorylation at Thr668 for the APP amyloidogenic processing. At the same time, NMDA stimulation for 30’-45’ led to an increase in Cdk5 activity and, in agreement with others, induced an increase of GSK-3β [41] as well as JNK activation [14,15]. It is important to note that APP phosphorylation and activation of these kinases preceded neuronal degeneration, which only became evident after 1 h of NMDA application. 3.3. Cellular Lysis Total protein extracts were obtained by washing cells twice in ice-cold PBS and lysed (20’-4 °C) in 1% Triton x-100 lysis buffer supplemented with proteases (1 × CPIK, Roche, 10634200) and phosphatases (1 μM 4-NPP, Roche, 10030536) inhibitors [47]. 3.1. Cortical Neuronal Culture Primary neuronal cultures were obtained from the cerebral cortex of two days post-natal rats, incubated with 200U of papain (Sigma Aldrich) (30’-34 °C), then with trypsin inhibitor (Sigma Aldrich) (45’-34 °C) and subsequently mechanically dissociated. All experimental procedures on animals were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and all efforts were made to minimise animal suffering. Neurons were plated in 35 mm dishes (∼7 × 105 cells/dish) pre-coated with 25 μg/mL poly-D-lysine (Sigma Aldrich). Plating medium was B27/neurobasal supplemented with 0.5 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. The experiments were performed 12 days from plating date, at which time neurons are considered differentiated. Neurons were treated with NMDA (100 μM, Sigma Aldrich), for 30’-45’-1 h. The inhibitors L803-mts (10 μM, Calbiochem), roscovitine (10 μM, Calbiochem), and D-JNKI1 (4 μM, Xigen SA, Lausanne, Switzerland) were administered to neurons 30’ before NMDA treatment. L803-mts and Roscovitine were diluted in DMSO. Treatment of neurons with DMSO only did not affect the results (data not shown). 2.7. Discussion In the second part of this study we investigated the role of Cdk5, GSK-3β and JNK in APP phosphorylation using specific kinase inhibitors. Although several studies have investigated the involvement of these kinases in APP phosphorylation their exact roles in models of excitotoxicity are still very unclear. We could show that inhibition of GSK-3β with a substrate competitive inhibitor (L803-mts) abolished NMDA induced APP phosphorylation after 45’ stimulation. Moreover we observed a positive effect of L803-mts on the APP amyloidogenic processing induced by NMDA treatment. In this short time window, the GSK-3β substrate competitive inhibitor led to a decrease of βAPPs production and an increase of αAPPs levels, even if it did not completely restore to control levels. This could be explained by the short-term treatment that is sufficient to significantly reduce P-APP levels but not all the APP processing. On the other hand, neither inhibition of Cdk5 by roscovitine, nor inhibition of JNK by D-JNKI reduced NMDA-induced APP hyperphosphorylation. Notably, inhibition of Cdk5 and/or JNK in NMDA stimulated neurons led to a further increase of APP hyperphosphorylation without affecting neuronal survival. Further studies are needed to elucidate the mechanisms behind such regulation. However we think that this data are not so surprising since several studies have reported cross-talks 53 Pharmaceuticals 2010, 3 among JNK, GSK-3β and Cdk5 pathways [42–44]. Plattner et al. described a cross-talk between GSK3 and Cdk5, where Cdk5 over-activation leads to GSK3 inhibition [45]. Similarly, the regulation of GSK-3β activity by JNK was recently demonstrated by Hu et al. [46]. We could thus speculate that inhibition of either Cdk5 or JNK could indirectly influence GSK-3 activity and thus APP hyperphosphorylation. Unfortunately dissecting further these signaling pathways is difficult: a) due to the rapidity of their response and b) because a combinational treatment with these inhibitors was toxic in our model. 3.2. Cytotoxicity Assay Neuronal death was evaluated by a Lactate dehydrogenase assay (LDH), (Cytotox 96 kit, Promega, WI). LDH assays were performed in triplicates. 3.7. Statistical Analysis All experiments were repeated using at least three independent culture preparations. Quantitative data were statistically analyzed by paired T-test with two-tailed distribution. A p value of < 0.05 was considered significant. 3.5. Western Blot Analysis Protein concentrations were quantified using Bradford Assay (Bio-Rad Protein Assay 500-0006) and 20 μg of whole cell proteins were separated by 8–10% SDS polyacrylamide gel. PVDF membranes were blocked in Tris-buffered saline (5% no fat milk powder, 0.1% Tween20) (1 h, room temperature). Primary antibodies were diluted in the same buffer (incubation overnight, 4 °C) using: 1:2000 anti APP clone 22C11 (APPs) (Chemicon, MAB348), 1:500 anti P-APP (a generous gift from Prof. P. Davis, Albert Einstein College of Medicine of Yeshiva University, NY, USA), 1:250 anti βAPPs (IBL, 18957), 1:1,000 anti αAPPs clone 6E10 (Signet, 9300), 1:2,000 anti P-GSK3β (Ser9) (Cell Signaling Technology, #9336), 1:2000 anti GSK-3β (27C10) (Cell Signaling Technology, #9315), 1:1,000 anti P-JNK (Cell Signaling Technology, #4671) 1:1000 anti JNK (Cell Signaling Technology, #9252). All blots were normalized to α-tubulin (Santa Cruz Biotechnology, sc-8035) and at least three independent experiments were performed. Western blots were quantified by densitometry using Quantity One software (Biorad). 3.6. Cdk5 Kinase Assay Cells were washed in ice-cold PBS and lysed (10 h - 4 °C) in RIPA buffer with proteases (1x CPIK, Roche, 10634200) and phosphatases (1 μM 4-NPP, Roche, 10030536) inhibitors. Three hundred μg of whole cell protein were incubated with 3 μg of anti Cdk5 (C-8) (Santa Cruz Biotechnology, sc-173) (2 h - 4 °C) and precipitated with Protein A Sepharose CL-4B (GE-Healthcare, 17-0780-01) (1 h - 4 °C). Immunoprecipitated complexes were incubated with 6 μg/μL of Histone H1 and 1 μCi/μL of γ-32P-ATP in kinase buffer (10 mM Tris, 1mM DTT, 2 mM EGTA, 10 mM MgCl2, 20 mM ATP) (30’, room temperature). Quantification of kinase assays was done using Quantity One software (Biorad) and based on at least three independent experiments. 4. Conclusions Altogether, these findings suggest that in adult differentiated stressed neurons GSK-3β is the kinase responsible for APP phosphorylation in excitotoxic conditions and an appropriate regulation of GSK- 3β activity can be useful for modulation of APP processing. Pharmaceuticals 2010, 3 54 Pharmaceuticals 2010, 3 3.4. Media Proteins Precipitation Four hundred μL of medium were incubated with 100 μL of TCA 50% (overnight, 4 °C). 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https://openalex.org/W2101733539	http://www.epj-conferences.org/articles/epjconf/pdf/2014/03/epjconf_inpc2013_01001.pdf	English	null	Structure and spin of the nucleon	EPJ web of conferences	2,014	cc-by	5,196	DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 2014 , / 01001 (2014) 201 66 epjconf EPJ Web of Conferences 46601001 DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences, 2014 , / 01001 (2014) 201 66 epjconf EPJ Web of Conferences 46601001 Abstract. Parton distribution functions, describing longitudinal momentum, helicity and transver- sity distributions of quarks and gluons, have been recently generalized to account also for transverse degrees of freedom. Two new sets of more general distributions, Trans- verse Momentum Distributions and Generalized Parton Distributions, were introduced to describe transverse momentum and space distributions of partons. Great progress has been made since then in measurements of diﬀerent Single Spin Asym- metries (SSAs) in semi-inclusive and hard exclusive processes providing access to TMDs and GPDs, respectively. Facilities world-wide involved in studies of the 3D structure of nucleon include HERMES, COMPASS, BELLE, BaBar, Halls A, B, and C at JLab, and PHENIX and STAR at RHIC (BNL). TMD studies in the Drell-Yan process are also becoming an important part of the program of hadron scattering experiments. Studies of TMDs are also among the main driving forces of the JLab 12-GeV upgrade project, several of the forward upgrade proposals of STAR and PHENIX at RHIC, and future fa- cilities, such as the Electron Ion Collider (EIC), FAIR in Germany, and NICA in Russia. In this contribution we present an overview of the latest developments in studies of parton distributions and discuss newly released results, ongoing activities, as well as some future measurements. H. Avakian1,a 1Thomas Jefferson National Accelerator Facility, 12000 Jefferson Ave. Suite 5,Newport News, VA 23606 H. Avakian1,a 1Thomas Jefferson National Accelerator Facility, 12000 Jefferson Ave. Suite 5,Newport News, VA 23606 efferson National Accelerator Facility, 12000 Jefferson Ave. Suite 5,Newport News, VA 23606 ae-mail: avakian@jlab.org This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Introduction The U,L,T correspond to unpolarized, longitudinally polarized, and transversely polarized nucleons (rows) and quarks (columns) Table 1. Leading twist TMD distribution functions (left) and GPDs (right). The U,L,T correspond to unpolarized, longitudinally polarized, and transversely polarized nucleons (rows) and quarks (columns) One of the most prominent applications is the Ji’s sum rule [4], providing access to the elusive or- bital angular momentum. The integral is weighted by x and calculated at any longitudinal momentum transfer ξ and vanishing momentum transfer t. J = lim t→0 X q=u,d,s 1 2 Z 1 −1 dx x {Hq(x, ξ, t) + Eq(x, ξ, t)} Recently there was a great progress in the proper deﬁnition of quark and gluon contributions to the proton spin. The gauge invariant interpretation of ∆g as the gluon spin contribution has been identiﬁed. It has been shown, however, that to access the canonical orbital angular momentum one has to extract experimentally either the phase space Wigner distribution [6] or particular twist-3 distributions [5]. The diﬀerence between diﬀerent decompositions can be interpreted as the change in the quark orbital angular momentum due to ﬁnal state interactions as the quark leaves the target in a DIS experiment [7]. Wide kinematic coverage of large acceptance detectors allows studies of exclusive (GPDs) and semi-inclusive (TMDs) processes providing complementary information on the transverse structure of nucleon. One of the cleanest processes to access GPDs is Deeply Virtual Compton Scattering (DVCS), in which one quark of the nucleon absorbs a virtual photon producing a real photon with the nucleon left intact. DVCS is most suitable for studying GPDs at moderate energies and in the valence quark regime. At low beam energies, the cross section for DVCS is small and masked by the more copious production of photons from the Bethe-Heitler (BH) process. However, DVCS ob- servables are only sensitive to chiral-even GPDs describing unpolarized and longitudinally polarized quarks. Hard exclusive production of light mesons allows to ﬁlter diﬀerent GPDs by diﬀerent ﬁnal state hadrons [8, 9]. In addition they provide a unique possibility to access “transversity” or chiral- odd GPDs through measurements of diﬀerent azimuthal moments of the cross section. 1 Introduction In the handbag approach developed recently by Liuti and Goldstein [10] and Kroll and Goloskokov [11, 12] the he- licity amplitudes depend on hard partonic subprocess and the GPD and the beam spin asymmetries of exclusive pions combined with other spin and azimuthal asymmetries, which can provide a unique possibility to access the elusive transversity GPDs. 1 Introduction Since the late 60s many theoretical and experimental groups dedicated their time to study the quark- gluon structure of nucleons and nuclei in terms of collinear ( “integrated”) parton distributions (PDFs) of hadrons. One of the most surprising results is the unexpectedly small fraction of the proton’s spin that is due to the contribution from quarks and antiquarks directing attention toward orbital motion of partons. Transverse space distributions of partons, encoded in Generalized Parton Distributions (GPDs), and transverse momentum dependent distributions, encoded in Transverse Momentum Dis- tributions (TMDs), have been widely recognized as key objectives of the JLab 12-GeV upgrade [1] and the polarized pp program at RHIC [2] as well as a driving force behind the construction of the EIC [3]. The information on QCD-dynamics inside hadrons, as encoded in GPDs and TMDs, is much richer than what one can learn from collinear PDFs. These 3D PDFs provide the information on the orbital structure, which is not accessible through regular PDFs and are expected to play a crucial role in explaining the spin structure of the nucleon. TMD and GPD distributions (see Table 1) describe partons with certain polarizations in nucleons in independence of the polarization state. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20146601001 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20146601001 EPJ Web of Conferences EPJ Web of Conferences N/q U L T U f1 h⊥ 1 L g1L h⊥ 1L T f ⊥ 1T g1T h1 , h⊥ 1T U L T U H ET L e H eET T E eE HT, e HT Table 1. Leading twist TMD distribution functions (left) and GPDs (right). The U,L,T correspond to unpolarized, longitudinally polarized, and transversely polarized nucleons (rows) and quarks (columns) U L T U H ET L e H eET T E eE HT, e HT N/q U L T U f1 h⊥ 1 L g1L h⊥ 1L T f ⊥ 1T g1T h1 , h⊥ 1T Table 1. Leading twist TMD distribution functions (left) and GPDs (right). INPC 2013 Most of the measurements of the polarized light sea-quark distribution have been performed in polarized semi-inclusive DIS (SIDIS) by the SMC, HERMES, and COMPASS collaborations by iden- tifying hadrons in the ﬁnal state. The analysis of SIDIS data relies on quantitative understanding of the fragmentation of quarks and antiquarks into observable ﬁnal-state hadrons and the uncertainties in the polarized antiquark PDFs separated by ﬂavor so far remain relatively large. Signiﬁcant parity-violating single-spin asymmetries (AL) in longitudinally polarized pp collisions have been observed recently at RHIC. The coupling of the Ws to the weak charge provides direct access to quark ﬂavor allowing studies of the spin-ﬂavor structure of sea quarks inside the proton. The STAR preliminary results on AW± L taken during 2012 shown in Figure 1 (right) have been already included in the pQCD-ﬁt and improved signiﬁcantly the determination of the polarization of the light sea quarks (DSSV++). (GeV/c) T Jet p 0 10 20 30 LL A 0 0.02 0.04 (GeV/c) T p 0 π 0 5 10 15 , Run 2005-2009 0 π PHENIX Prelim. PHENIX shift uncertainty 0 π DSSV++ for STAR Prelim. jet, Run 2009 STAR shift uncertainty DSSV++ for jet PHENIX / STAR scale uncertainty 6.7% / 8.8% from pol. not shown (GeV/c) T Jet p 0 10 20 30 LL A 0 0.02 0.04 (GeV/c) T p 0 π 0 5 10 15 , Run 2005-2009 0 π PHENIX Prelim. PHENIX shift uncertainty 0 π DSSV++ for STAR Prelim. jet, Run 2009 STAR shift uncertainty DSSV++ for jet PHENIX / STAR scale uncertainty 6.7% / 8.8% from pol. not shown η lepton -2 -1 0 1 2 -1 -0.5 0 0.5 =1 pdf error 2 χ ∆ DSSV08 L0 with DSSV08 RHICBOS DSSV08 CHE NLO STAR Preliminary Run 2012 - W + W ν + ± e → ± W → +p p =510 GeV s < 50 GeV e T 25 < E L A Rel lumi syst 3.4% beam pol scale uncertainty not shown Remaining syst <10% of stat errors Figure 1. Preliminary 2009 data compared to the DSSV++ ﬁt (left) and the single spin asimmetry, AL, for W± as measured by STAR in 2012 (right). The η is the charged lepton pseudorapidity in the CMS frame (η = ln[tan(θl/2)], where θl is the polar angle). INPC 2013 η lepton -2 -1 0 1 2 -1 -0.5 0 0.5 =1 pdf error 2 χ ∆ DSSV08 L0 with DSSV08 RHICBOS DSSV08 CHE NLO STAR Preliminary Run 2012 - W + W ν + ± e → ± W → +p p =510 GeV s < 50 GeV e T 25 < E L A Rel lumi syst 3.4% beam pol scale uncertainty not shown Remaining syst <10% of stat errors L A L A Figure 1. Preliminary 2009 data compared to the DSSV++ ﬁt (left) and the single spin asimmetry, AL, for W± as measured by STAR in 2012 (right). The η is the charged lepton pseudorapidity in the CMS frame (η = ln[tan(θl/2)], where θl is the polar angle). 2 Collinear parton distributions Due to the dominance of gluon induced hard scattering processes at RHIC, longitudinally polarized proton-proton collisions studied by PHENIX and STAR are currently the best source of information on the gluon polarization, ∆g. Measurements of the double helicity asymmetry, ALL, of neutral pions and jets, at PHENIX and STAR respectively, indicate a sizable positive ∆g (see Figure 1) in the accessible range of x (0.05 < x < 0.2). The uncertainties for ∆g(x), however, remain signiﬁcant in the presently unmeasured small x region, preventing a reliable determination of the full integral. 01001-p.2 3 Spin-azimuthal asymmetries 0 0.02 0.04 0.06 0.08 0.2 0.3 0.4 (a) x A LU sinφ e p → e' π+ X 0 0.02 0.04 0.06 0.08 0.1 0.4 0.6 0.8 (b) z 0.2 0.4 0.6 -0.05 0 0.05 0.1 0.2 z A UL sinφ π0 π+ π- x P⊥ (GeV) 0.25 0.5 0.75 1 Figure 2. First measurements of SSAs in SIDIS with longitudinally polarized target [13] (left) and beam [18] (right). Two fundamental QCD mechanisms giving rise to single-spin asymmetries were identiﬁed. First the Collins mechanism [29, 30], where the asymmetry is generated in the fragmentation of trans- versely polarized quarks, and second the Sivers mechanism [31–33], that arises due to ﬁnal state interactions at the distribution function level. The ﬁrst interpretation of observed signiﬁcant SSA with longitudinally polarized target, AUL, at HERMES[13] (see Fig. 2) was based on the convolution of the higher twist distribution function hL, related to the production of transversely polarized quarks due to quark-gluon interactions and the leading-twist Collins fragmentation function [34, 35], H⊥ 1 , de- scribing the fragmentation of transversely polarized quarks to unpolarized hadrons (pions). The beam SSA, ﬁrst measured by CLAS collaboration [18] (see Fig. 2, right panel) have also been interpreted in terms of the Collins mechanism [36–39] and used for a ﬁrst determination of the twist-3 distribution function e(x) [37]. The magnitude of the extracted e(x) is also consistent with predictions using the chiral quark soliton model [40–42]. Collinear higher-twist distribution function e(x) later on has been interpreted in terms of average transverse forces acting on a transversely polarized quark at the instant after absorbing the virtual photon [43]. The analysis [37, 44–46] of sub-leading single-spin asymmetries observed at HERMES [16] and later on at JLab [18] led to the introduction of a complete set of twist-3 distribution functions [33, 47]. Observation of the SSA in pion SIDIS has opened a new avenue to study the spin-orbit correlations and quark-gluon interactions, triggering studies of SSAs worldwide. The ﬁrst experimental evidence of a non-zero Collins function was obtained in SIDIS by the HERMES [15] and COMPASS collaborations [48] (see Fig. 3), where the convolution of the Collins function and the parton transversity distribution function h1 was measured. Measurements performed at very diﬀerent beam energies are consistent and indicate a signiﬁcant asymmetry in the valence region with opposite signs for positive and negative pions. 3 Spin-azimuthal asymmetries In recent years, measurements of azimuthal moments of polarized hadronic cross sections in hard processes have emerged as a powerful tool to probe nucleon structure. Several experimental groups are currently trying to pin down various eﬀects related to the nucleon structure through studies of spin- azimuthal asymmetries in SIDIS (HERMES at DESY [13–16], COMPASS at CERN [17], Jeﬀerson Lab [18–20]) polarized proton-proton collisions (PHENIX, STAR and BRAHMS at RHIC) [21, 22], and electron-positron annihilation (Belle at KEK and BaBar at SLAC) [23]. Azimuthal distributions of ﬁnal state particles in SIDIS, in particular, are sensitive to the orbital motion of quarks and play an important role in the study of transverse momentum distributions (TMDs) of quarks in the nucleon. φ p y ( ) q Signiﬁcant azimuthal moments in leptoproduction (Acos φ UU ), which have been measured in SIDIS already by EMC collaboration [24], were reproduced by latest measurements at CERN, HERMES and JLab [19, 20, 25, 26]. The amplitudes of these azimuthal modulations depend on the kinematic variables relevant for the SIDIS process, namely the Bjorken scaling variable x, the 4-momentum of the virtual photon squared Q2, the energy fraction of the hadron z, and the transverse momentum of the hadron PT. The ﬁrst unambiguously measured single-spin phenomena in hard scattering, which triggered im- portant theoretical developments, were transverse asymmetries measured in pp collisions and siz- 01001-p.3 01001-p.3 EPJ Web of Conferences able longitudinal target (Asin φ UL ) and beam (Asin φ LU ) spin asymmetries observed at HERMES and JLab [13, 14, 16, 18, 27, 28] in SIDIS. Signiﬁcant left-right asymmetries observed in the inclusive measure- ment of pions produced in the collision of transversely polarized (anti)protons with an unpolarized hydrogen target measured by the E-704 collaboration at center-of-mass energies of about 20 GeV, were conﬁrmed at center-of-mass energies up to 200 GeV by the STAR and BRAHMS collaboration at RHIC [21, 22]. 0.2 0.4 0.6 -0.05 0 0.05 0.1 0.2 z A UL sinφ π0 π+ π- x P⊥ (GeV) 0.25 0.5 0.75 1 0 0.02 0.04 0.06 0.08 0.2 0.3 0.4 (a) x A LU sinφ e p → e' π+ X 0 0.02 0.04 0.06 0.08 0.1 0.4 0.6 0.8 (b) z Figure 2. First measurements of SSAs in SIDIS with longitudinally polarized target [13] (left) and beam [18] (right). 3 Spin-azimuthal asymmetries Recent measurements of multiplicities and double spin asymmetries as a function of the ﬁnal transverse momentum of pions in SIDIS at JLab [19, 49] suggest that transverse momentum distributions may depend on the polarization of quarks and 01001-p.4 01001-p.4 INPC 2013 possibly also on their ﬂavor. Kinematic dependencies of single- and double-spin asymmetries have been measured in a wide range in x and PT with CLAS using a longitudinally polarized proton target. Measurements of the PT-dependence of the double-spin asymmetry, performed for the ﬁrst time, indicate the possibility of diﬀerent average transverse momenta for quarks aligned or anti-aligned with the nucleon spin [49], consistent with latest lattice calculations of ratios of transverse momentum distributions of quarks anti-aligned (q−) and aligned (q+) with proton spin [50] (see Fig. 4). x −2 10 −1 10 p Coll A −0.1 0 0.1 z 0.5 1 −0.1 0 0.1 positive hadrons negative hadrons )c (GeV/ h T p 0.5 1 1.5 −0.1 0 0.1 Figure 3. Dedicated measurements of Collins asymmetry with transversely polarized targets by the HER- MES [15] (left plot) and COMPASS [48] (right plot) collaborations as a function of relevant kinematic variables. x −2 10 −1 10 p Coll A −0.1 0 0.1 z 0.5 1 −0.1 0 0.1 positive hadrons negative hadrons )c (GeV/ h T p 0.5 1 1.5 −0.1 0 0.1 Figure 3. Dedicated measurements of Collins asymmetry with transversely polarized targets by the HER- MES [15] (left plot) and COMPASS [48] (right plot) collaborations as a function of relevant kinematic variables. -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.5 1 π+ A1 0 0.5 1 π- PT (GeV) 0 0.5 1 π0 Figure 4. Transverse momentum dependence of ratio of densities of u quarks anti-aligned (q−) and aligned (q+) with the proton spin [50] (left panel). The solid curve and the statistical error band in blue have been obtained from the Gaussian, with gray band at the bottom showing the uncertainty due to the renormalization [50]. The dashed curve and the band were obtained using alternative Gaussian parametrizations. Two diﬀerent ways to use Gaussians for the parametrization of lattice data, when positively deﬁned q+ = f1 + g1 and q−= f1 −g1 were parametrized, as opposed to f1 and g1 in the ﬁrst case, measure possible model dependence. 3 Spin-azimuthal asymmetries Assuming the thrust axis deﬁnes the q¯q direction and by selecting pions in opposite hemispheres with respect to the thrust axis, the asymmetry as a function 01001-p.5 EPJ Web of Conferences of azimuthal angles φ1 and φ2 of produced hadrons gives access to ratios of polarized and unpolarized fragmentation functions. Figure 5. Kinematics of hadron production in e+e−collisions (left) and corresponding measurements of asym- metry related to the analyzing power of Collins fragmentation from BaBar and Bell experiments, and z1 and z2 are corresponding pion fractional energies. Figure 5. Kinematics of hadron production in e+e−collisions (left) and corresponding measurements of asym- metry related to the analyzing power of Collins fragmentation from BaBar and Bell experiments, and z1 and z2 are corresponding pion fractional energies. Figure 6. Expected statistical error of the Sivers asymmetry versus xF assuming two years of data taking (280 days) at COMPASS (left) and Fermilab (right) for a dimuon mass range of 4.2 < M < 8.5 GeV. The bands shows the theoretical predictions of the asymmetry from Anselmino et al. [52] with shaded areas showing the √ 20-sigma error band [53]. Figure 6. Expected statistical error of the Sivers asymmetry versus xF assuming two years of data taking (280 days) at COMPASS (left) and Fermilab (right) for a dimuon mass range of 4.2 < M < 8.5 GeV. The bands shows the theoretical predictions of the asymmetry from Anselmino et al. [52] with shaded areas showing the √ 20-sigma error band [53]. 3 Spin-azimuthal asymmetries The right panel shows CLAS measurement of the PT-dependence of the double spin asymmetry for pion SIDIS on a longitudinally polarized proton [49]. -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.5 1 π+ A1 0 0.5 1 π- PT (GeV) 0 0.5 1 π0 Figure 4. Transverse momentum dependence of ratio of densities of u quarks anti-aligned (q−) and aligned (q+) with the proton spin [50] (left panel). The solid curve and the statistical error band in blue have been obtained from the Gaussian, with gray band at the bottom showing the uncertainty due to the renormalization [50]. The dashed curve and the band were obtained using alternative Gaussian parametrizations. Two diﬀerent ways to use Gaussians for the parametrization of lattice data, when positively deﬁned q+ = f1 + g1 and q−= f1 −g1 were parametrized, as opposed to f1 and g1 in the ﬁrst case, measure possible model dependence. The right panel shows CLAS measurement of the PT-dependence of the double spin asymmetry for pion SIDIS on a longitudinally polarized proton [49]. Figure 4. Transverse momentum dependence of ratio of densities of u quarks anti-aligned (q−) and aligned (q+) with the proton spin [50] (left panel). The solid curve and the statistical error band in blue have been obtained from the Gaussian, with gray band at the bottom showing the uncertainty due to the renormalization [50]. The dashed curve and the band were obtained using alternative Gaussian parametrizations. Two diﬀerent ways to use Gaussians for the parametrization of lattice data, when positively deﬁned q+ = f1 + g1 and q−= f1 −g1 were parametrized, as opposed to f1 and g1 in the ﬁrst case, measure possible model dependence. The right panel shows CLAS measurement of the PT-dependence of the double spin asymmetry for pion SIDIS on a longitudinally polarized proton [49]. Transverse momentum dependence of ratio of densities of u quarks anti-aligned (q−) and aligned (q+ The direct information on the Collins function has been ﬁrst obtained from e+e−annihilation experiments via the study of the semi-inclusive processes e+e−→q¯q →ππX at Belle [23] and later on conﬁrmed by the BaBar experiment at SLAC [51]. Collins asymmetry can be extracted using the thrust reference frame (Fig. 5). 4 Future measurements of DY and SIDIS One of the most remarkable features of the Sivers distribution function, responsible for the signiﬁcant SSA with transversely polarized targets in SIDIS (see Fig. 3), where it appears in convolution with the unpolarized fragmentation function D1, is the sign change from SIDIS to DY. An experimental proof of the sign-reversal property of the Sivers function is a crucial test of QCD in the non-perturbative regime. Several experiments are currently planning to measure polarized Drell-Yan either with a polar- ized beam or a polarized target including COMPASS at CERN [54], Fermilab E1207 [53], Panda at FAIR [55], NICA at JINR [56], and RHIC. The main goal of COMPASS is to measure the interaction 01001-p.6 01001-p.6 INPC 2013 between valence quarks and valence antiquarks focusing on the pion-proton (deuteron) collisions, col- lecting a suﬃcient amount of data in the so-called safe dimuon invariant mass region from 4 to 9 GeV, where the theoretical formalism using TMDs is very well elaborated and the interpretation of the data is straightforward. The Fermilab E-906/SeaQuest spectrometer accommodates a large coverage in parton momentum fraction x, covering the valence quark region (0.35–0.85), and the sea quark region (0.1–0.45) for the beam and target polarizations, respectively (see Fig. 6). between valence quarks and valence antiquarks focusing on the pion-proton (deuteron) collisions, col- lecting a suﬃcient amount of data in the so-called safe dimuon invariant mass region from 4 to 9 GeV, where the theoretical formalism using TMDs is very well elaborated and the interpretation of the data is straightforward. The Fermilab E-906/SeaQuest spectrometer accommodates a large coverage in parton momentum fraction x, covering the valence quark region (0.35–0.85), and the sea quark region (0.1–0.45) for the beam and target polarizations, respectively (see Fig. 6). Several proposals have been already approved by the JLab PAC to study GPDs and TMDs at JLab12 and were awarded the highest physics rating. The full program involves measurements us- ing the CLAS12 and SOLID large acceptance detectors combined with precision measurements us- ing Hall-C and Hall-A spectrometers. Precision measurements using the upgraded CLAS detector (CLAS12) with polarized NH3 and ND3 targets will allow access to the kT-distributions of u and d quarks aligned and anti-aligned with the spin of the nucleon. Integrated over transverse momentum, the data will also be used to extract the kT-integrated standard PDFs. 4 Future measurements of DY and SIDIS A wider range in Q2 provided by the CLAS12 detector at JLab would also allow studies of the Q2-evolution, important for under- standing and controlling possible higher-twist contributions. By using QCD evolved TMDs one can explain the observed discrepancies between HERMES [57] and COMPASS [58, 59] data, and predic- tions have been made for the non-trivial behavior of the Sivers asymmetry as a function of Q2 [60]. 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https://openalex.org/W2804317271	http://agritrop.cirad.fr/587970/1/Chaintreuil_et_al_2018.pdf	English	null	Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics	BMC plant biology	2,018	cc-by	12,101	Abstract Background: Among semi-aquatic species of the legume genus Aeschynomene, some have the unique property of being root and stem-nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the production of Nod factors. These species provide an excellent biological system with which to explore the evolution of nodulation in legumes. Among them, Aeschynomene evenia has emerged as a model legume to undertake the genetic dissection of the so-called Nod-independent symbiosis. In addition to the genetic analysis of nodulation on a reference line, natural variation in a germplasm collection could also be surveyed to uncover genetic determinants of nodulation. To this aim, we investigated the patterns of genetic diversity in a collection of 226 Nod-independent Aeschynomene accessions. Results: A combination of phylogenetic analyses, comprising ITS and low-copy nuclear genes, along with cytogenetic experiments and artificial hybridizations revealed the richness of the Nod-independent Aeschynomene group with the identification of 13 diploid and 6 polyploid well-differentiated taxa. A set of 54 SSRs was used to further delineate taxon boundaries and to identify different genotypes. Patterns of microsatellite diversity also illuminated the genetic basis of the Aeschynomene taxa that were all found to be predominantly autogamous and with a predicted simple disomic inheritance, two attributes favorable for genetics. In addition, taxa displaying a pronounced genetic diversity, notably A. evenia, A. indica and A. sensitiva, were characterized by a clear geographically-based genetic structure and variations in root and stem nodulation Conclusion: A well-characterized germplasm collection now exists as a major genetic resource to thoroughly explore the natural variation of nodulation in response to different bradyrhizobial strains. Symbiotic polymorphisms are expected to be found notably in the induction of nodulation, in nitrogen fixation and also in stem nodulation. Subsequent genetic analysis and locus mapping will pave the way for the identification of the underlying genes through forward or reverse genetics. Such discoveries will significantly contribute to our understanding of the molecular mechanisms underpinning how some Aeschynomene species can be efficiently nodulated in a Nod-independent fashion. Keywords: Aeschynomene, Diversity, Genotype, Legume, Nodulation, Ploidy, Species, Symbiosis Naturally occurring variations in the nod- independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics Clémence Chaintreuil1,8, Xavier Perrier2,3, Guillaume Martin2,3, Joël Fardoux1,8, Gwilym P. Lewis4, Laurent Brottier1,8, Ronan Rivallan2,3, Mario Gomez-Pacheco5, Mickaël Bourges5, Léo Lamy1,8, Béatrice Thibaud2,3, Heriniaina Ramanankierana6, Herizo Randriambanona6, Hervé Vandrot7, Pierre Mournet2,3, Eric Giraud1,8 and Jean-François Arrighi1,8* * Correspondence: jean-francois.arrighi@ird.fr 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM, Campus International de Baillarguet, F-34398 Montpellier, France 8LSTM, Univ. Montpellier, CIRAD, INRA, IRD, Montpellier SupAgro, Montpellier, France Full list of author information is available at the end of the article Chaintreuil et al. BMC Plant Biology (2018) 18:54 https://doi.org/10.1186/s12870-018-1260-2 Chaintreuil et al. BMC Plant Biology (2018) 18:54 https://doi.org/10.1186/s12870-018-1260-2 Background To broaden our understanding of the molecular mechanisms underlying the nitrogen- fixing symbiosis, there has arisen a fast growing interest in uncovering the diversity of nodulation processes that are found in other legume species [5, 6]. p g Forward genetics are now expected to allow the identi- fication of the specific molecular determinants of the Nod-independent process in A. evenia. To optimize re- search effort, a reference line was inbred and success- fully used to generate an SSR-based genetic map of A. evenia [9]. This genetic map uncovered the genome structure and the distribution of symbiotic genes. It also provides a basis for a genome sequencing project and paves the way of the genetic dissection of nodulation in this reference line. This does not exclude exploring the naturally occurring variations in nodulation, as a com- plementary genetic approach, in order to increase our understanding of symbiotic gene functions and of the genetic control of symbiosis as successfully performed in other legumes such as Medicago, Lotus and soybean [22]. But exploiting genetic diversity requires prior knowledge of the extent and structure of the variations occurring in the species of interest. Although the genetic relationships among the Nod-independent Aeschynomene species have been analysed using molecular markers, only two studies have included a small set of accessions for the diploid A. evenia and the related polyploid A. indica [17, 18]. As a consequence, the variations within and among the species of the Nod-independent clade remain largely uncharacterized. g In this line, the mainly tropical legume genus Aeschynomene represents a group of prime interest as it contains several original symbiotic features. The genus Aeschynomene was ori- ginally known for the ability of different species to develop stem nodules in addition to the typical root nodules. Stem nodulation is uncommon in legumes, being shared with a very few hydrophytic species of the genera Sesbania, Neptunia and Discolobium, but it is widespread among the semi-aquatic Aeschynomene species [7–9]. In addition, some bradyrhizobia isolated from Aeschynomene stem nodules exhibit a photosyn- thetic activity that was shown to play a key role in stem nodules by directly furnishing energy to the bacterium that can be used for biological nitrogen fixation [10, 11]. Background taxonomic revision of American Aeschynomene was pub- lished in 1955 by Rudd [15], but it predated phylogenetic studies in plants and included no species native outside the Neotropics. First molecular studies of Aeschynomene pointed to new cryptic taxa differing by their ploidy levels [16, 17]. The knowledge gained from the study of the Nod- independent clade was also used to select Aeschynomene evenia as a new model legume for the purpose of decipher- ing the molecular mechanisms of the Nod-independent sym- biosis [16, 18]. Key attributes of this species include its small, diploid genome (2n = 20, 415 Mb/1C), its selfing nature and its prolific seed production. Several tools have been devel- oped including artificial hybridization and the Agrobacterium rhizogenes-mediated root transformation, rendering this spe- cies ideal for molecular genetic studies. First insights were obtained from RNAseq analysis and reverse genetics by re- vealing that some symbiotic determinants identified in Medicago and Lotus are recruited in the Nod-independent process but several key genes involved in bacterial recogni- tion, symbiotic infection and nodule functioning were found not to be expressed during root nodulation [9, 19–21]. g The legume family (Leguminosae) accounts for ~ 27% of the world’s primary crop production and is second only to cereals in economic and nutritional value. It includes many crops of agronomic importance for grain production, pas- ture and agroforestry. Many legumes are pioneers plants improving soil fertility and moderating harsh environments. Such economic and ecological success of the legume family is, in large part, due to the ability of the vast majority of its 20,000 species to develop symbiotic interactions with nitrogen-fixing bacteria collectively referred as rhizobia [1]. In this symbiosis, the rhizobia produce signal molecules, the Nod factors, whose specific recognition by the host plant is necessary to activate the formation of root nodules that correspond to symbiotic organs where the rhizobia are hosted. Inside the nodules, the rhizobia reduce atmospheric nitrogen (N2) into ammonium (NH4 +), a form of nitrogen that is usable by the plant for its development. Historically, two model legumes, Medicago truncatula and Lotus japoni- cus, have been used to genetically investigate this nodula- tion process. Such studies have resulted in the identification and elucidation of the role of many genes that are essential for the different steps of nodule development and its infec- tion by the symbiont [2–4]. * Correspondence: jean-francois.arrighi@ird.fr 1 Full list of author information is available at the end of the article © 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. 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. Page 2 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 2 of 15 Background Even more outstanding was the discovery that some of these photo- synthetic bradyrhizobia lack both the canonical nodABC genes required for the synthesis of the key Nod factors and a type III secretion system (T3SS) that is known in other rhizobia to activate or modulate nodulation [12–14]. This led to a new paradigm in nodulation studies in which an al- ternative symbiotic process between rhizobia and le- gumes efficiently triggers nodule formation in a Nod (factor)-independent fashion. To enable an efficient use of the natural variation in genetic studies of nodulation, we surveyed the genetic diversity occurring in a collection of 226 Nod-independent Aeschynomene accessions spanning the whole distributional range of this clade. As a first step, genetic relationships and differentiation between Nod-independent Aeschynomene taxa were established using a combination of molecular phylogenies, cytogenetics and hybridization experiments. Phylogenetic analysis of the genus Aeschynomene revealed that all the species endowed with a Nod-independent nodulation process cluster in a single clade where no species using a Nod-dependent symbiotic process are found [8]. A Page 3 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 This information then served as supportive data for the analysis of genotype data obtained for the germplasm col- lection using a set of 54 SSR markers. Patterns of microsat- ellite diversity illuminated the genetic basis of the Aeschynomene taxa and uncovered their genetic differenti- ation. The presence of an underlying genetic structure was then compared with geographical distribution data. with A. deamii, a third one comprising A. evenia and its sister species, and a fourth one containing A. sensitiva and related species (Fig. 1a). p g In this phylogenetic tree, a number of putative species displayed the same ITS sequence. In particular, two rare species, A. magna and A. pluriarticulata, tightly clus- tered with two widespread species, A. denticulata and A. evenia, respectively (Fig. 1a). Similarly, A. evenia and A. indica shared the same ITS signature but they were pre- viously shown to form a species complex containing three cytotypes (2×, 4× and 6×) [17]. To sort these, accessions of this species complex were genotyped with SSR markers that are indicators of their genome consti- tution [17]. This led to the additional discovery that the A. indica 6× accessions of African and Australian origin displayed different SSR profiles, justifying their distinc- tion in the phylogenic tree (Fig. 1a, data not shown). Species identification and relationships A collection of 233 accessions was developed to investi- gate the phylogenetic relationships and the genetic dif- ferentiation in the Nod-independent Aeschynomene clade (Additional file 1: Table S1). It included all the known species included in the clade and aimed to cover their distributional range [15, 23, 24]. The nuclear ribo- somal ITS region was used as a marker of the species identity and served to reconstruct a phylogeny of the whole group based on the Neighbor Joining (NJ) method (Additional files 2 and 3: Tables S2 and S3). To simplify the resulting tree, accessions showing less than 1% of di- vergence in their sequence were grouped in the same clade (Fig. 1a, Additional file 4: Doc. S1). The ITS tree was composed of four lineages: one grouping A. filosa, A. rostrata and A. tambacoundensis, a monospecific one a b Fig. 1 Phylogenetic and genetic relationships in the Nod-independent Aeschynomene clade. Phylogenetic reconstructions were obtained using the Neighbor Joining method. a ITS phylogeny. Accessions with ITS sequence divergence < 1% were clustered together. Numbers of accessions per taxon are indicated in brackets. b Phylogeny based on 5 concatenated low-copy nuclear gene fragments: CYP1, eiF1α, SUI1, SuSy and a gene homolog to Glyma.07G136800 and Glyma.18G187300. -A, -B and -C indicate the different copies found in polyploid species. The four gene pools are identified with a circled number and the A. evenia and A. sensitiva groups are framed in a box bordered with a dashed red line. Diploid taxa are in black and polyploid taxa in blue with ploidy level indicated. Numbers at nodes represent bootstrap values (% of 1000 replicates) b a b b Fig. 1 Phylogenetic and genetic relationships in the Nod-independent Aeschynomene clade. Phylogenetic reconstructions were obtained using the Neighbor Joining method. a ITS phylogeny. Accessions with ITS sequence divergence < 1% were clustered together. Numbers of accessions per taxon are indicated in brackets. b Phylogeny based on 5 concatenated low-copy nuclear gene fragments: CYP1, eiF1α, SUI1, SuSy and a gene homolog to Glyma.07G136800 and Glyma.18G187300. -A, -B and -C indicate the different copies found in polyploid species. The four gene pools are identified with a circled number and the A. evenia and A. sensitiva groups are framed in a box bordered with a dashed red line. Diploid taxa are in black and polyploid taxa in blue with ploidy level indicated. Background It is also noteworthy that two accessions did not fit well with the description of any known Aeschynomene species and so we refer to these as Aeschynomene sp. (328) and A. sp. (353) (Fig. 1a, Additional file 1: Table S1). To clarify the genetic status of these putative new taxa, they were both in- cluded in a flow cytometry analysis and a chromosome count (Additional file 1: Table S1, Additional file 5: Figure S1). Cytogenetic data were mapped onto the phylogeny, showing Genetic patterns and behaviours g To further uncover genetic relationships between taxa, five low copy nuclear genes -CYP1 (Cyclophilin 1), eiF1α (eukaryotic translation initiation factor α), SUI1 (transla- tion factor), SuSy (Sucrose Synthase) and a gene homolog to Glyma.07G136800 and Glyma.18G187300 identified in Glycine max - were cloned and sequenced in selected ac- cessions (Additional file 2: Table S2). For diploid species, single sequences were obtained, while for polyploid spe- cies homeologous sequences were isolated for almost all genes (Additional file 3: Table S3). The five genes treated separately gave similar NJ trees where the homeologous sequences for polyploid taxa could be differentiated based on the differential clustering with the sequences of diploid taxa (not shown). To provide a unique and well-resolved NJ phylogeny, the gene sequences were concatenated to- gether (Fig. 1b). In the resulting tree, the topology of the branches containing the diploid species was similar to that of the ITS tree, corroborating the distinctness of A. sp. (328) from other known Aeschynomene species (Fig. 1a,b). For the polyploid taxa, the different genome components were scattered different part of the phylogeny, revealing that the two taxa A. sp. (353) and A. virginica, and the African and Australian A. indica 6× had the same or a similar genomic constitution (Fig. 1b). To assess the gen- etic differentiation of these related taxa, they were manu- ally crossed: A. sp. (353) with A. virginica, the African A. indica 6× with the Australian A. indica 6× and A. indica 4× with A. indica 6× for comparison (Fig. 2). In all cases, hybrid plants were obtained but they greatly differed in their fertility compared to their respective parental acces- sions, as seen by a drastic reduction of both the number of developed pods per flowering axis and of seeds per pod. To determine the genetic structure of the genomes of dif- ferent taxa, we conducted a SSR genotyping of the collec- tion of Aeschynomene accessions. For this, we tested the set of 500 SSRs previously used when developing a genetic map for A. evenia [9]. These markers were screened for polymorphism in four samples that are genetically differ- ent: two accessions of A. evenia 2× (the reference line CIAT 22838 and the mapping parent CIAT 8232), one ac- cession of A. indica 4× (PI 196206) and one of A. indica 6× (LSTM19). Species identification and relationships Numbers at nodes represent bootstrap values (% of 1000 replicates) Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 4 of 15 As a result, these data suggest that the tested taxa are truly different Aeschynomene species. that Aeschynomene sp. (328) represented a new 2× taxon while A. sp. (353) was a 4× taxon, similar to the closely related A. virginica, and confirming the hexaploid status of the Australian A. indica 6× (Fig. 1a). Genetic patterns and behaviours sensitiva group was compensated by the use of an additional set of SSR markers. Of the 64 SSR markers, 54 generated clearly interpretable allele profiles in at least one of the two groups, 38 SSR markers for the A. evenia group and 46 SSR markers for the A. sensitiva group, with 30 of them in common (Additional file 6: Figure S2). Allelic diversity, estimated by the number of alleles Na, varied im- portantly among the SSR markers used with an average of 12.8 alleles per locus in the A. evenia group (n = 186 acces- sions) and 4.21 in the A. sensitiva group (n = 40 accession) (Additional file 10: Table S6). Observed heterozygosities were very low for each SSR (Ho < 0.1 with one exception at 0.2), indicating that they amplified at single locus for diploid accessions and homeologous loci for polyploid accessions (Additional file 10: Table S6). the Aeschynomene taxa, one of the highest being found in A. evenia (mean 5.2 alleles per SSR) (Table 1, Additional file 10: Table S6). These values reflected the genetic diversity of the taxa, but they were likely to have been influenced by the marked variation in sample sizes and by the non-random selection of the SSR markers. The mean number of alleles detected for the SSR markers in each taxon were congruent with their ploidy levels, with (i) 1.00 to 1.06 alleles observed for diploid taxa, (ii) 1.42 to 1.92 alleles for tetraploid taxa and (iii) 2.63 to 2.84 alleles for hexaploid taxa (Table 1, Additional file 12: Table S8). Accessions displaying more al- leles than expected from their ploidy level were considered as heterozygous (Additional files 1 and 13: Tables S1, S9). Selfing rates based on the observed heterozygosities, ranged from 94.5% to 100%, with an average 98.7% score for A. evenia (Table 1, Additional file 13: Table S9). This provided genetic support for previous observations that the Nod- independent Aeschynomene species are preferentially autog- amous [17, 18]. These results supported the appropriateness of these SSRs for investigating the genetic properties of species. Therefore they were analysed in a second step to characterize the taxa. Transferability level of the SSR markers from A. evenia, ranged from 92 to 100% for other taxa of the A. evenia group and from 72 to 78% for those of the A. sensitiva group (Table 1, Additional file 11: Table S7). Genetic patterns and behaviours Of these, 64 markers were selected using two main criteria: (1) the requirement to be polymorphic between the two A. evenia accessions so as to avoid the use of invariant SSRs, (2) to amplify a single allele in the 2× accessions, 2 and 3 alleles in the 4× and 6× accessions, respectively, with the assumption that the distinct alleles came from the different genomic components of the polyploid species (Additional files 6, 7 and 8: Figure S2, Tables S4 and S5). Forty nine out of the 64 selected markers were previously positioned on different A. evenia linkage groups, with their distribution representing a reasonable coverage of the A. evenia genetic map (Additional file 7: Table S4). Preliminary genotyping experiments revealed that the SSRs developed from A. evenia sequences had a transfer- ability rate qualified as (i) modest with A. deamii and A. tambacoundensis, (ii) good with A. sensitiva and A. pratensis and (iii) high with A. denticulata and A. scabra. These data were in accordance with the phylogenetic distance of the different gene pools relative to A. evenia (Fig. 1), but this prompted us to restrict our analysis to the A. evenia and A. sensitiva groups. The corresponding accessions were then subjected to high-throughput SSR Fig. 2 Hybridization experiments between related taxa. Manual crosses were performed between A. virginica and A. sp (353), A. indica 4× and A. indica 6× -Africa-, A. indica 6× -Africa- and A. indica 6× -Australia. Plant fertility was evaluated for the parental taxa and two independently obtained F1 hybrids based on the number of seeds produced per pod and the number of developed pods per flowering axis. Error bars represent s.d. (n = 30) Fig. 2 Hybridization experiments between related taxa. Manual crosses were performed between A. virginica and A. sp (353), A. indica 4× and A. indica 6× -Africa-, A. indica 6× -Africa- and A. indica 6× -Australia. Plant fertility was evaluated for the parental taxa and two independently obtained F1 hybrids based on the number of seeds produced per pod and the number of developed pods per flowering axis. Error bars represent s.d. (n = 30) Page 5 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 5 of 15 genotyping by capillary sequencing (Additional file 9: Figure S3). The absence of amplification for some of them in the A. Genetic patterns and behaviours Analysing the Na par- ameter revealed different levels of genetic variation among Geographical structure of the Aeschynomene species Geographical structure of the Aeschynomene species The Nod-independent Aeschynomene clade is mainly a trop- ical/subtropical group, but the species show distinct geo- graphical distributions [15, 23, 24]. Out of the 19 taxa considered here (Table 1), 13 are strictly American, while three taxa (A. evenia, A. indica 4× and A. sensitiva) have a wider distribution; two are African (A. indica 6× - Africa- and A. tambacoundensis) and one Australian (A. indica 6× - Australia-). This confirmed previous conclusions that Amer- ica is the centre of origin and diversification of this clade but that several outliers have subsequently evolved in other con- tinents [8]. Because some species showed a pronounced gen- etic differentiation and are part of polyploid species complexes, we investigated to what extent the corresponding cytotypes and genotypes are geographically structured. In the A. sensitiva-A. pratensis species complex, A. sensitiva has a transatlantic distribution. When mapping accessions globally, a clear geographical separation of the four A. sensitiva geno- types was observed. One occupies the Caribbean region, one found in Colombia, another one the central area of South America and, more noticeably a fourth one being present both in coastal East Brazil and in Africa, suggesting a recent dispersal event (Fig. 5a, Additional file 9: Figure S3, Additional file 1: Table S1). In contrast to A. sensitiva, the low genetic diversity observed in A. pratensis 4× revealed no consistent geographical pattern (Fig. 5A, Additional file 9: Figure S3, Additional file 1: Table S1). In a search for other relationships, a Factorial Ana- lysis (FA) was also carried out in DARwin v5 [25]. This approach is more informative regarding distances among different groups and it also allows comparison of accessions of different ploidy levels. We focused on A. evenia and A. sensitiva to get a more detailed view of the genetic relatedness between the 2× genotypes and the derived polyploid taxa. FA clearly distin- guished A. sensitiva and A. pratensis when using the factorial axes 1 and 2 (Fig. 4a). Factorial axes 2 and 3 separated the four A. sensitiva genotypes and but the central position of A. pratensis was interpreted has an absence of a preferential relationship with any of the A. sensitiva genotypes (Fig. 4b). Therefore, either the parental A. sensitiva genotype that contributed to the polyploid genome of A. pratensis was missing or A. In the pantropically distributed A. evenia-A. Genetic diversity and genotype delineation Based on the geographical distributions of the accessions making up the different Nod-independent Aeschynomene taxa, most of their genetic diversity and structure were ex- pected to be uncovered. Therefore, the genotyping data Table 1 Summary of the data obtained for the Aeschynomene taxa Taxa/ ploidy level n samples n genotypes Cross-species transferability N Table 1 Summary of the data obtained for the Aeschynomene taxa Table 1 Summary of the data obtained for the Aeschynomene taxa Taxa/ ploidy level n samples n genotypes Cross-species transferability NA n co-present alleles/SSR Ho Comment 2× taxa A. ciliata 5 100% 1.2 1.00 0.000 A. deamii 3 – – – – A. denticulata 24 3 92% 2.7 1.06 0.055 A. magna conspecific A. evenia ssp. evenia 44 7 – 5.2 1.01 0.013 A. pluriarticulata conspecific A. evenia ssp. serrulata 15 2 100% 2.3 1.02 0.019 A. filosa 2 – – – – A. rostrata 1 – – – – A. rudis 7 95% 1.9 1.00 0.004 A. scabra 8 100% 1.6 1.00 0.000 A. selloi 3 78% 1.1 1.00 0.000 A. sensitiva 27 4 78% 2.6 1.03 0.028 A. sp (328) 1 72% 1 1.00 0.000 new taxon A. tambacoundensis 1 – – – – 4× taxa A. indica 4× 41 6 100% 4.9 1.92 0.019 A. pratensis 9 78% 2.1 1.60 0.000 A. sp (353) 1 100% 1.6 1.66 0.000 new taxon A. virginica 1 100% 1.5 1.42 0.000 6× taxa A. indica 6× Africa 16 2 100% 4.8 2.84 0.005 A. indica 6× Australia 24 3 100% 6.9 2.63 0.002 new taxon Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 6 of 15 Page 6 of 15 obtained for the A. evenia and A. sensitiva groups were combined and used to estimate pair-wise distances be- tween all accessions and thereby generate a dissimilarity matrix in DARwin v5 [25]. From this, NJ trees were calcu- lated for the 2×, 4×, 6× ploidy levels separately. Indeed, al- leles of the genomic components of polyploid accessions cannot be analysed separately and so their inclusion in the analysis of the diploid accessions would result in their grouping with only one of the potential progenitors. Considering the 2× NJ tree, well-separated clades were evident and corresponded to the known species A. evenia, A. ciliata, A. denticulata, A. rudis, A. scabra, A. serrulata, A. selloi and A. sensitiva, as well as the newly identified A. sp. (328) (Fig. Genetic diversity and genotype delineation 3a,Additional file 9: Figure S3). Interestingly, A. pluriarticulata was found to be nested within the A. evenia accessions and the accession of A. magna tightly clustered with accessions of A. denticulata. This information together with the phylogenetic and genetic relationships makes their taxonomic distinctness uncertain. Conversely, the tree topology showed a clear separation of the two A. evenia subspecies, evenia and serrulata, and several 2× taxa (A. denticulata, A. evenia ssp. evenia, A. evenia ssp. serrulata and A. sensitiva) could be subdivided in different clusters de- lineating genotypes (Fig. 3a,Additional file 9: Figure S3). Among these, genetic diversity and differentiation was the highest for A. evenia ssp. evenia (here after A. evenia s.s. in the text), with 7 genotypes identified. Regarding the 4× tree, A. virginica and A. sp (353) were found to form sister line- ages and contrasted patterns of genetic differentiation were observed with a very low genetic variability noted for A. pratensis whereas A. indica 4× was composed of several well-diverged clusters (Fig. 3b,Additional file 9: Figure S3). In the 6× tree, the African and Australian A. indica 6× were genetically distant with the African set subdivided in two homogenous genotypes while the Australian set contained three genotypes that formed far more diverse assemblages, indicative of distinct evolutions (Fig. 3c, Additional file 9: Figure S3). pratensis was formed before the intraspecific differen- tiation of A. sensitiva as suggested by the sequence divergence of the nuclear genes (Fig. 1b). For A. evenia s.s., the FA separated the 7 identified geno- types but grouped the three A. indica taxa together when using factorial axes 1 and 2 (Fig. 4c). These polyploid taxa could be separated along factorial axis 4 and showed preferential affinity with the two un- separated genotypes (1) and (2) of A. evenia along factorial axis 2 (Fig. 4d). These observations suggested a common origin of the three A. indica taxa that would derive from the same A. evenia genome donor, this latter being potentially ancestral to the genotypes 1 and 2. obtained for the A. evenia and A. sensitiva groups were combined and used to estimate pair-wise distances be- tween all accessions and thereby generate a dissimilarity matrix in DARwin v5 [25]. From this, NJ trees were calcu- lated for the 2×, 4×, 6× ploidy levels separately. Genetic diversity and genotype delineation Indeed, al- leles of the genomic components of polyploid accessions cannot be analysed separately and so their inclusion in the analysis of the diploid accessions would result in their grouping with only one of the potential progenitors. Considering the 2× NJ tree, well-separated clades were evident and corresponded to the known species A. evenia, A. ciliata, A. denticulata, A. rudis, A. scabra, A. serrulata, A. selloi and A. sensitiva, as well as the newly identified A. sp. (328) (Fig. 3a,Additional file 9: Figure S3). Interestingly, A. pluriarticulata was found to be nested within the A. evenia accessions and the accession of A. magna tightly clustered with accessions of A. denticulata. This information together with the phylogenetic and genetic relationships makes their taxonomic distinctness uncertain. Conversely, the tree topology showed a clear separation of the two A. evenia subspecies, evenia and serrulata, and several 2× taxa (A. denticulata, A. evenia ssp. evenia, A. evenia ssp. serrulata and A. sensitiva) could be subdivided in different clusters de- lineating genotypes (Fig. 3a,Additional file 9: Figure S3). Among these, genetic diversity and differentiation was the highest for A. evenia ssp. evenia (here after A. evenia s.s. in the text), with 7 genotypes identified. Regarding the 4× tree, A. virginica and A. sp (353) were found to form sister line- ages and contrasted patterns of genetic differentiation were observed with a very low genetic variability noted for A. pratensis whereas A. indica 4× was composed of several well-diverged clusters (Fig. 3b,Additional file 9: Figure S3). In the 6× tree, the African and Australian A. indica 6× were genetically distant with the African set subdivided in two homogenous genotypes while the Australian set contained three genotypes that formed far more diverse assemblages, indicative of distinct evolutions (Fig. 3c, Additional file 9: Figure S3). Geographical structure of the Aeschynomene species indica species complex, a prominent geographical division be- tween the different taxa was observed. A. evenia s.s. (2×) grows both in America and Africa, A. indica 4× is wide- spread in Asia, including India, and it is also present in North Eastern America, while the two A. indica 6× taxa distinctly occur in Africa and Australia (Fig. 5b). At the intraspecific level, the identified genotypes were found to represent geographically defined groups. A. evenia displayed a high level of genetic structure with 5 Ameri- can and 2 African genotypes. As far as phylogenetic rela- tionships can be inferred from an SSR-based NJ tree, the Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 7 of 15 a b c Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk a b a a c c c Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk African genotypes appeared to have diverged after a re- cent transatlantic migration from the Neotropics (Fig. 5b, Additional file 9: Figure S3, Additional file 1: Table S1). A. Geographical structure of the Aeschynomene species Two pairss of factorial axes (with the percentage of variation they account for indicated in parenthesis) are used for each species complex so as to show genetic distinctness and relationships. Taxon colours and genotype numbers are the same as in Fig. 3 standpoint. For the polyploid taxa (4 tetraploids and 2 hexaploids), low-copy nuclear genes and SSR analysis supported an allopolyploid origin and several genome donors could be identified [17; this study]. This must fa- cilitate the identification and distinction of the different subgenomic components of the polyploid genomes and this indicated that these polyploid taxa must behave gen- etically as diploids. Segregation of molecular markers, however, has not been performed to date at a genome- wide scale and we therefore cannot exclude that they might behave as segmental allopolyploids just as recently reported for peanut and chrysanthemum [27, 28]. Such dual structure may be found notably in A. pratensis for which the two expected homeologous versions of the tested low-copy nuclear genes were not always detected. reactions characterized by the accumulation of brown compounds, most probably of polyphenol nature as already described for incompatible interactions [26], were obvious in nodules of A. indica 6× -Africa- inoculated with BTAi1 (Fig. 6b, Additional file 14: Figure S4a). This incompatibility was accompanied by a low nitrogen-fixing activity and an overall reduction in plant development (Fig. 6c, Additional file 14: Figure S4b). Geographical structure of the Aeschynomene species indica 4× contained 3 distinct Indian lineages, one genotype spanning Eastern Asia and the North Eastern America, a second widespread in Northern Australia but also occurring in Asia, and a third throughout South Asia and extending to the Pacific (Fig. 5b). The Australian A. indica 6× was found to have a wide distribution extend- ing from Eastern to Western Australia. Missing collec- tion data limited the analysis of the three 6× genotypes but they did not overlap in distributional area with A. indica 4× (Fig. 5b, Additional file 9: Figure S3, Additional file 1: Table S1). In contrast, distribution of the African A. indica 6× was restricted to the Subsahelian zone and the two identified genotypes clearly could be distin- guished based on their location, one in the western part of the zone, the other one with a more central position (Fig. 5b, Additional file 9: Figure S3, Additional file 1: Table S1). Noteworthy, at a macroscale, there exists an overlap in distribution of African A. indica 6× with A. evenia s.s, but the latter was more widespread in Africa. To test whether this genetic diversity could support poly- morphism in nodulation traits, a number of accessions of A. evenia s.s. and A. indica were submitted to root or stem in- oculation with two photosynthetic nodABC gene-lacking Bradyrhizobium strains, ORS278 and BTAi1. A high vari- ation in stem nodule development was observed in A. evenia when it was inoculated with ORS278 (Fig. 6a). Conversely, root nodulation was more homogenous but plant defense Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 8 of 15 b d a b c d Fig. 4 Factorial analysis of Nod-independent Aeschynomene taxa. (a) and (b) for the A. evenia-A. indica species complex, (c) and (d) for the A. sensitiva-A. pratensis species complex. Two pairss of factorial axes (with the percentage of variation they account for indicated in parenthesis) are used for each species complex so as to show genetic distinctness and relationships. Taxon colours and genotype numbers are the same as in Fig. 3 a a b a c d Fig. 4 Factorial analysis of Nod-independent Aeschynomene taxa. (a) and (b) for the A. evenia-A. indica species complex, (c) and (d) for the A. sensitiva-A. pratensis species complex. Discussion This in-depth characterization of a Nod-independent Aeschynomene germplasm collection identified four main lineages and uncovered genetic diversity and struc- ture at different scales: cytotypes, species and genotypes (as summarised in Table 1). Interestingly, this group of Aeschynomene is composed of mainly diploid taxa (13 out of 19), including the model legume A. evenia, which are expected to be the easiest to handle at a genetic In addition to shedding light on the genetic basis of the different Aeschynomene species, our analyses were used to identify different taxa and to delimit their Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 9 of 15 b a Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. 3 a a b Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. 3 b g. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. dica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 4 Table S1 and Fig re S4 Ta on colo rs and genot pe n mbers are the same as in Fig 3 b Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and 14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. Discussion 6 Variation of nodulation traits observed in accessions of A. evenia and A. indica. (a)Stem nodulation observed in various accessions of A. evenia, 3 weeks post inoculation with ORS278. (b) Root nodule development in accessions of A. evenia and A. indica following inoculation with Bradyrhizobium ORS278 and BTAi1. 14-dpi nodules were cut to observe the leghemoglobin color and reaction defense (arrow). (c) Comparison of plant growth (aerial part) after inoculation with Bradyrhizobium ORS278 and BTAi1, at 14 dpi. Scale bar in (a): 5 mm, in (b): 1 mm All these data taken together make our germplasm collection a valuable genetic resource for the Nod- independent Aeschynomene group. So how best to fur- ther exploit it? To decipher the molecular mechanisms underlying the different original nodulation properties found in the Nod-independent symbiosis, A. evenia was recently selected as a model species [16, 18]. The species is currently being subjected to full genome sequence analysis and an ongoing mutagenesis project is predicted to identify new symbiotic genetic determinants in the near future. Such approaches applied to the historical model legumes M. truncatula and L. japonicus led to major advances in the study of the nitrogen-fixing sym- biosis with the identification of a set of symbiotic genes involved in the recognition of rhizobial signals, transduc- tion, infection and nodule organogenesis [2, 4]. But mutants are usually screened for the loss of their ability to establish a symbiosis, due to the disruption of gene function, and they are developed in the frame of the study of a single plant line-rhizobial strain system. Conversely, the screening of natural populations with multiple rhizobial strains can reveal some symbiotic phe- notypes that depend on both the host genetic back- ground and the rhizobial strain. In fact, studying natural variation approach has been shown to be a powerful tool for gaining insights into the genetic basis underlying the specificity of the symbiotic interaction in three legumes, be reliably considered as a new species belonging to the A. sensitiva group, notwithstanding that this conclusion is based on a single accession of what is apparently a rare taxon. Given their high variability, the SSR markers are a power- ful tool to highlight putative subdivisions in different Aeschynomene species that served to define genotypes. Strik- ing is that these genotypes, delineated solely on the basis of marker data, corresponded to geographically based sub- groups. Discussion 3 were found to form sister clades in the ITS and low-copy nu- clear gene phylogenies (in accordance with their current grouping into a single species) but are clearly distinct entities in the SSR tree (Figs. 1,3). Concordant with this strong genetic differentiation, the two subspecies of A. evenia previ- ously were demonstrated not to be cross-compatible [18]. Similarly, A. virginica and A. sp. 353, along with the African and Australian 6× A. indica taxa, formed separate groups in the SSR tree and their hybridization generated F1 hybrids with a marked reduction in fertility (Figs. 2,3). This raises the question of whether these taxa should be treated at the subspecies level or as separate species. On the other hand, the situation was clear for Aeschynomene sp. (328); this taxon was consistently found to be divergent in the ITS, low-copy nuclear gene and the SSR trees (Figs. 1,3). Therefore, it can taxonomic boundaries. Indeed, the approach of genotyp- ing a germplasm collection already has shown to be a powerful tool resolving taxonomic issues and providing the basis of a good taxonomic classification in the leg- ume genus Lens that includes the cultivated lentil [29]. Here, the rare species Aeschynomene pluriarticulata and A. magna were found to have ITS and low-copy nuclear gene sequences very similar to those of A. evenia and A. denticulata, respectively (Fig. 1). The NJ SSR trees further revealed that these two pairs of taxa clustered closely together. This strongly suggested that A. pluriarticulata and A. magna are more likely morphological variants of A. evenia and A. denticulata than distinct species (Fig. 3). But for A. magna, living material is now necessary to confirm this. Conversely, A. evenia ssp. evenia and A. evenia ssp. serrulata Page 10 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 a b c Fig. 6 Variation of nodulation traits observed in accessions of A. evenia and A. indica. (a)Stem nodulation observed in various accessions of A. evenia, 3 weeks post inoculation with ORS278. (b) Root nodule development in accessions of A. evenia and A. indica following inoculation with Bradyrhizobium ORS278 and BTAi1. 14-dpi nodules were cut to observe the leghemoglobin color and reaction defense (arrow). (c) Comparison of plant growth (aerial part) after inoculation with Bradyrhizobium ORS278 and BTAi1, at 14 dpi. Scale bar in (a): 5 mm, in (b): 1 mm b c Fig. Discussion Thus, the genetic structure of the Aeschynomene taxa appeared to mirror the eco-geographic distribution of the associated genotypes, a situation also described for other plants including lentil, tomato, pigeonpea and switchgrass [30–33]. Although not a major aim of this study, such genetic structure can help to understand the origin and the migration of some populations. This is notably the case for A. indica whose natural distribution range is obscure. The identified genetic clusters, which most prominently corre- sponded with geographical distribution patterns, likely reflected real differences within each species. Among the species studied, A. evenia s.s. had the highest genetic diver- sity with 7 genotypes, some of them previously being shown to be fully cross-compatible [18]. It is also noteworthy that the different Aeschynomene taxa displayed very high selfing rates. Using lines that tend to be mostly homozygous facili- tates artificial hybridizations and analysis of confidently seg- regation patterns in the progeny in order to investigate the genetic determinism underlying the nodulation traits. Page 11 of 15 Page 11 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 genetic analysis of this differential susceptibility. Variations in symbiotic traits with naturally-nodulating photosyn- thetic Bradyrhizobium strains have not been thoroughly surveyed yet, but the observations made in the present study using the strains ORS278 and BTAi1 yield promis- ing preliminary results. By evaluating different genotype- rhizobial combinations, we predict that symbiotic poly- morphisms will be found, notably in the induction of nodulation, nitrogen fixation and also in stem nodulation. Crossable accessions exhibiting polymorphic symbiotic phenotypes can then be selected for hybridization experi- ments, subsequent genetic analysis and locus mapping. This will pave the way for the identification of the genes underpinning these symbiotic responses through forward or reverse genetics. To assist in these studies, genomic and genetic data, together with a number of molecular tools, are being accumulated for the model species A. evenia and it thus represents the easiest system to work with [16, 20]. Despite the high genetic diversity observed in A. evenia, the number of available germplasm samples remains relatively modest since it has not been as extensively sampled as other legumes of interest [22]. Therefore, the accessions of other Nod-independent taxa in the same gene pool or in the related one containing A. sensitiva, represent a good complement. In addition, the expected high level of microsynteny with A. Discussion evenia will facilitate a synteny-based positional approach, as has been successfully performed between Pea and Medicago to iden- tify the gene underlying the SYM2 locus [34, 45]. As a re- sult, genetic resources developed for the Nod-independent Aeschynomene clade can be fully exploited for the search of natural variation in nodulation with Bradyrhizobium. Medicago, Lotus or soybean. The identified symbiotic polymorphisms were mostly of two types: some plant- rhizobial strain combinations resulted in non-nodulating phenotypes (Nod−), others in the production of small white infected nodules proved to be defective in nitrogen fixation (Fix−) [34–38]. This indicated that the control of host-rhizobial strain compatibility occurred at two differ- ent levels in the symbiotic process. y p It is noteworthy that the genetic analysis of natural vari- ation shed light on the function of some key symbiotic genes. The LysM-RLK receptors, which were identified using the mutant approach to be the probable Nod factor receptors, represent a well-known example. Indeed, a synteny-based positional cloning identified LYK3 in Medi- cago as corresponding to the SYM2 gene that controls the symbiotic infection in a Nod factor structure dependent manner in the pea ‘Afghanistan’ ecotype [34, 38]. The diver- sity information has also been exploited in Lotus to further substantiate that the LysM-RLK receptors mediate specific recognition of Nod factors [39]. More recently, our current understanding of the symbiotic mechanisms has been chal- lenged for the supposed role of NCR peptides that were ini- tially shown as important effectors of endosymbiont’s differentiation to nitrogen-fixing bacteroids. Making use of the differential ability of Sinorhizobium meliloti Rm41 to form functional or non-nitrogen fixing nodules depending on the Medicago accession used, two genes, NFS1 and NFS2, were identified and shown to code for NCR peptides [36–40]. This broadened the role of the NCR peptides in the fixation stage by revealing that some of them also con- trol discrimination against incompatible microsymbionts. A thorough survey of naturally occurring variation also pro- vided ground for the discovery of new genetic determinants of nodulation. This was notably the case in soybean where this approach led to the identification of two dominant genes that restrict nodulation in a strain-specific manner, Rj2/Rfg1 encoding a TIR-NBS-LRR resistance (R) gene and Rj4 that codes for a thaumatin-like protein [41, 42]. Discussion This revealed that some host genotypes are able to trigger gene- for-gene resistance, which is found in plant-pathogen interactions, to selectively interact with certain symbiotic strains but to exclude others. Genome size estimation and chromosome counting g Flow cytometry measurement was performed on leaf material to estimate genome sizes of various accessions as already explained [16]. These estimates were based on the measurements of three plants per accession using Lycopersicum esculentum (Solanaceae) cv “Roma” (2C = 1.99 pg) as the internal standard. For chromosome num- ber counts, metaphasic chromosomes were prepared from root-tips, spread on slides and stained with DAPI (4′,6-diamidino-2-phenylindole). Chromosomes were counted from images obtained with a fluorescent micro- scope as previously described [16]. Plant DNA extraction underlying genetic determinants. Discoveries of alterna- tive functions in symbiotic genes identified in other model legumes or of new genes involved in the recogni- tion of the still unknown non-Nod bacterial signal, nod- ule functioning and in the restriction of compatibility would add a new dimension to our understanding of the genetic control of nodulation in the Nod-independent symbiosis. Genomic DNA was extracted from fresh leaves using the CTAB (Cetyl Trimethyl Ammonium Bromide) method improved by the addition of β-mercaptoethanol 2% and PVPP 2%. For herbarium material, a protocol adapted for fragmented DNA was used, with increased length of the incubation (90 min), centrifugation (20 min) and precipitation (15 min) steps was used. DNA quantity was evaluated by spectrophotometer and DNA samples normalized to a uniform concentration of 10 ng/μL. Gene sequencing and sequence analysis All the accessions of Aeschynomene used in this study, their geographical origin and source data are listed in Additional file 1: Table S1. Seeds were scarified with sulphuric acid for germination and plants were grown in pots filled with compost under greenhouse conditions (temperature: 26-36 °C, relative humidity: 70%-80%, insect-proof screens) as detailed [16]. Interspecific crosses were performed according to the protocol developed earl- ier and the nature of the resulting hybrids was checked using SSR markers (data not shown) [16]. Fertility of the F1 plants was assessed by recording the number of successfully developed pods per flowering axis and the number of seeds in each pod. The nuclear ribosomal internal transcribed spacer region (ITS: ITS1-5.8S rDNA gene-ITS2) and five low copy nuclear genes CYP1, eiF1α, Sucrose Synthase, SUI1 and a gene homolog to Glyma.07G136800 and Glyma.18G187300 identified in Glycine max were amplified with the primers listed in Additional file 2: Table S2. PCR amplifica- tions, cloning and sequencing of PCR products were performed as already described [17, 18]. The DNA se- quences generated in this study were deposited in Genbank (Additional file 3: Table S3) and additional sequences are available in Additional file 4: Doc. S1. For the phylogenetic analyses, the gene sequences were aligned in ClustalX, version 1.81b and the align- ments were checked in Genedoc v2.7. Phylogenetic reconstructions were performed with the MEGA v7 program using the Neighbor Joining approach and the Tamura 3-parameter model with a 1000 x bootstrap. SSR marker selection and genotyping SSR marker selection and genotyping A total of 500 primer pairs were initially defined to de- velop SSR markers for genetic mapping in A. evenia (Additional file 7: Table S4) [9]. In the present study, they were again tested on two polymorphic accessions of A. evenia and two cytotypes of A. indica (Additional file 8: Table S5). Forward primers all contained a 5′-end M13 tail (5′-CACGACGTTGTAAAACGAC-3′), enabling the tagging the PCR products during the PCR amplification with 4 M13 primer-fluorescent dyes 6-FAM™, NED®, VIC®, or PET® (Applied Biosystems, CA, USA). Amplicon sizes were analysed using an ABI 3700 automatic capillary se- quencer (Applied Biosystems) as previously described [9]. 54 SSR markers were organized in 4-SSR multiplexes and used to genotype the accessions belonging to the A. evenia and the A. sensitiva groups as detailed in Additional file 6: Figure S2. Allele scorings were analysed using GeneMapper 4.0 software (Applied Biosystems) and exported as data ta- bles for two groups of genotyping. Conclusions With the goal of discovering natural variation in the Nod-independent Aeschynomene legumes, we developed a large collection of 226 accessions that spans the geo- graphical distribution of the different taxa in this group, including the model species A. evenia. These accessions were subjected to combined analyses of gene sequen- cing, cytogenetics, hybridization experiments and SSR genotyping. This work resulted in the delineation of taxon boundaries and in the discovery of new genotypes. Taxa displaying a significant genetic diversity were char- acterized by a clear geographically-based genetic struc- ture. In addition, low-copy nuclear genes and patterns of microsatellite diversity illuminated the genetic basis of the Aeschynomene diploid and polyploid taxa that are all predominantly autogamous and have a predicted simple disomic inheritance, two attributes favorable for genet- ics. Such a well-characterized collection of accessions constitutes a major genetic resource for exploring the natural variation of nodulation in response to different Bradyrhizobia strains and for searching for their These illuminating examples show that there is a mileage to be gained from exploring the natural variation in nodulation in the Nod-independent Aeschynomene group. To screen our germplasm collection, many Bradyr- hizobium strains are available, including photosynthetic and non-photosynthetic ones [14, 43, 44]. A recent report revealed important variations in the ability of different Nod-independent Aeschynomene species to be nodulated in a T3SS-dependent fashion by the non-photosynthetic Bradyrhizobium strains STM6978 and USDA61 [14]. Such marked variations were also observed between different accessions of A. evenia s.s., providing the basis for a Page 12 of 15 Page 12 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Plant nodulation and ARA Nodulation tests were carried out using Bradyrhizobium sp. strains ORS278 and BTAi1 [12]. The strains were cultivated for 7 days in yeast-mannitol liquid medium at 34 °C. Root inoculation was performed in an in vitro growth chamber on 7-day-old plants using 1 mL of bac- terial culture with an optical density at 600 nm adjusted to 1. Stem inoculation was carried out in a greenhouse by wrapping the stem of 5-week-old plants with a paper soaked with bacterial culture for 24 h. Stem nodules were observed at 21dpi and root nodules at 14dpi. Nitrogen-fixing activity was estimated on the entire plant by measurement of acetylene reducing activity (ARA) and microscopic observations were performed using a stereo-microscope (Nikon AZ100, Champigny- sur-Marne, France) as previously published [46]. Competing interests Additional file 10: Table S6. Allelic diversity of the SSR markers (XLSX 22 kb) Additional file 10: Table S6. Allelic diversity of the SSR markers (XLSX 22 kb) Funding g This work was supported by a grant from the French National Research Agency (ANR-AeschyNod-14-CE19-0005-01) that served for the design of the study, experimentation and analysis of the data. Availability of data and materials The gene sequences generated in this study have been deposited in GenBank (accession numbers listed in Additional file 3: Table S3) and additional sequences are available in Additional file 4: Doc. S1. All the SSR marker characteristics are included as supplementary information files (Additional files 7, 8, 10, 11, 12 and 13: Table S4-S9). SSR data analysis Genotyping data files were assembled in a single database that was used to determine genetic relationships among the accessions. For this, a distance-based approach was Page 13 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 13 of 15 applied by calculating with a shared allele index a genetic dissimilarity matrix in DARwin v5 software [25]. Then, individual relations were separately ana- lyzed for each ploidy level (2×, 4×, 6×) with a tree construction based on an unweighted Neighbor Join- ing method while genetic affinities between diploid and polyploid species were investigated by a factorial analysis as implemented in DARwin v5. The genetic diversity was evaluated by computing the number of alleles per locus (Na) and the observed heterozygos- ities Ho for each SSR locus and for different accession groups (Additional files 12 and 13: Tables S8, S9). USDA (USA). We are also very grateful to the many herbaria and botanists who provided additional relevant plant material that significantly contributed our sampling. The present work has benefited from the facilities and expertise of the cytometry facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr/spip.php?rubri- que184) and of the molecular cytogenetic facilities of the AGAP laboratory (https:// umr-agap.cirad.fr/en/platforms/plateformes/genotyping). Authors’ contributions CC was responsible for obtaining the plant material and the SSR genotyping data, and together with LB carried out the gene sequencing. RR and PM participated in the SSR marker development and genotyping. XP, GM, LL and BT performed computational analysis of the SSR genotyping data. MGP and MB carried out cytometry analyses. JF and EG handled the nodulation tests. HV, H. Randriambanona and H. Ramanankierna organized field collection of plant material and contributed to the acquisition and analysis of diversity data. GPL provided expertise in legume taxonomy and biogeography, and together with EG reviewed the manuscript. J.F.A. conceived the study, analyzed the data and wrote the paper. All authors read and approved the final manuscript. Additional file 2: Table S2. Nuclear genes used for the phylogenetic analyses. (XLSX 11 kb) Additional file 3: Table S3. GenBank numbers for the sequences used in the phylogenetic analyses. (XLSX 12 kb) Additional file 4: Doc. S1. ITS sequences obtained for the Nod- independent Aeschynomene accessions. (DOCX 30 kb) Additional file 5: Figure S1. Chromosome numbers in new Aeschynomene taxa. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 1008 kb) Additional file 6: Figure S2. Schematic representation of the different steps of the genotyping process from marker selection to data treatment. (PPTX 64 kb) Additional files Additional file 1: Table S1. Accessions used in this study, origin and characteristics. (XLSX 23 kb) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 11: Table S7. Cross-species transferability of the SSR markers (XLSX 13 kb) Additional file 11: Table S7. Cross-species transferability of the SSR markers (XLSX 13 kb) Ethics approval and consent to participate The sources of the Aeschynomene accessions used in this study are indicated in Additional file 1: Table S1. In addition to seedbank and herbaria supply, some accession were collected for purpose of this study in Madagascar (with authorization from the Malagasy Ministry of Scientific Research) and in New-Caledonia (licence n°60,912 from the Northern Province). Collection and identification of these new accessions were performed by Heriniaina Ramanankierana, Herizo Randriambanona, Hervé Vandrot and Jean-François Arrighi. Additional file 7: Table S4. Origin, location and primer sequences for the SSR markers used for genotyping. (XLSX 13 kb) Additional file 8: Table S5. Repeat motif and allelic amplification profiles of the SSR selected for genotyping. (XLSX 13 kb) Additional file 9: Figure S3. Detailed NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 65 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly colored. Identified genotypes are marked with a red dot and numbered. Accessions are designated with their LSTM code mentioned in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb) Additional file 9: Figure S3. Detailed NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 65 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly colored. Identified genotypes are marked with a red dot and numbered. Accessions are designated with their LSTM code mentioned in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb) Author details 1 Additional file 12: Table S8. Scoring of mean allele number per SSR and species (XLSX 15 kb) Additional file 12: Table S8. Scoring of mean allele number per SSR and species (XLSX 15 kb) 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM, Campus International de Baillarguet, F-34398 Montpellier, France. 2CIRAD, Amélioration Génétique et Adaptation des Plantes Méditerranéennes et Tropicales, UMR AGAP, Campus de Lavalette, F-34398 Montpellier, France. 3AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France. 4Comparative Plant and Fungal Biology Department, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK. 5Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud. Université Paris-Saclay, 91198 Gif-sur-Yvette, France. 6Laboratoire de Microbiologie de l’Environnement/Centre National de Recherche sur l’Environnement, 101 Antananarivo, Madagascar. 7IAC, Laboratoire de Botanique et d’Ecologie Végétale Appliquée, UMR AMAP, 98825 Pouembout, Nouvelle-Calédonie, France. 8LSTM, Univ. Montpellier, CIRAD, INRA, IRD, Montpellier SupAgro, Montpellier, France. 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM, Campus International de Baillarguet, F-34398 Montpellier, France. 2CIRAD, Amélioration Génétique et Adaptation des Plantes Méditerranéennes et Tropicales, UMR AGAP, Campus de Lavalette, F-34398 Montpellier, France. 3AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France. 4Comparative Plant and Fungal Biology Department, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK. 5Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud. Université Paris-Saclay, 91198 Gif-sur-Yvette, France. 6Laboratoire de Microbiologie de Additional file 13: Table S9. Observed heterozygosity (Ho) in Aeschynomene species (XLSX 14 kb) Additional file 13: Table S9. Observed heterozygosity (Ho) in Aeschynomene species (XLSX 14 kb) Additional file 14: Figure S4. Comparison of the nodulation properties of A. evenia s.s. and A. indica. Different accessions were root inoculated with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a) Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb) Additional file 14: Figure S4. Comparison of the nodulation properties of A. evenia s.s. and A. indica. Different accessions were root inoculated with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a) Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb) Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Acknowledgements f We are grateful to the different germplasm banks for provision of seeds: AusPGRIS (Australia), CIAT (Colombia), ILRI (Ethiopia), IRRI (Philippines), Kew Gardens (UK) and Page 14 of 15 Page 14 of 15 Page 14 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Received: 27 October 2017 Accepted: 6 March 2018 References Liu J, Yang S, Zheng Q, Zhu H. Identification of a dominant gene in Medicago truncatula that restricts nodulation by Sinorhizobium meliloti strain Rm41. BMC Plant Biol. 2014;14:167. 15. Rudd VE. The American species of Aeschynomene. Contributions of the United States National Herbarium. 1955;32:1–172. 37. Yang S, Wang Q, Fedorova E, Liu J, Qin Q, Zheng Q, et al. 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BMC Plant Biology (2018) 18:54 • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2996746440	https://europepmc.org/articles/pmc7227094?pdf=render	English	null	The accuracy of species-specific allometric equations for estimating aboveground biomass in tropical moist montane forests: case study of Albizia grandibracteata and Trichilia dregeana	Carbon balance and management	2,019	cc-by	9,481	© The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. 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 in a credit line to the data. Abstract Generally, the accumulation of a great deal of carbon stock in the aboveground biomass of tropical forests was verified [5, 6]. However, the biomass information is uncertain for The accuracy of species‑specific allometric equations for estimating aboveground biomass in tropical moist montane forests: case study of Albizia grandibracteata and Trichilia dregeana Damena Edae Daba* and Teshome Soromessa Backgroundh The tropical forest ecosystem has been playing a signifi- cant role in mitigating the atmospheric carbon dioxide concentration and associated climate change impacts. Correspondence: damenae2011@gmail.com Center for Environmental Science, College of Natural and Computational Sciences, Addis Ababa University, Addis Ababa, Ethiopia Correspondence: damenae2011@gmail.com Center for Environmental Science, College of Natural and Computational Sciences, Addis Ababa University, Addis Ababa, Ethiopia Correspondence: damenae2011@gmail.com Center for Environmental Science, College of Natural and Computational Sciences, Addis Ababa University, Addis Ababa, Ethiopia Abstract Background: Application of allometric equations for quantifying forests aboveground biomass is a crucial step related to efforts of climate change mitigation. Generalized allometric equations have been applied for estimating biomass and carbon storage of forests. However, adopting a generalized allometric equation to estimate the biomass of different forests generates uncertainty due to environmental variation. Therefore, formulating species-specific allometric equations is important to accurately quantify the biomass. Montane moist forest ecosystem comprises high forest type which is mainly found in the southwestern part of Ethiopia. Yayu Coffee Forest Biosphere Reserve is categorized into Afromontane Rainforest vegetation types in this ecosystem. This study was aimed to formulate species-specific allometric equations for Albizia grandibracteata Tuab. and Trichilia dregeana Sond. using the semi- destructive method. Results: Allometric equations in form of power models were developed for each tree species by evaluating the statistical relationships of total aboveground biomass (TAGB) and dendrometric variables. TAGB was regressed against diameter at breast height (D), total height (H), and wood density (ρ) individually and in a combination. The allometric equations were selected based on model performance statistics. Equations with the higher coefficient of determina- tion (adj.R2), lower residual standard error (RSE), and low Akaike information criterion (AIC) values were found best fit- ted. Relationships between TAGB and predictive variables were found statistically significant (p ≤ 0.001) for all selected equations. Higher bias was reported related to the application of pan-tropical or generalized allometric equations. Conclusions: Formulating species-specific allometric equations is found important for accurate tree biomass estima- tion and quantifying the carbon stock. The developed biomass regression models can be applied as a species-specific equation to the montane moist forest ecosystem of southwestern Ethiopia. Keywords: Afromontane rainforest, Model comparisons, Scatter plots, Semi-destructive, Species-spe Particularly, this ecosystem is known for its highest car- bon pool when compared to other biomes of the world [1–3]. It is the most productive ecosystem accounting for over 60% of global terrestrial photosynthesis and one-third of global primary productivity [4]. Generally, the accumulation of a great deal of carbon stock in the aboveground biomass of tropical forests was verified [5, 6]. However, the biomass information is uncertain for Particularly, this ecosystem is known for its highest car- bon pool when compared to other biomes of the world [1–3]. It is the most productive ecosystem accounting for over 60% of global terrestrial photosynthesis and one-third of global primary productivity [4]. Carbon Balance and Management Carbon Balance and Management Daba and Soromessa Carbon Balance Manage (2019) 14:18 https://doi.org/10.1186/s13021-019-0134-8 Daba and Soromessa Carbon Balance Manage (2019) 14:18 https://doi.org/10.1186/s13021-019-0134-8 Open Access Correspondence: damenae2011@gmail.com Center for Environmental Science, College of Natural and Computational Sciences, Addis Ababa University, Addis Ababa, Ethiopia © The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. 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 in a credit line to the data. Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 2 of 13 Daba and Soromessa Carbon Balance Manage 85% of the country. The forestry sector alone contributes a total emission of about 37% CO2 emission in the coun- try. The total contribution of other sectors like power, transport, industry, and buildings is less than 15% [30, 31]. many tropical forests due to the paucity of site-specific allometric equations [5, 7]. Therefore, applying a robust method for carbon stock estimation is a crucial step for the successful implementation of climate change mitiga- tion strategies like REDD+ [8]. To reduce the current rate of carbon dioxide emis- sion, the country has devised forestry-based strategies like REDD+ in potential forest areas. However, the esti- mation of biomass and carbon stock change depends on generic pan-tropical allometric equations, which gen- erate bias when applied to individual tree species [28]. Species-specific equations were not formulated for many tree species in Ethiopia. Therefore, this study is intended to formulate species-specific TAGB allometric equations considering selected tree species (A. grandibracteata and T. dregeana). The selected tree species have a wide range of ecological distribution across different parts of Africa [32]. A. grandibracteata species has multiple socio-eco- nomic benefits and ecological services. It is fast-growing species on forest soils with high moisture-holding capac- ity. A. grandibracteata is a medium-sized deciduous tree with a straight trunk to 20 m, and a flattened or layered crown [33]; it can grow up to 30 m [34]. T. dregeana is also a very large evergreen tree to 30 m, with a large straight trunk dividing into large branches and a rounded crown [32]. These attributes of the selected species are important for storing a high amount of biomass and car- bon. The semi-destructive method of data collection was found environmentally sound approach in the situation of the biosphere reserve. The core and buffer zones of the Yayu coffee-forest biosphere reserve were established mainly for scientific research, biodiversity conservation, and for monitoring the ecological processes. Allometric equations are important for their applica- tion to local and national forest carbon assessments, as well as for global carbon balance assessments [9]. Primar- ily, the current issue of global carbon cycles is the promi- nent factor for the formulation of biomass regression models [7, 10, 11]. As a result, generalized pantropical allometric equations were developed by many research- ers [5, 7, 12, 13]. The development of a generalized allo- metric equation was approached by measuring multiple tree species and it was intended to be applied to a broad range of tropical forests [12]. However, a great error is generated related to adopting generic pantropical allo- metric equations for many forests [14, 15]. Biomass error that can be generated at individual tree level is also regularly propagated bias at forest stand and country- level during the assessment of biomass and carbon stock change when the appropriate allometric equation is not used. Environmental variations among different forests are the ultimate factors for the variation of their biomass. Climatic regimes are the prominent factors that affect the growth of woody plants and biomass accumulation of different forest stands [16, 17]. Also, environmental variability in the context of physiographic and edaphic conditions plays a significant role in the variation of spe- cies composition and biomass difference among different forest sites [18, 19]. Within-stand variation of biomass for different tree species is related to tree architecture, growth strategies and its dynamic interplay with the bio- physical environments [20–22]. The difference of TAGB across a forest landscape is mostly related to the variation in slope, elevation, and aspect [18, 23]. Generally, tropical forests are known for their high diversity of woody plants. The application of multispecies pan-tropical equations to individual tree species generates uncertainty of TAGB [9, 24]. Therefore, formulating a species-and site-specific biomass regression model was found the best approach to accurately quantify biomass and carbon storage of for- ests [25–27].f Species description semi-destructive procedures for biomass, diameters of the trunk and large branches were measured at every 1 m length directly in the field. The fresh biomass of trimmed branches was also directly measured in the field. The vol- ume and dry weight of the wood aliquots were measured in the laboratory which was later used for wood density calculation. Three small branches for every tree sampled (3 × 60 = 180) were trimmed for the determination of trimmed biomass. The fresh biomass of small untrimmed branches was calculated based on the relationship between BD and dry biomass of trimmed branches. The fresh biomass of large untrimmed branches and trunk was calculated from volume and wood density measure- ment. The tree sections were considered to be cylinder; whereas, density was considered to be the same for all compartments of the tree. The assumption is that along every 1 m length of a tree there is no tapering (variation in diameter is insignificant), and every section is consid- ered to have cylinder shape [38]. Albizia grandibracteata species belongs to the plant fam- ily Fabaceae; whereas, T. dregeana species belongs to the plant family Meliaceae. Both tree species have a wide range of ecological distributions [32]. A. grandibracteata species mostly grow in the upland rainforest and river- ine forest areas, with preference in moist and wet sites. This tree species is known for its multiple provisional services [35]. It provides multiple socio-economic ben- efits (firewood, farm tools, medicine from the root, bee forage, ornamental, and soap from its bark); it is also known for nitrogen fixation which is one of its ecologi- cal services [32]. In Ethiopia, A. grandibracteata species grow in moist agroclimatic zones within an altitude range of 1200–1700 m asl. [33]. It is a medium-sized deciduous tree with a straight trunk to 20 m and flattened crown; it can also attain a maximum height of 30 m. In Ethiopia, T. dregeana Sond. species occurs in the moist and wet montane rainforest of southwestern and eastern highland between 1100 and 2200 m asl. altitude ranges [33]. This tree species is well known for provi- sional services like firewood, timber (construction, fur- niture), and coffee shade under natural forest. The T. dregeana Sond. is a large evergreen tree grows up to 35 m height [36]. Biomass calculations The procedures of semi-destructive methodology in “Manual for building tree volume and biomass allomet- ric equations” prepared by Food and Agriculture Organi- zation (FAO) [38] were followed. A random sampling technique where all individuals have an equal probabil- ity of being involved in the study was applied. Therefore, the selection of individuals from different diameters at breast height (DBH) classes was used. The random sam- pling technique was conducted with a quick screening of vegetation variability across the landscape or particular locality; to delimit the vegetation type in mind [20]. The guideline by [10] suggests the relevance of considering some biophysical factors of the forest stand to have rep- resentative sample sets. Detail information about sam- pling trees, field-based measurements, and laboratory analysis can be found in [28, 38, 39]. All procedures of laboratory analysis related to wood and leaves aliquots and calculation of trimmed and untrimmed biomass can be found in the FAO manual [38] was used. Comparison between species‑specific and pan‑tropical allometric equationshi The species-specific equation and pan-tropical allomet- ric equations were compared in this study for accuracy assessment. The pan-tropical allometric equations poten- tially applicable in tropical moist forests were used for the comparison. In Ethiopia, these equations have been most frequently used for biomass estimation in the mon- tane moist forest ecosystem. The datasets for biomass comparisons were generated form: (1) Measured bio- mass- generated based on semi-destructive procedures; (2) Specific Equation-equation which was developed for A. grandibracteata and T. dregeana; (3) Equations devel- oped by [5, 12, 13, 41] for tropical forests. Site descriptionh The study was conducted in Yayu Coffee-Forest Bio- sphere Reserve which is located in Illubabor Zone, southwestern Ethiopia. The biosphere extends between latitude 8° 15′ 0′′–8° 35′ 0′′ N and longitude 35° 30′ 0′′–36° 0′ 0′′ E of zone 36 (Fig. 1). Detailed informa- tion regarding the study area extent, soil types, climatic condition, altitudinal range, and establishment of the biosphere reserve can be found in [28]. Celtis africana, Diospyros abyssinica, Albizia grandibracteata, Ehretia cymosa, Trichilia dregeana, Vangueria apiculata. Argom- uellera macrophylla, Antiaris toxicaria, Millettia fer- ruginea, and Cordia africana are dominant tree species of the study forest. In addition, Albizia grandibracteata and Trichilia dregeana contributes significant amount of basal area (BA) M2 ha−1, which has great implication to storing high amount of biomass and carbon (Daba and Yayu Coffee-Forest Biosphere has comprised Afromon- tane Rainforest Vegetation types, which is home for the endemic Coffea arabica in Southwestern Ethiopia [28]. This forest is known for storing a good deal of carbon stock and high species diversity. According to [29], the forest is categorized into Eastern Afromontane Biodiver- sity Hotspots which has global significance. In Ethiopia, forestry and agricultural sectors are the major sources of carbon dioxide CO2 emission, contributing more than Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 3 of 13 Daba and Soromessa Carbon Balance Manage soromessa: Species composition, stand structure and regeneration status of tree species in Yayu coffee forest biosphere reserve, Illubabore zone, southwestern Ethio- pia, unpublished). Fig. 1 Map of Ethiopia with Oromia region, and the study area. The study area is marked green and the lines with blue color are rivers ap of Ethiopia with Oromia region, and the study area. The study area is marked green and the lines with blue color are rivers soromessa: Species composition, stand structure and regeneration status of tree species in Yayu coffee forest soromessa: Species composition, stand structure and regeneration status of tree species in Yayu coffee forest biosphere reserve, Illubabore zone, southwestern Ethio- pia, unpublished). Page 4 of 13 Daba and Soromessa Carbon Balance Manage (2019) 14:18 Daba and Soromessa Carbon Balance Manage Measuring trimmed and untrimmed fresh biomassh The diameter at the bases of each trimmed branch was measured and the leaves of each branch were fully har- vested from the wood. Only basal diameter was meas- ured for small untrimmed branches. A section about one-meter length was preferred for diameter measure- ment of the trunk and main branches [40]. Detail infor- mation regarding measurement of trimmed branches in the field, measurement of wood and leaf aliquots in the laboratory, measurement of small and large untrimmed branches, and trunk can be found in [28, 38]. Species description It has a straight trunk that can attain higher DBH size that divides into large branches and forms a rounded crown [33]. The selected tree species have a sig- nificant contribution in terms of stocking density and BA m2 ha−1; which indicates that these species have great potential in storing TAGB and carbon [37]. Scatter plots of TAGB against dendrometric variables for A. grandibracteata As depicted in (Fig. 2b, 3a, and 4a) that the dependent variable (TAGB) increases with a unit increase of inde- pendent variables (D, H, and D2H). This indicates the existence of a strong relationship between TAGB and the predictive variables. However, a significant relationship was not observed between TAGB and ρ (Fig. 4b). The relationship between wood density and tree dendromet- ric variables is quite complex for a tree species related to microsite variation and tree maturity as well as due to different site factors. Allometric equations and their performanceh The allometric equations relating the dependent variable TAGB against the predictor variables (D, H, and ρ) were formulated for A. grandibracteata and T. dregeana tree species. The descriptive summary of these main variables for TAGB regression models formulation was presented in (Table 1). Selected allometric equationsh The allometric equations were formulated by relating TAGB against independent variables individually and in combination. The selected allometric equations were tested for goodness of fit based on different performance statistics. The coefficients for all selected allometric equa- tions were found statistically significant (p ≤ 0.001); indi- cating the strong relationships between the TAGB and its predictor variables (Table 2). Tree biomass proceduresh The biomass calculation has also followed the proce- dure in “Manual for building tree volume and biomass allometric equations” prepared by FAO [38]. During Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 5 of 13 Daba and Soromessa Carbon Balance Manage (2019) 14:18 Daba and Soromessa Carbon Balance Manage Data analysis and model selection linear relationship between the paired variable (D & H, D & ρ). The correlation coefficient between D and H is 0.92 indicating a strong relationship between the variables for A. grandibracteata and also was found statistically signifi- cant at (p < 0.05). The correlation coefficient between D & H is 74.3, and statistically significant at (p < 0.05) for T. dregeana. However, the correlation coefficient between predictor variables (D & ρ) was found statistically not significant at (p > 0.05) for both tree species. Scatter plot depicted in (Fig. 2a, b) verifies the relationship between D & H of A. grandibracteata and T. dregeana species respectively; indicating an increment of tree height for a unit increment of its DBH. The DBH versus tree height plot was constructed for the area of interest; also, it has a purpose to compare and determine how appropriate the biomass regression model for a given site. Points regarding the summarized data and statistical package used during the data analysis was can be found in [28]. Eight allometric equations were developed by evaluating the relationships among the considered vari- ables i.e. TAGB against single predictor variables (D, H, ρ); TAGB against single compound variables (D2H, DH, ρDH); TAGB against multiple variables (D + H + ρ; D2H + ρ; D + H). These models were fitted based on log- transformed data and all have achieved model goodness of fit statistics. The relationship between TAGB and wood density was found statistically not significant (p > 0.05). Biomass regression models selections and evaluation were tested based on performance statistics including coefficient of determination (adj.R2), residual standard error (RSE), Akaike information criterion (AIC), and p-value. AIC is an estimator of the relative quality of sta- tistical models for a given set of data. AIC estimates the quality of each model relative to each other [42]. All for- mulated models used natural logarithm transformation; to minimize the systematic bias during the back transfor- mation a correction factor (CF) was calculated for each equation [43]. TAGB aboveground biomass (in kg), D diameter at breast height (in cm), H total height (in m), ρ wood density (in g cm−3) Scatter plots of TAGB against dendrometric variables for T. dregeanah Scatter plots of TAGB against dendrometric variables for T. dregeanah model shows strong relationships between the TAGB and the main predictive variables. The value of adj. R2 describes that 99.36% variation in TAGB was explained by the predictor variables in this model. The 2nd best- performing equation was (AgEq2) which was formu- lated by relating TAGB against D2H and wood density. It is the biomass regression model with a lower AIC value (− 50.96) based on statistical criteria for model selec- tion. A strong relationship was observed between TAGB and the predictive variables. The value of adj. R2 also describes that the predictor variables explain 98.30% var- iation of the TAGB. The scatter plots displayed in (Fig. 5a, b and 6a) shows the increase of TAGB against a unit variation of the pre- dictor variables (D, H, & D2H). The scatter plots of TAGB against D exhibits the linear relationship. The relation- ship between TAGB and H also shows an increment of TAGB for an increment of tree height. However, the rela- tionship between TAGB and ρ has shown a weak asso- ciation, due to the complex relationship of wood density with site factors and stand structure. The scatter plot of TAGB against D2H shows linear- ity; however, a significant relationship was not observed between TAGB and ρ as displayed in (Fig. 5). The 3rd best equation (AgEq3) was formulated by relating TAGB with D. The adj. R2 value for this bio- mass regression equation has shown (97.97%); indicat- ing that D is a single tree dendrometric variable that best explains variation in TAGB of A. grandibracteata species. Other selected equations (AgEq4, 5, 6, 7, and 8) have also achieved models performance statistics and also listed in decreasing order of importance based on AIC value (− 44.49, − 32.89, 12.59, 16.97, 29.77) respectively. There is a lower value of adj.R2 and a higher value of RSE for these models compared to the above models (1, 2, and 3). Generally, a strong relationship was found between TAGB and the dendrometric variables for all models. However, H has explained (adj. R2 = 81.9%) variation in TAGB of A. grandibracteata tree species. TAGB against predictive variable (H) has achieved a strong relationship which is statistically significant at (p ≤ 0.001). In contrast, a model relating TAGB with ρ was found statistically insignificant and then rejected. Selected biomass regression models for A. The relationship between the predictor variables of A. grandibracteata and T. dregeanahfi The correlation coefficient between the predictor vari- ables was calculated using Pearson’s correlation coeffi- cient at a 95% confidence interval. Pearson’s correlation coefficient is the statistical measure of the strength of a Table 1 Descriptive summary of dendrometric variables for A. grandibracteata and T. dregeana TAGB aboveground biomass (in kg), D diameter at breast height (in cm), H total height (in m), ρ wood density (in g cm−3) Tree species Variables Minimum Maximum Mean Standard deviation A. grandibracteata TAGB 6.26 2268 587.1 629.17 DBH 5.2 70.8 31.5 18.92 H 4 38 22.97 11.14 ρ 0.3559 0.5824 0.4709 0.0703 T. dregeana TAGB 2.87 5502 939.6 1587.66 D 5.2 105 36.37 28.94 H 3.5 38 25.15 11.76 ρ 0.2406 0.5799 0.4179 0.0745 Table 1 Descriptive summary of dendrometric variables for A. grandibracteata and T. dregeana Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 6 of 13 Fig. 2 Scatter plot of Diameter-Height relationships for: a A. grandibracteata, b T. dregeana tree species Fig. 2 Scatter plot of Diameter-Height relationships for: a A. grandibracteata, b T. dregeana tree species Scatter plots of TAGB against dendrometric variables for T. dregeanah grandibracteata The species-specific biomass regression models were developed for the selected tree species based on log- transformed data. All selected allometric equations have achieved the model goodness of fit. The best performing regression models of A. grandibracteata and T. dregeana were listed in decreasing order of importance based on AIC value (Table 2).h The formulated allometric equations were listed in decreasing order of importance from (AgEq1–AgEq8 and TdEq1–TdEq8) respectively for A. grandibracteata and T. dregeana to estimate TAGB (Table 2). The equations were ordered based on their AIC values, where the lower the AIC vale comparatively is the best equation and vice versa. Therefore, comparisons among the selected allo- metric equations of A. grandibracteata shows that AgEq1 was found best based on its AIC (− 79.49) value. This Daba and Soromessa Carbon Balance Manage (2019) 14:18 Daba and Soromessa Carbon Balance Manage Page 7 of 13 Trichilia dregeana biomass regression model (TdEq1) lower value of RSE is the parameter that has proved the Table 2 Best fitted regression models for predicting aboveground biomass of A. grandibracteata and T. dregeana Where TAGB: aboveground tree biomass (kg); D: diameter at breast height of tree (cm); H: total tree height (m); ρ: Wood Density (g cm−3); α: intercept; β1, β2, β3: are slopes; adj.R2: adjusted R square; RSE: Residual Standard Error; AIC: Akaike Information Criterion; CF: correction factor; AgEq: A. grandibracteata Equation; TdEq: T. dregeana equation Equation No. Scatter plots of TAGB against dendrometric variables for T. dregeanah Allometric equations Coefficients Model performance statistics Symbol Value Adj.R2 RSE AIC CF p-value AgEq1 TAGB = exp[α + β1ln(D) + β2ln(H) + β3ln(ρ)] α − 0.793 0.9936 0.1347 − 79.49 1.0091 ≤ 0.001 β1 2.117 β2 0.062 β3 0.991 AgEq2 TAGB = exp[α + β1 ln(D2H) + β2ln(ρ)] α − 0.810 0.983 0.2198 − 50.96 1.0245 ≤ 0.001 β1 0.749 β2 1.030 AgEq3 TAGB = exp[α + β1ln(D)] α − 1.744 0.9797 0.2408 − 46.41 1.0294 ≤ 0.001 β1 2.241 AgEq4 TAGB = exp[α + β1ln(D) + β2ln(H)] α − 1.755 0.979 0.2449 − 44.49 1.0304 ≤ 0.001 β1 2.199 β2 0.049 AgEq5 TAGB = exp[α + β1ln(D2H)] α − 1.834 0.9681 0.3016 − 32.89 1.0465 ≤ 0.001 β1 0.775 AgEq6 TAGB = exp[α + β1ln(H)] α − 1.363 0.8545 0.6437 12.59 1.2302 ≤ 0.001 β1 2.286 AgEq7 TAGB = exp[α + β1ln(ρDH)] α − 0.699 0.9682 0.3007 16.97 1.0462 ≤ 0.001 β1 1.129 Ag Eq8 TAGB = exp[α + β1ln(DH)] α − 1.803 0.9514 0.3722 29.77 1.0717 ≤ 0.001 β1 1.172 TdEq1 TAGB = exp[α + β1 ln(D) + β2 ln(H) + β3 ln(ρ)] α − 2.526 0.975 0.3204 22.55 1.0560 ≤ 0.001 β1 2.029 β2 0.593 β3 0.648 TdEq 2 TAGB = exp[α + β1ln(D2H) + β2ln(ρ)] α − 2.756 0.973 0.3302 23.49 1.0560 ≤ 0.001 β1 0.897 β2 0.562 TdEq 3 TAGB = exp[α + β1ln(D2H)] α − 3.168 0.972 0.3408 24.48 1.0598 ≤ 0.001 β1 0.888 TdEq 4 TAGB = exp[α + β1ln(D) + β2ln(H)] α − 3.032 0.972 0.3371 24.74 1.0585 ≤ 0.001 β1 1.964 β2 0.641 TdEq 5 TAGB = exp[α + β1ln(D)] α − 2.563 0.962 0.3911 32.74 1.0795 ≤ 0.001 β1 2.427 TdEq 6 TAGB = exp[α + β1ln(DH)] α − 3.356 0.958 0.4121 35.87 1.0886 ≤ 0.001 β1 1.377 TdEq 7 TAGB = exp[α + β1ln(ρDH)] α − 2.220 0.951 0.4467 40.71 1.1049 ≤ 0.001 β1 1.393 TdEq 8 TAGB = exp[α + β1ln(H)] α − 3.088 0.819 0.8565 79.77 1.4431 ≤ 0.001 β1 2.771 Table 2 Best fitted regression models for predicting aboveground biomass of A. grandibracteata and T. dregeana n models for predicting aboveground biomass of A. grandibracteata and T. dregeana Table 2 Best fitted regression models for predicting aboveground biomass of A. grandibracteata and T. dregeana ound biomass of A. grandibracteata and T. Scatter plots of TAGB against dendrometric variables for T. dregeanah dregean e biomass (kg); D: diameter at breast height of tree (cm); H: total tree height (m); ρ: Wood Density (g cm−3); α: intercept; β1, β2, β3: are e; RSE: Residual Standard Error; AIC: Akaike Information Criterion; CF: correction factor; AgEq: A. grandibracteata Equation; TdEq: T. Where TAGB: aboveground tree biomass (kg); D: diameter at breast height of tree (cm); H: total tree height (m); ρ: Wood Density (g cm−3); α: intercept; β1, β2, β3: are slopes; adj.R2: adjusted R square; RSE: Residual Standard Error; AIC: Akaike Information Criterion; CF: correction factor; AgEq: A. grandibracteata Equation; TdEq: T. dregeana equation lower value of RSE is the parameter that has proved the fitness of this model. The allometric equation (TdEq2) has also shown the model goodness of fit well among the selected biomass regression models. This model Trichilia dregeana biomass regression model (TdEq1) was found as the best model with the least AIC value (22.55). The predictor variables in this model have explained 97.5% of the variation in TAGB. Also, the Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 8 of 13 predicts the relationship of TAGB against the main var- iables (D2H + ρ) and each predictive variable was inde- pendently fitted. The predictive variables explain adj. R2 (97.3%) variation of TAGB for this tree species. The l ti hi b t TAGB d th di t i bl AIV values. The single predictive variable D in (TdEq5) explains adj.R2 (96.2%) variation in TAGB. Tree diam- eter is among the dendrometric variable that can be accurately and easily measured in the field. However, t h i ht (H) i l di t i bl i TdE 8 Fig. 3 Linear regression for log-transformed data: a aboveground biomass against D; b aboveground biomass against height Fig. 4 Linear regression for log-transformed data: a aboveground biomass against D2H, b aboveground biomass against wood density Fig. 3 Linear regression for log-transformed data: a aboveground biomass against D; b aboveground biomass against height Fig. 3 Linear regression for log-transformed data: a aboveground biomass against D; b aboveground biomass against height ig. 3 Linear regression for log-transformed data: a aboveground biomass against D; b aboveground biomass against height Fig. 4 Linear regression for log-transformed data: a aboveground biomass against D2H, b aboveground biomass against wood density Fig. Scatter plots of TAGB against dendrometric variables for T. dregeanah 4 Linear regression for log-transformed data: a aboveground biomass against D2H, b aboveground biomass against wood density ession for log-transformed data: a aboveground biomass against D2H, b aboveground biomass against wood density AIV values. The single predictive variable D in (TdEq5) explains adj.R2 (96.2%) variation in TAGB. Tree diam- eter is among the dendrometric variable that can be accurately and easily measured in the field. However, tree height (H) as a single predictor variable in TdEq8 was found to explain adj. R2 (81.9%) of TAGB varia- tion which is lowest compared to all other formulated equation. The highest value of RSE (0.8565) was also recorded for this equation when compared to the other selected models. AIV values. The single predictive variable D in (TdEq5) explains adj.R2 (96.2%) variation in TAGB. Tree diam- eter is among the dendrometric variable that can be accurately and easily measured in the field. However, tree height (H) as a single predictor variable in TdEq8 was found to explain adj. R2 (81.9%) of TAGB varia- tion which is lowest compared to all other formulated equation. The highest value of RSE (0.8565) was also recorded for this equation when compared to the other selected models. predicts the relationship of TAGB against the main var- iables (D2H + ρ) and each predictive variable was inde- pendently fitted. The predictive variables explain adj. R2 (97.3%) variation of TAGB for this tree species. The relationship between TAGB and the predictor variable was also found highly significant at (p ≤ 0.001). i The other equations (TdEq3, 4, 5, 6, 7, & 8) were listed in decreasing order of importance considering their AIV values (24.48, 24.74, 32.74, 35.87, 40.71, 79.71) respectively. Generally, these models have achieved the goodness of fit statistics considering the adj.R2, RSE, & Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 9 of 13 Comparison of species‑specific with pan‑tropical equations The pan-tropical equations that have been used for esti- and T. dregeana. When compared to pan-tropical equa- tion. The higher value of PBIAS shows the poor perfor- Fig. 5 Scatter plots for: a aboveground biomass against diameter, b aboveground biomass against height for T. dregeana Fig. 6 Scatter plots for: a TAGB against D2H, b TAGB against wood density for T. dregeana Fig. 5 Scatter plots for: a aboveground biomass against diameter, b aboveground biomass against height for T. dregeana Fig. Scatter plots of TAGB against dendrometric variables for T. dregeanah 5 Scatter plots for: a aboveground biomass against diameter, b aboveground biomass against height for T. dregeana Fig. 6 Scatter plots for: a TAGB against D2H, b TAGB against wood density for T. dregeana Fig. 6 Scatter plots for: a TAGB against D2H, b TAGB against wood density for T. dregeana and T. dregeana. When compared to pan-tropical equa- tion. The higher value of PBIAS shows the poor perfor- mance of pan-tropical equations in predicting TAGB of a specific species. This proofs the application of species specific allometric equation is fundamental to accurately estimate TAGB of tree species. Comparison of species‑specific with pan‑tropical equations The pan-tropical equations that have been used for esti- mation of forest biomass and carbon stocks in Ethiopia were compared with species-specific equations of A. grandibracteata and T. dregeana. Summary of the statis- tical parameters (paired t-test, mean difference of TAGB, percent bias, and root mean square error) for equations comparison was presented in (Table 3). The statistical parameters calculated were based on observed and pre- dicted TAGB. The species-specific equation found bet- ter in accurately predicting TAGB of A. grandibracteata The allometric equation TAGB = 0.0509(ρD2H) by [12] is potentially applicable equation in tropical moist for- ests. In addition, this equation has been most frequently used for biomass estimation in Afromontane rainforests of Ethiopia. Species-specific equation with input variable Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 10 of 13 Table 3 Comparison of species-specific to pan-tropical equations in predicting biomass of A. grandibracteata and T. dregeana PT: Pan-tropical, TFM: Tropical Forests Moist, SS: Species-specific, PBIAS: percent bias, RMSE: root mean square error, D: diameter at breast height, H: total height, ρ: wood density, AgEq: A. grandibracteata Equation; TdEq: T. dregeana Equation Input variable Source Type Equations Mean biomass difference (kg) PBIAS RMSE Pared t-test T-value p-value D Chave et al. [12] PT AGB = ρ × 0.223 × (D)2.148 × (D2)0.207 × (D3)0.028 1117.20 190.29 1859.62 4.047 0.000 AgEq3 SS TAGB = 0.175 × D2.241 6.45 1.09 213.94 0.163 0.872 D, H, ρ Chave et al. [5] PT TAGB. Scatter plots of TAGB against dendrometric variables for T. dregeanah = 0.0673 × (ρD2H)0.976 457.88 77.99 776.72 3.931 0.000 AgEq2 SS TAGB = 0.445 × (D2H)0.749 × ρ1.030 63.21 − 10.77 216.44 − 1.644 0.111 AgEq5 SS TAGB = 0.159 × (D2H)0.787 41.99 − 7.15 212.58 − 1.085 0.287 AgEq7 SS TAGB = 0.497 × (ρDH)1.129 95.61 − 16.29 269.68 − 2.042 0.050 Brown et al. [41] TFM TAGB = 0.0899 × (D2Hρ)0.9522 499.95 85.16 822.14 4.125 0.000 AgEq1 SS TAGB = 0.452 × D2.117 × H0.062 ×ss ρ0.991 33.85 − 5.77 179.34 − 0.035 0.309 D Brown [13] TFM TAGB = 0.118 × D2.53 1235 210.36 2073.59 3.993 0.000 AgEq3 SS TAGB = 0.175 × D2.241 6.45 1.09 213.94 0.163 0.872 D, H, ρ Chave et al. [12] TFM AGB = 0.0509 × ρD2H 430.28 73.29 752.12 3.756 0.001 AgEq1 SS TAGB = 0.452 × D2.117 × H0.062 × ρ0.991 33.85 − 5.77 179.34 − 0.035 0.309 AgEq4 SS TAGB = 0.173 × D2.199 × H0.049 3.6292 0.62 211.03 0.093 0.927 AgEq2 SS TAGB = 0.445 × (D2H)0.749 × ρ1.030 63.21 − 10.77 216.44 − 1.644 0.111 AgEq7 SS TAGB = 0.497 × (ρDH)1.129 95.61 − 16.29 269.68 − 2.042 0.050 D Chave et al. [12] PT TAGB = ρ × 0.223 × (D)2.148 × (D2)0.207 × (D3)0.028 1990.50 211.85 4322.19 − 2.794 0.009 TdEq 5 SS TAGB = 0.077 × D2.427 89.89 9.57 552.83 − 0.889 0.381 D,H, ρ Chave et al. [5] PT TAGB. = 0.0673 × (ρD2H)0.976 555.82 59.15 1099.35 − 3.156 0.004 TdEq2 SS TAGB = 0.064 × (D2H)0.897 × ρ0.562 130.80 − 13.92 575.44 1.257 0.219 TdEq3 SS TAGB = 0.042 × (D2H)0.888 124.93 − 13.29 534.76 1.294 0.206 Brown et al. [41] TFM TAGB = 0.0899 × (D2Hρ)0.9522 592.60 63.07 1128.80 − 3.322 0.002 TdEq1 SS TAGB = 0.0799 × D2.029 × H0.593 × ρ0.648 85.33 − 9.08 493.59 0.945 0.352 TdEq2 SS TAGB = 0.064 × (D2H)0.897 × ρ0.562 130.80 − 13.92 575.44 1.257 0.219 TdEq7 SS TAGB = 0.109 × (ρDH)1.393 236.08 − 25.13 907.31 1.451 0.157 D Brown [13] TFM TAGB = 0.118 × D2.53 1532.30 163.08 3409.18 − 2.709 0.011 TdEq5 SS TAGB = 0.077 × D2.427 89.89 9.57 552.83 − 0.889 0.381 D, H, ρ Chave et al. Scatter plots of TAGB against dendrometric variables for T. dregeanah [12] TFM TAGB = 0.0509ρD2H 539.11 57.38 1101.95 − 3.021 0.005 TdEq1 SS TAGB = 0.0799 × D2.029 × H0.593 × ρ0.648 85.325 − 9.08 493.59 0.945 0.352 TdEq2 SS TAGB = 0.064 × (D2H)0.897 × ρ0.562 130.80 − 13.92 575.44 1.257 0.219 TdEq3 SS TAGB = 0.042 × (D2H)0.888 124.93 − 13.29 534.76 1.294 0.206 TdEq7 SS TAGB = 0.109 × (ρDH)1.393 236.08 − 25.13 907.31 1.451 0.157 Table 3 Comparison of species-specific to pan-tropical equations in predicting biomass of A. grandibracteata and T. dregeana PT: Pan-tropical, TFM: Tropical Forests Moist, SS: Species-specific, PBIAS: percent bias, RMSE: root mean square error, D: diameter at breast height, H: total height, ρ: wood density, AgEq: A. grandibracteata Equation; TdEq: T. dregeana Equation Discussion (ρD2H) was formulated (i.e. TAGB = 0.3274 × (ρD2H)0.759 for A. grandibracteata and TAGB = 0.0832 × (ρD2H)0.899 for T. dregeana) based on measured dataset of total aboveground biomass. Therefore, Chave’s equation was compared with the species-specific equation as depicted in (Fig. 7a, b).The power models were plotted by regress- ing tree DBH against TAGB (Measured and predicted biomass). The predicted TAGB was obtained using spe- cies-specific equations (Species Eq.) and generalized equation (Generalized Eq.). The biomass regression model formulated by relating TAGB against multiple variables (D, H, and ρ) was found statistically the best performing equation for the selected regression models of A. grandibracteata and T. dregeana tree species. This model has considered important pre- dictive variables that improve the accuracy of estimat- ing TAGB. Several studies also suggest the importance of considering the dendrometric variables (D, H, and ρ) in formulating biomass regression models [25, 44]. Stud- ies explain that TAGB estimation is inaccurate when tree height is not available as the predictor variable. Allomet- ric equations are more likely to vary across vegetation Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 11 of 13 Daba and Soromessa Carbon Balance Manage Fig. 7 Species specific and pan-tropical allometric equations comparison for: a A. grandibracteata, b T. dregeana TAGB. Measured biomass: was obtained based on the semi-destructive methodology for allometric equation; Specific Equation: equation which was developed for A. grandibracteata and T. dregeana; Generalized Equation: was taken from [12] for pantropical tropical moist forest stands (ρD2H) was found the best fit model in many studies [5, 12]. Wood density is the best dendrometric variable that converts tree volume into biomass. However, A signifi- cant relationship was not established when data of TAGB was regressed against the wood density of both A. grandi- bracteata and T. dregeana. Generally, wood density var- ies among individuals of the same tree species due to the variability of environmental conditions. Similarly, the study of [46] explains the variation of specific wood grav- ity of a tree species with spatial variation across a forest landscape. Also, the comprehensive study of [47] reveals the variation of tree wood density is most likely corre- lated with increasing elevation, the coarseness of soil tex- ture, and drought stress. The importance of wood density as a single variable in yielding the best fitted TAGB model is a great point of debate in several recent studies. Discussion The comprehensive study of [48] explains that xylem density which is the physical property of wood varies between individuals, species, and environments. Since, it reflects the physiological strategies of trees that lead to growth and survival, wood density as a single predictor variable will not be correlated with TAGB. Several other studies confirm the regional variation of stand-level wood spe- cific gravity that can significantly affect the variation of TAGB. Overall, the great importance of wood density is reported in carbon accounting of tropical forests. How- ever, its variation among different species is correlated with morphological, mechanical, physiological and eco- logical properties [21, 49]. The biomass regression model relating TAGB against D has achieved the model goodness of fit and found statistically significant in this study. This has confirmed that there is a strong relationship between TAGB and D. Basically, D measurement is accurate and practical when compared to other dendrometric variables. Several stud- ies have also reported the significance of D in predict- ing TAGB [9, 12, 50]. In this study, many of the biomass regression models formulated for TAGB against predic- tors (DH) or (D + H) were also found the best performing models. These equations are robust in predicting TAGB of the tree species considered in the study. Similar stud- ies also clarify that the addition of diameter and height as a predictive variable in biomass measurement shows improvement in the TAGB variation. Fig. 7 Species specific and pan-tropical allometric equations comparison for: a A. grandibracteata, b T. dregeana TAGB. Measured biomass: was obtained based on the semi-destructive methodology for allometric equation; Specific Equation: equation which was developed for A. grandibracteata and T. dregeana; Generalized Equation: was taken from [12] for pantropical tropical moist forest stands types since diameter and height are influenced by the environmental condition.h The compound variable of diameter and height (D2H) as a single predictor or in combination with ρ is robust in prediction TAGB of the tree in this study. Allometric equations with such predictor variables are mostly pro- posed for its wide range of applications. The study of [45] also reports that the combination of predictor variables (D & H) is used to capture volume variation. The impor- tant predictor variables (D & H) can be directly modu- lated with climatic and physiographic factors, hence affect the biomass. Authors’ contributions DED, conceived the research; contributed to data analysis and wrote the draft manuscript; TS, edited and improved the manuscript. Both authors read and approved the final manuscript. The existence of a few allometric equations for sub- Saharan Africa is reported by several studies [10, 54, 55]. Many of adopted generalized equations generate great uncertainty of biomass. The study of [14] reports that higher bias was observed related to the Chave’s model II largely overestimating by approximately 300% to 400% for two tropical forest sites. This confirms the signifi- cance of formulating species-and site-specific allometric equations for tropical forests. Such an approach avoids a systematic error generated related to the generalized equation which possibly propagates to the national and global carbon budget. Generally, in response to global climate change mitigation, the monitoring and assess- ment of carbon dioxide from forests is essential. Ethio- pia is known for its diverse vegetation ecosystems and associated high diversity of woody plants. However, the assessment of biomass and carbon stock of forests has been practiced by adopting the generic pan-tropical allo- metric equations that cause great uncertainty. Therefore, the development and application of species-specific allo- metric equation is inevitable for accurate estimation of biomass. Formulating allometric equations for all woody plants in Ethiopia is quite desirable for accurately quanti- fying the biomass and carbon stock of forests to achieve accurate national and international reporting of carbon dioxide emission inventories. Funding This research was supported by Addis Ababa University Thematic Research Fund. Conclusions l Allometric equations for estimating the above-ground biomass in tropical lowland Dipterocarp forests. For Ecol Manag. 2009;257(8):1684–94. 10. Jara MC, Henry M, Réjou-Méchain M, Wayson C, Zapata-Cuartas M, Piotto D, et al. Guidelines for documenting and reporting tree allometric equa- tions. Ann For Sci. 2014;72(6):763–8. 11. Wang C. Biomass allometric equations for 10 co-occurring tree species in Chinese temperate forests. For Ecol Manag. 2006;222(1–3):9–16. 12. Chave J, Andalo C, Brown S, Cairns MA, Chambers JQ, Eamus D, et al. Tree allometry and improved estimation of carbon stocks and balance in tropical forests. Oecologia. 2005;145:87–99. Consent for publication h f ll Authors give full permission for the publication, reproduction, and broadcast. References 1. Riutta T, Malhi Y, Kho LK, Marthews TR, Huasco WH, Khoo M, et al. Logging disturbance shifts net primary productivity and its allocation in Bornean tropical forests. Glob Change Biol. 2018;24(7):2913–28. g 2. Townsend AR, Cleveland CC, Houlton BZ, Alden CB, White JWC. Multi- element regulation of the tropical forest carbon cycle. Front Ecol Environ 2011;9(1):9–17. 2. Townsend AR, Cleveland CC, Houlton BZ, Alden CB, White JWC. Multi- element regulation of the tropical forest carbon cycle. Front Ecol Environ. 2011;9(1):9–17. 3. Foley JA, Asner GP, Costa MH, Coe MT, DeFries R, Gibbs HK, et al. Ama- zonia Revealed: Forest Degradation and Loss of Ecosystem Goods and Services in the Amazon Basin. Front Ecol Environ. 2007;5(1):25–322. 4. Moore S, Adu-Bredu S, Duah-Gyamfi A, Addo-Danso SD, Ibrahim F, Mbou AT, et al. Forest biomass, productivity and carbon cycling along a rainfall gradient in West Africa. Glob Change Biol. 2018;24(2):496–510. Received: 17 May 2019 Accepted: 7 December 2019 Received: 17 May 2019 Accepted: 7 December 2019 Received: 17 May 2019 Accepted: 7 December 2019 Discussion For instance, the biomass regression model that relates TAGB against the compound variable Also, such models have anticipated to increases the accuracy at a multiregional scale [20, 51] related to its application. On the other hand, the relationship between D and H is modulated by multiple environmental fac- tors of forests [52, 53]. The report of [5] also suggests the consideration of the diameter-height relationship for locally developed allometric equations. The ultimate rea- son is that the variation of the relationship between these Daba and Soromessa Carbon Balance Manage (2019) 14:18 Page 12 of 13 predictive variables depends directly on the bioclimatic variables. Conclusions l Formulating an allometric equation is an important approach for the estimation of tree biomass. 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https://openalex.org/W2989787748	https://europepmc.org/articles/pmc6882150?pdf=render	English	null	Hierarchical clustering of maximum parsimony reconciliations	BMC bioinformatics	2,019	cc-by	9,412	Hierarchical clustering of maximum parsimony reconciliations Ross Mawhorter and Ran Libeskind-Hadas* (2019) 20:612 (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics https://doi.org/10.1186/s12859-019-3223-5 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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: Maximum parsimony reconciliation in the duplication-transfer-loss model is a widely-used method for analyzing the evolutionary histories of pairs of entities such as hosts and parasites, symbiont species, and species and genes. While efficient algorithms are known for finding maximum parsimony reconciliations, the number of such reconciliations can be exponential in the size of the trees. Since these reconciliations can differ substantially from one another, making inferences from any one reconciliation may lead to conclusions that are not supported, or may even be contradicted, by other maximum parsimony reconciliations. Therefore, there is a need to find small sets of best representative reconciliations when the space of solutions is large and diverse. Results: We provide a general framework for hierarchical clustering the space of maximum parsimony reconciliations. We demonstrate this framework for two specific linkage criteria, one that seeks to maximize the average support of the events found in the reconciliations in each cluster and the other that seeks to minimize the distance between reconciliations in each cluster. We analyze the asymptotic worst-case running times and provide experimental results that demonstrate the viability and utility of this approach. Conclusions: The hierarchical clustering algorithm method proposed here provides a new approach to find a set of representative reconciliations in the potentially vast and diverse space of maximum parsimony reconciliations. Keywords: Phylogenetic trees, Maximum parsimony reconciliation, Duplication-transfer-loss model Moreover, while parsimony methods are appropriate for relatively simple evolutionary histories, they are likely to be less accurate for complex ones. *Correspondence: hadas@cs.hmc.edu Department of Computer Science, Harvey Mudd College, Claremont, California, USA Maximum parsimony reconciliations An instance of the DTL-MPR problem is a 6-tuple (S, G, φ, d, t, ℓ) where S = (VS, ES) and G = (VG, EG) are binary trees, φ is a function that maps the leaves of G to the leaves of S. This function need not be one-to-one nor onto. Parameters d, t, and ℓare non-negative event costs for duplication, transfer, and loss events, respectively. These events are explained in detail below. The trees S and G are assumed to be undated, but all results in this paper can be easily adapted to dated trees as well. A reconciliation mapping for a given instance is a func- tion that maps the vertices of G to the vertices of S such that (g) = φ(g) for each leaf g of G and, if g is an internal vertex of G with children g′ and g′′, then (1) (g) cannot be a descendant of either (g′) or (g′′) and (2) at least one of (g′) or (g′′) is equal to or a descendant of (g). A reconciliation mapping induces four types of events. Each internal vertex g ∈VG induces one speciation, dupli- cation, or transfer event. In addition, an internal vertex may induce zero or more loss events. For an internal gene tree vertex g, with children g′ and g′′, the events induced by are as follows: A reconciliation mapping for a given instance is a func- tion that maps the vertices of G to the vertices of S such that (g) = φ(g) for each leaf g of G and, if g is an internal vertex of G with children g′ and g′′, then (1) (g) cannot be a descendant of either (g′) or (g′′) and (2) at least one of (g′) or (g′′) is equal to or a descendant of (g). A reconciliation mapping induces four types of events. Each internal vertex g ∈VG induces one speciation, dupli- cation, or transfer event. In addition, an internal vertex may induce zero or more loss events. For an internal gene tree vertex g, with children g′ and g′′, the events induced by are as follows: In this paper, we describe an efficient and practical method for clustering the space of MPRs using agglom- erative hierarchical clustering. The hierarchical cluster- ing method described here has a number of important properties. Maximum parsimony reconciliations First, it is applicable to a variety of differ- ent objectives and linkage criteria. Second, the clusters are compactly represented as reconciliation graphs [14], which permits efficient algorithms to compute statistics on these clusters and to find one or more representative reconciliations in each cluster including median reconcil- iations [11] and maximum event support reconciliations [12], among others. Third, the asymptotic worst-case run- ning time is practical for large trees, large values of k, and is not dependent on the number of MPRs. We demon- strate the viability of this approach on a large Tree of Life dataset [15] in which some trees induce more than 1012 MPRs. Speciation event: Vertex g induces a speciation event if one of (g′) and (g′′) is in the left subtree and the other is in the right subtree of (g). Duplication event: Vertex g induces a duplication event if each of (g′) and (g′′) is either equal to or a descendant of (g) but does not satisfy the require- ments for a speciation event. Transfer event: Vertex g induces a transfer event if exactly one of (g′) and (g′′) is either equal to or a descendant of (g) and the other is neither an ancestor nor a descendant of (g). Transfer event: Vertex g induces a transfer event if exactly one of (g′) and (g′′) is either equal to or a descendant of (g) and the other is neither an ancestor nor a descendant of (g). Loss events: Each non-root vertex g (including leaf ver- tices) may induce zero or more loss events as follows: Let p(g) denote the parent of g in tree G. If (p(g)) is ancestral to (g), then each species vertex s on the path from (p(g)) to (g) induces a loss event, except for (g) and also not (p(g)) if p(g) induces a speciation event. For each loss induced by a ver- tex s on the path from (p(g)) to (g), we say that g passes through s. In summary, the contributions of this paper are: 1 A general framework for agglomerative hierarchical clustering of MPR space; 2 Application of this method for two specific linkage criteria, one seeking to maximize the average event support in each cluster and the other seeking to minimize the distance between MPRs in each cluster; 3 Experimental results on a large biological dataset that demonstrate the viability and utility of this approach. Background Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics Page 2 of 12 (2019) 20:612 of notation and definitions, this material is taken directly from [9, 10] with permission. may even be contradicted, by other maximum parsimony reconciliations. A fundamental problem, therefore, is that of identi- fying a set of best representative reconciliations. Prior work has included efforts to sample MPRs uniformly at random [8] and to find a single median MPR [11]. Recent work has demonstrated that MPR space is, in general, too diverse to be represented by a single MPR [9, 12]. Algorithms have been developed to implic- itly cluster MPR space using k-medoids and k-centers [13], but these algorithms have several limitations. First, the asymptotic running times of these algorithms are O(nk+3 log k) where n is the size of the trees and k is the desired number of clusters. Thus, these algorithms are generally impractical except for very small datasets and numbers of clusters. Moreover, these clustering algorithms provide a representative reconcliation for each cluster but do not provide the clustering itself. Thus, it is not possible to compute various statistics on the clusterings nor to determine to which cluster an MPR belongs. Background Moreover, while parsimony methods are appropriate for relatively simple evolutionary histories, they are likely to be less accurate for complex ones. Phylogenetic tree reconciliation is a widely-used tech- nique for studying the evolutionary history of pairs of enti- ties such as hosts and parasites, pairs of symbionts, and species and genes. In the duplication-transfer-loss (DTL) model, the biological events that are used to explain the possible discordance between pairs of tree are speciation, duplication, transfer, and loss. In the maximum parsimony framework, each type of event has an associated cost and the objective is to find a mapping of one tree (e.g., the gene tree) onto the other tree (e.g., the species tree) that minimizes the total cost of the events induced by that mapping. The maximum parsimony reconciliation problem in the DTL model has received considerable attention over the last decade due to its broad applicability. Efficient algorithms have been developed for the reconciliation problem [1, 4, 5] and have been implemented in a number of popular software tools [1, 4, 6, 7]. Hundreds of published studies in the life sciences have used these tools in their analyses. Typically, reconciliation is performed using a maximum parsimony formulation. Maximum parsimony has been shown to accurately reconstruct simulated data where ground truth is known [1]. While alternative statistical approaches have also been explored, they have many more parameters that must be estimated and the algorithms are generally prohibitively slow [2, 3]. Nonetheless, it must be noted that all reconciliation methods are inherently limited by the evolutionary processes that they model. Unfortunately, the number of maximum parsimony rec- onciliations (MPRs) can grow exponentially in the size of the trees [8]. Moreover MPRs often differ substan- tially from one another [9, 10]. In such cases, making inferences from a single maximum parsimony reconcilia- tion can lead to conclusions that are not supported, and © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Maximum parsimony reconciliations The cost of a reconciliation mapping is defined to be the sum of the costs of all of the induced events. Typically, speciations events are considered null events and thus have cost zero. A minimum cost reconciliation mapping is called a maximum parsimony reconciliation (MPR). Figure 1a shows an example of a DTL-MPR instance and Fig. 1b, c shows two different MPRs for that instance We provide an easily-extensible Python tool, called cluMPR (www.cs.hmc.edu/~hadas/clumpr), that imple- ments this clustering method. al [9] with permission • (S(g,s), {(g′, s′), (g′′, s′′)}) for each speciation in which g is mapped to s, g′ is mapped to s′ or one of its descendants, and g′′ is mapped to s′′ or one of its descendants, where s′ and s′′ denote the children of s; • (S(g,s), {(g′, s′), (g′′, s′′)}) for each speciation in which g is mapped to s, g′ is mapped to s′ or one of its descendants, and g′′ is mapped to s′′ or one of its descendants, where s′ and s′′ denote the children of s; using duplication, transfer, and loss costs of 1, 4, and 1, respectively. Using existing algorithms, a maximum parsimony rec- onciliation can be found in time O(\|G\|\|S\|) [1, 2]. The problem becomes NP-complete, however, if the reconcil- iation is required to be temporally feasible which means that there exists a total ordering of the events such that an event involving a gene vertex g comes earlier in the ordering than any event involving a descendant of g. Fortunately, temporal infeasiblity can be detected when it occurs [2, 16]. • (D(g,s), {(g′, s), (g′′, s)}) for each duplication in which g is mapped to s. • (T(g,s), {(g′, s), (g′′, ˆs)}) for each transfer in which g is mapped to s and one child, wlog g′′, is mapped to a vertex ˆs that is not ancestrally related to s; y • (L(g,s), {(g, s′)}) for each loss in which g passes through s, and s′ is the vertex that follows s on the path from (p(g)) to (g); and • (C(g,s), ∅) for a contemporaneous leaf association where g and s are leaves and φ(g) = s. We provide an easily-extensible Python tool, called cluMPR (www.cs.hmc.edu/~hadas/clumpr), that imple- ments this clustering method. We provide an easily-extensible Python tool, called cluMPR (www.cs.hmc.edu/~hadas/clumpr), that imple- ments this clustering method. We provide an easily-extensible Python tool, called cluMPR (www.cs.hmc.edu/~hadas/clumpr), that imple- ments this clustering method. The next several subsections provide definitions that will be used to describe our algorithm. For consistency Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Page 3 of 12 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Page 3 of 12 Fig. 1 DTL reconciliation. a An instance of the DTL reconciliation problem comprising a species tree (black), a gene tree (gray), and a leaf mapping. Duplication, transfer and loss costs are 1, 4, and 1, respectively. b and c Two different MPRs, each with total cost 4. d The associated reconciliation graph. Mapping nodes are indicated with double line borders. Event nodes are designated with S (speciation event), D (duplication event), T (transfer event), or L (loss event). The reconciliation traversal indicated by solid edges corresponds to the MPR in (b) and the reconciliation traversal indicated by dashed edges corresponds to the MPR in (c); bold edges indicate shared elements of the two MPRs. Figure adapted from Haack et. al [9] with permission Fig. 1 DTL reconciliation. a An instance of the DTL reconciliation problem comprising a species tree (black), a gene tree (gray), and a leaf mapping. Duplication, transfer and loss costs are 1, 4, and 1, respectively. b and c Two different MPRs, each with total cost 4. d The associated reconciliation graph. Mapping nodes are indicated with double line borders. Event nodes are designated with S (speciation event), D (duplication event), T (transfer event), or L (loss event). The reconciliation traversal indicated by solid edges corresponds to the MPR in (b) and the reconciliation traversal indicated by dashed edges corresponds to the MPR in (c); bold edges indicate shared elements of the two MPRs. Figure adapted from Haack et. Reconciliation graphs and traversals The space of all MPRs can be represented in polynomial space using a reconciliation graph (Fig. 1d). This repre- sentation was originally developed by Scornavacca et al. [14] for dated trees and later modified and adapted for undated trees [17]. For completeness, this representation is summarized below. Next, we make several observations about this represen- tation. First, if g is mapped to s as a speciation event, the children of g, denoted g′ and g′′, are mapped to descen- dents of s. However, the speciation event is represented by associating g′ with one child of s (denoted s′) and asso- ciating g′′ with the other child of s (denoted s′′). Loss events are introduced for each loss incurred as g′ (or g′′) passes through species vertices on the path from s′ (or s′′) to (g′) (or (g′′)). Similarly, for a duplication event in Consider a DTL-MPR instance (S, G, φ, d, t, ℓ). Let denote the set of all MPRs for this instance. For a gene vertex g, let the children of g be denoted by g′ and g′′. Then, events(g, s) is the set of the following tuples induced by each MPR ∈: Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Page 4 of 12 Mawhorter and Libeskind-Hadas BMC Bioinformatics which g is mapped to s, the children of g may be mapped to s or descendants of s. However, the duplication event is represented by associating both g′ and g′′ with s and then loss events are introduced for each loss on the path from s to (g′) and on the path from s to (g′′). Finally, if g is mapped to s as a transfer event, then one child of g, wlog g′, is mapped to g or one of its descendants while the other child, g′′ is mapped to a vertex ˆs that is not ancestrally related to s. The transfer event is represented by associ- ating g′ with s (and associating g′′ with ˆs); loss events are introduced for each loss on the path from s to (g′). For a reconciliation graph R, a reconciliation traversal (abbreviated as traversal) is a subgraph of R whose root is a mapping node in sources(R). Methods In this section, we describe a general method for hierar- chical clustering of MPR space and then provide examples of two specific applications of this method, one that seeks clusterings that maximize the average event support of the MPRs in each cluster and the other that seeks to minimize the average distance between MPRs in each cluster with respect to a given distance metric on MPRs. Typically, agglomerative clustering algorithms are ini- tialized with each item (e.g., MPR) forming its own cluster. Subsequently, pairs of clusters are merged according to the particular linkage criterion until the desired number of clusters is obtained. Since the appropriate number of clusters is often difficult to ascertain a priori, the pair- ing may continue until all the items are in a single cluster. By recording the intermediate clusterings, an appropriate number of clusters can be selected according to one of many different criteria [18, 19]. The representation is compact by merit of the fact that, while a mapping (g, s) and its events may arise in many different MPRs, they are shared in this graph represen- tation. Therefore, the size of the reconciliation graph is easily seen to be polynomial in the size of the two trees. Ma et al. give a formal description of the algorithm for constructing undated reconciliation graphs, a derivation of its O(\|G\|\|S\|2) running, and show that undated rec- onciliation graphs are acyclic [17]. Figure 1d shows the reconciliation graph for the DTL-MPR instance in Fig. 1a when duplication and loss have cost one and transfer has cost four. However, the initialization step for agglomerative clus- tering is, in general, not viable for MPRs since the number of such reconciliations can grow exponentially with the sizes of the trees [20]. Therefore, our approach is to begin the agglomerative clustering process with a small num- ber of clusters, where each MPR is represented in one of those clusters. In other words, in the interest of com- putational efficiency, rather than starting the clustering process with a very large number of singleton clusters, we begin the process with a much smaller number of larger Reconciliation graphs and traversals Each non-leaf mapping node added to the traversal has exactly one of its event node children added to the traversal. Each event node added to the traversal has all of its mapping node chil- dren added to the traversal. Figure 1d shows two traversals corresponding to the two MPRs in Fig. 1b, c. There is a straightforward bijection between the set of MPRs and the set of traversals in the reconciliation graph [17]. A traversal, in turn, can be represented as the set of event nodes that it comprises. Thus, we may represent an MPR as the set of event nodes in the corresponding traver- sal. For an MPR R, let E(R) denote the set of event nodes in that reconciliation. For each such tuple e, let type(e) denote its first element, namely the event type and the ordered pair (g, s), and let associations(e) denote its second element, namely a set of zero or more ordered pairs. Note that if e cor- responds to a speciation, duplication, or transfer event, then associations(e) is a set containing two ordered pairs, each representing an association between a gene tree ver- tex and a species tree vertex. If e is a loss event, then associations(e) is a set containing one such ordered pair indicating where the loss is incurred. A reconciliation graph represents the space of all MPRs for a given pair of trees G and S their leaf associations, and their DTL event costs. We will represent subsets of that space, corresponding to clusters, using subgraphs of the reconciliation graph. A reconciliation subgraph is a sub- graph of the reconciliation graph comprising the union of one or more traversals. Thus, a reconciliation subgraph includes at least one source node of the reconciliation graph, all of the sink nodes of the reconciliation graph, and some subset of the mapping and event nodes. Reconciliation graph The reconciliation graph contains a mapping node for each (g, s) pair where g is mapped to s in some MPR and, if not already included, a node (g, s) is also introduced if g passes through s due to a loss event. The reconciliation graph also contains an event node corresponding to each tuple in events(g, s). There is a directed edge from each mapping node (g, s) to each event node in events(g, s) and a directed edge from each event node e to a mapping node corresponding to an ordered pair in associations(e). (Throughout this paper, we use the term vertex for an ele- ment of the gene or species tree and the term node for an element of the reconciliation graph.) Reconciliation traversal Next, we define reconciliation traversals, which corre- spond to MPRs. Let sources(R) denote the set of source nodes of reconcilation graph R which, by definition, are mapping nodes of the form (rg, ·) where rg represents the root of tree G. Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Page 5 of 12 Mawhorter and Libeskind-Hadas BMC Bioinformatics Proof The number of mapping nodes is bounded by O(nm) since each mapping node associates a gene tree vertex with a species tree vertex. Each mapping node has a number of event node children bounded by O(n) since a mapping node may induce a speciation event in one of two ways, depending on which child of g is mapped to which child of s, it may induce a single duplication event, it may induce O(n) transfer events since one of the two children of g may be transferred to a different node of S, and may induce up to two loss event children depending on whether the loss occurs on the left or right child of s. Therefore, the total number of event nodes is bounded by O(n2m) and the total number of mapping and event nodes is bounded by O(n2m). Since each of the O(nm) mapping nodes has a number of children bounded by O(n) and each of the O(n2m) event nodes has at most two children, the number of edges is bounded by O(n2m). clusters. These initial clusters are constructed from the reconciliation graph and are represented by reconciliation subgraphs. Subsequently, when two clusters are agglom- erated, their reconciliation subgraphs are merged. The number of initial clusters in our agglomerative cluster- ing algorithm is denoted N; in the next section we show experimentally that this approach is effective for small val- ues of N. In other words, the shortcut that is used to start the clustering with a small number of large clusters is both efficacious and computationally viable. In the remainder of this section we describe the method for initializing the clusters, describe two linkage criteria, show that these criteria can be computed in polynomial time, and describe a method for identifying the presence of clusters. Representing and initializing clusters h l l To generate the initial clustering, we begin by selecting a depth level L to descend in the reconciliation graph. The set of sources of the reconciliation graph is said to be the set of level 0 subtraversals. For each source node in that set, we consider all of its event node children. Each source node, a single child event node, and the event node’s children (which are, by definition, mapping nodes) forms a level 1 subtraversal. In general, given the set of all level i subtraversals, we construct the set of all level i + 1 sub- traversals as follows: For each level i subtraversal, consider the set of all of its mapping node leaves. For each such mapping node, select one event node child and that event node’s children (which are, again, mapping nodes) to form a level i + 1 subtraversal. This process is repeated, each time constructing all subtraversals at a given level, until we reach the set of all level L subtraversals. For each level L subtraversal, we add all of the nodes reachable from its leaves to form a reconciliation subgraph. These reconcili- ation subgraphs form the set of initial clusters. Note that this process has the desirable property that at the largest possible level, the subtraversals become complete traver- sals and we construct an initial clustering in which each cluster is a single MPR. Lemma 2 The construction of the reconciliation sub- graphs corresponding to the initial clusters takes time O(Nn2m). Proof The subtraversals can be constructed using breadth-first search starting from the sources of the rec- onciliation graph. By Lemma 1, the reconciliation graph has O(n2m) nodes and O(n2m) edges. Therefore, this process takes time O(n2m). Next, each of the N subtraver- sals is expanded into a subgraph of the reconciliation graph corresponding to an initial cluster, which takes time O(Nn2m). In the next two sections, we discuss linkage criteria for merging the initial clusters. Lemma 1 The number of nodes and edges in a reconcil- iation graph is bounded by O(n2m). Criterion 1: minimizing average distance h k f d The weighted average distance (WAD) of C is defined to be support with respect to the MPRs in each cluster is higher than in the entire space of MPRs. By selecting a repre- sentative MPR in each cluster, we can again obtain a set of MPRs that better represent the diversity of MPR space than could be done by selecting a single MPR drawn from the entire space. WAD(C) = k i=1 \|Ci\|μi k i=1 \|Ci\| To optimize this objective function, a natural linkage criterion is to agglomerate the pair of clusters that gives the largest reduction in the weighted average distance, which is effectively a gradient descent heuristic on this objective function. The computation of the average dis- tances between MPRs in a reconcilation graph can be performed in polynomial time [10] in spite of the fact that the number of MPRs may be exponentially large. Let C = {C1, C2, . . . , Ck} denote a k-clustering of MPR space. Let σi denote the average event support in cluster Ci. The weighted average support (WAS) of C is defined to be WAS(C) = k i=1 \|Ci\|σi k i=1 \|Ci\| To optimize this objective function, a natural linkage criterion selects the pair of clusters whose agglomeration gives the largest increase in the weighted average sup- port, which is a gradient ascent heuristic on this objective function. The computation of event frequencies can be computed in polynomial time [11]. Lemma 3 The running time of the clustering algorithm for weighted average distance is O(N2n4m2 log m). Proof Computing the number of MPRs in the reconcil- iation subgraph can be performed in time O(nm) [1] and computing the average distance between all pairs of MPRs can be performed in time O(n4m2 log m) [10]. Lemma 4 The running time of the clustering algorithm for weighted average support is O(N2n2m). By Lemma 2, construction of the initial clustering can be performed in time O(Nn2m). We then compute the aver- age distance and number of MPRs for each of the N initial clusters in time O(Nn4m2 log m). Proof The analysis is identical to that in Lemma 3 except that the computation of average distance is replaced by the computation of support values, which can be computed in time O(n2m) [12]. Thus, the initial construction of the clustering takes time O(Nn2m) and the subsequent clus- tering takes time O(N2n2m). Criterion 1: minimizing average distance h k f d In this section we seek to find a set of clusters that minimizes the average distance between MPRs within each cluster with respect to a given distance metric. Let d(R1, R2) be a distance metric for any pair of MPRs, R1 and R2. For example, in the symmetric distance metric, the dis- tance is the number of events that are in exactly one of the two MPRs, that is \|E(R1) ⊕E(R2)\| where E(R) denotes the set of events in reconciliation R and ⊕is the symmetric set difference operator [11]. In the path distance metric, the distance is defined as the sum, over all gene nodes g, of the length of the path from s1 to s2, where g is mapped to s1 in R1 and g is mapped to s2 in R2 [12, 21]. A number of other distance metrics for MPRs have been proposed as well [21, 22]. For concreteness, we use the symmetric dis- tance metric here, although these results are applicable to other distance metrics as well. In our implementation of this algorithm, the user selects a desired number of initial clusters and the algorithm finds the smallest value of L that results in at least that many initial clusters. Let N denote the number of initial clus- ters actually found by this initialization step. Note that N may be larger than the desired number since the smallest level that generates at least the desired number of clusters depends on the reconciliation graph. Henceforth, let N denote the number of initial clusters and let n and m denote the number of vertices in the species and gene trees, respectively. Let C = {C1, C2, . . . , Ck} denote a k-clustering of MPR space. Let \|Ci\| denote the number of MPRs in cluster Ci and let μi denote the average distance between all pairs of Let C = {C1, C2, . . . , Ck} denote a k-clustering of MPR space. Let \|Ci\| denote the number of MPRs in cluster Ci and let μi denote the average distance between all pairs of Lemma 1 The number of nodes and edges in a reconcil- iation graph is bounded by O(n2m). Page 6 of 12 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics MPRs in Ci with respect to the given distance metric. Criterion 1: minimizing average distance h k f d Thus, the total running time is bounded by O(N2n2m). Next, we compute and record the weighted average dis- tance when merging each pair of initial clusters. This requires O(N2) computations of the average distance and number of MPRs, for a total of O(N2n4m2 log m) time. On each of the O(N) successive iterations, it takes O(N) time to identify the pair of clusters to merge. Computing the average distance and number of MPRs in that cluster takes time O(n4m2 log m) and merging the two reconcil- iation subgraphs takes time O(n2m) since, by Lemma 1, each of the two subgraphs being merged has O(n2m) nodes and edges. Finally, we must compute the distance and number of MPRs between the new (merged) graph and the other O(N) graphs, which requires O(N) com- putations of the average distance and number of MPRs. Thus, each merging iteration requires O(Nn4m2 log m) time, and the O(N) iterations take O(N2n4m2 log m) time in total. The total running time of the clustering algorithm is, therefore, bounded by O(N2n4m2 log m). The improvement score T l h f To analyze the performance of the hierarchical clustering method, we define an improvement score. For the average event support criterion, which is a maximization prob- lem, the improvement score for a given clustering is the weighted average support for the clustering divided by the weighted average support for the entire MPR space, which is simply the average event support. The improvement score indicates the improvement in intracluster support values using clustering versus using no clustering. For the average distance criterion, which is a minimization prob- lem, we invert this ratio: The improvement score for a given clustering is the average pairwise distance between all MPRs divided by the weighted average distance for the clustering. In this case, the improvement score indi- cates the improvement in the intracluster distances using clustering versus using no clustering. Criterion 2: maximizing average event support Another objective of interest is to find a clustering that maximizes the average event support in each cluster. For each event found in an MPR in a given cluster, the support (or frequency) for that event is the fraction of MPRs in that cluster that include that event [11]. In many cases, a sig- nificant fraction of events have very low support over the space of all MPRs [12]. Thus, it may be desirable to par- tition MPR space into clusters, where the average event Note that the improvement score compares a cluster- ing of size k to no clustering or, equivalently, a clustering of size k to a clustering of size 1. A related measure of interest is the improvement achieved by going from k −1 clusters to k clusters, for k ≥2. Let Ca and Cb be Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics Page 7 of 12 Table 1 Running times for the 100 gene families that were clustered using weighted average support and distance linkage criteria Objective Mean runtime (s) Standard deviation (s) # Timeouts Support 25.70 61.99 5 Distance 266.72 272.71 13 Table 1 Running times for the 100 gene families that were clustered using weighted average support and distance linkage criteria the clusters agglomerated by the algorithm when k clus- ters are reduced to k −1 clusters or, equivalently, when k −1 clusters are split into k clusters. Let Cab denote the agglomeration of those two clusters. The local improve- ment at k, k ≥2, denoted WASlocalk for weighted average support, is defined to be: WASlocalk = WAS({Ca, Cb}) WAS({Cab}) spaces. Some gene families in this set induced over 1012 MPRs. The 100 selected gene trees had between 20 and 299 leaves. spaces. Some gene families in this set induced over 1012 MPRs. The 100 selected gene trees had between 20 and 299 leaves. Similarly, for weighted average distance, the local improvement at k is denoted WADlocalk and is defined to be: WADlocalk = WAD({Cab}) WAD({Ca, Cb}) Running times We used a commodity server (AMD Opteron 6276 2.3 GHz, 503 GB RAM) for our experiments. We used N = 25 for the minimum initial number of clusters since we found little benefit to using a larger value of N as discussed below. We set a 20-min timeout for each tree which resulted in some timeouts. Running times and the number of timeouts are summarized in Table 1. Note that when the local improvement is relatively small (e.g., close to 1), there is little improvement in the objec- tive function due to splitting Cab into Ca and Cb. Con- versely, when this score is relatively large, the objective function improves due to the splitting. Therefore, by iden- tifying the value(s) of k where the local improvement score is relatively large, we can identify potentially appropriate number(s) of clusters. Impact of the number of initial clusters The efficiency of this clustering method depends on using a relatively small number of initial clusters, denoted by the parameter N. In theory, starting with a very large number of clusters (e.g., singleton clusters, each compris- ing a single MPR) should produce better final clusterings than starting with a small number of larger initial clusters since those initial clusters are simply constructed from the topology of the reconciliation graph and not iteratively by applying the linkage criterion beginning with clusters of size one. Thus, we investigated the relationship between the improvement score and the number of initial clusters. Specifically, we measured improvement as a function of Results We applied our algorithm to a widely-used Tree of Life dataset comprising 100 primarily prokaryotic species and 4849 gene trees [15] using duplication, transfer, and loss costs of 2, 3, 1, respectively [15, 23]. While these costs have been used in many studies, the xScape algorithms and tools provide a systematic approach for selecting event costs for a given dataset and we recommend using those in practice [24]. We randomly sampled 100 of the 4849 gene trees that induced at least 1000 MPRs since the clustering problem is of particular interest in large MPR Fig. 2 Improvement of two clusters versus no clustering as a function of the size of the initial clustering, N. Each color represents a different gene tree. The sizes of the initial clusterings varies among gene trees due to differences in their reconciliation graphs Fig. 2 Improvement of two clusters versus no clustering as a function of the size of the initial clustering, N. Each color represents a different gene tree. The sizes of the initial clusterings varies among gene trees due to differences in their reconciliation graphs Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics Page 8 of 12 Fig. 3 The average change in improvement from Fig. 2 as a function of the size of the initial clustering, N. For each value of N, each gene family that gives rise to an initial clustering of size less than N and one of size greater than N is considered. The improvement between those two clustering sizes is measured and averaged over all such gene families. For large values of N, the number of families that had a clustering of size less than N and also one of size greater than N but less than our maximum of 200 is very small. Thus, for values of N near 200 the number of samples is small and any statistic for those values is therefore susceptible to error Fig. 3 The average change in improvement from Fig. 2 as a function of the size of the initial clustering, N. For each value of N, each gene family that gives rise to an initial clustering of size less than N and one of size greater than N is considered. The improvement between those two clustering sizes is measured and averaged over all such gene families. Results For large values of N, the number of families that had a clustering of size less than N and also one of size greater than N but less than our maximum of 200 is very small. Thus, for values of N near 200 the number of samples is small and any statistic for those values is therefore susceptible to error Improvement as a function of k N for k = 2 because k = 2 is the last iteration of the algorithm and thus incorporates the agglomeration choices from all previous iterations. The results are sum- marized in Fig. 2. Figure 3 shows the change in improve- ment as a function of N. Note that the sharp spike and drop-off at the right ends of the two plots are due to very small sample sizes for those values of N and thus should not be considered in this evaluation. The average change in improvement is very small across this range of N, indi- cating that the quality of the clusterings is not strongly dependent on the number of initial clusters. We explored how improvement (the ratio between the objective function at k clusters versus 1 cluster) changes as a function of k. These results are summarized in Fig. 4. For some gene families, improvements were consistently close to 1.0, meaning that there is not evidence of clus- ters in their MPR spaces. However, cases in which the improvement score is relatively large suggest that clusters exist. Figure 5 shows local improvement of k (the improve- ment resulting from splitting k−1 clusters into k clusters). The values of k that are relatively large indicate Fig. 4 Improvement as a function of the number of clusters, k. Each curve represents a single gene tree. The improvement is relative to the score for a single cluster Fig. 4 Improvement as a function of the number of clusters, k. Each curve represents a single gene tree. The improvement is relative to the score for a single cluster Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Page 9 of 12 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics Page 9 of 12 Fig. 5 Local improvement as a function of the number of clusters, k. Each curve represents a single gene tree Fig. 6 Number of MPRs in the original reconciliation graph versus improvement after forming two clusters Fig. 5 Local improvement as a function of the number of clusters, k. Each curve represents a single gene tree Fig. 5 Local improvement as a function of the number of clusters, k. Each curve represents a single gene tree Fig. 6 Number of MPRs in the original reconciliation graph versus improvement after forming two clusters Fig. 7 Relationship between the two linkage criteria for two clusters. Improvement as a function of k On the left, clusters were generated for each family using the WAS objective, then evaluated using both. On the right, clusters were generated using the WAD objective Fig. 5 Local improvement as a function of the number of clusters, k. Each curve represents a single gene tree Fig. 6 Number of MPRs in the original reconciliation graph versus improvement after forming two clusters Fig. 6 Number of MPRs in the original reconciliation graph versus improvement after forming two clusters Fig. 6 Number of MPRs in the original reconciliation graph versus improvement after forming two clusters Fig. 7 Relationship between the two linkage criteria for two clusters. On the left, clusters were generated for each family using the WAS objective, then evaluated using both. On the right, clusters were generated using the WAD objective Fig. 7 Relationship between the two linkage criteria for two clusters. On the left, clusters were generated for each family using the WAS objective, then evaluated using both. On the right, clusters were generated using the WAD objective Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics Page 10 of 12 Page 10 of 12 (2019) 20:612 clusterings using weighted average support and weighted average distance, using improvement scores to measure the strength of the relationship. The plot at left in Fig. 7 shows the results of finding two clusters using the average weighted support criterion and evaluating the improve- ment in both weighted average support and weighted average distance on the resulting clusters. The plot at right in Fig. 7 shows the analogous results when clustering using the average weighted distance criterion. There is a small but statistically significant positive correlation between the two improvement scores (r = 0.43, p = 9.38 × 10−6, n = 95 for clusters obtained using event support, and r = 0.51, p = 3.87 × 10−7, n = 85 for clusters obtained using pairwise distance). These results indicate that in some cases, clusters arise regardless of which of the two linkage criteria are used. However, in general, the two link- age criteria are sufficiently different that the presence of candidates for an appropriate number of clusters. Collectively, these results indicate that while some gene families do not give rise to clusters, a number of gene fam- ilies appear to have two clusters and some have an even larger number of clusters. Impact of the number of mPRs Figure 6 shows the relationship between the improvement score (from no clustering to two clusters) and the number of MPRs. We found that there is no correlation between improvement score and the number of MPRs, implying that the presence of clusters is not dependent on the size of MPR space for this dataset. cluMPR software tool A Python implementation of the agglomerative hierar- chical clustering algorithm is available in the cluMPR tool (www.cs.hmc.edu/~hadas/clumpr). This tool sup- ports clustering using the weighted average support and the weighted average distance linkage criteria, allows for a median reconciliation to be generated from each clus- ter as the representative of that cluster, and is extendible to other linkage criteria. The tool generates various types of analyses and plots such as those shown in the previous section. Fig. 9 Local improvement for k using weighted average distance for gene family COG1230. The relatively high local improvement at k = 2 and low local improvement for larger values of k suggests that there are two clusters in this space We conclude with an example of how the cluMPR tool can be useful and how the results can be interpreted. For this example, we chose the gene tree (COG1230) from the 100 trees in our sample that gave the largest improvement for weighted average distance (1.48) for k = 2. This tree induced 718848 MPRs. way to identify a best set of representative MPRs when MPR space is too diverse to be adequately represented by a single MPR. We used the hierarchical clustering algorithm to cluster the MPR space using the weighted average distance link- age criterion. Figure 8 shows the distribution of distances between all MPRs at the top left. The second row shows the distribution of pairwise distances for two clusters (k = 2) and the third row shows the pairwise distances for three clusters (k = 3). A number of challenges remain for future work. First, determining the appropriate number of clusters in an MPR space remains an important problem. We have offered one approach using local improvement scores, but other techniques such as silhouettes [18] and gap statistics [19] are potentially applicable and merit inves- tigation. Second, the relative merits of the two linkage criteria described here, as well as other possible criteria, also merit exploration and evaluation. Finally, while the Tree of Life dataset used here is large and diverse, exper- imental studies using other datasets and event costs are also of potential interest. There is strong evidence for two clusters in this exam- ple since the original bimodal distribution resolves into two clusters with unimodal distance distributions. Abbreviations DTL: Duplication-transfer-loss; MPR: Maximum parsimony reconciliation; PDV: Pairwise distance vector; WAD: Weighted average distance; WAS: Weighted average support Conclusion and future work The authors thank Yi-Chieh Wu for valuable discussions and the anonymous reviewers for feedback and suggestions that improved this manuscript. In this paper we have described an agglomerative hierar- chical clustering methodology for the space of maximum parsimony reconciliations in the duplication-transfer-loss model. We have demonstrated this method for two dif- ferent linkage criteria and have shown that the worst-case asymptotic running time is polynomial in the sizes of the trees and the size of the initial clustering. Using the improvement score measure, we have shown experimen- tally that this method is effective even for small initial clusterings. Thus, this approach provides an efficient way to identify clusters in MPR space. From each cluster, we can then select one or more representative MPRs (e.g, median MPRs or maximum average event support MPRs). Therefore, we believe that this method provides a useful Correlation between linkage criteria Different linkage criteria may result in different cluster- ings. Figure 7 summarizes the relationship between the Fig. 8 Results of clustering using the weighted average distance criteria for gene family COG1230 from the Tree of Life data set using DTL costs 2, 3, 1, respectively with N = 27. On the top are the pairwise distances for the entire MPR space. The second row shows the pairwise distances for k = 2 clusters. The third row shows the pairwise distances for k = 3 clusters obtained using the same method. In this case, the initial distribution is bimodal, suggesting the presence of multiple clusters. For k = 2, the local improvement is 1.48 and both distance distributions are unimodal, indicating that two clusters were identified. For k = 3, the local improvement is 1.10 and the distributions are remain unimodal, suggesting the presence of just two clusters Fig. 8 Results of clustering using the weighted average distance criteria for gene family COG1230 from the Tree of Life data set using DTL costs 2, 3, 1, respectively with N = 27. On the top are the pairwise distances for the entire MPR space. The second row shows the pairwise distances for k = 2 clusters. The third row shows the pairwise distances for k = 3 clusters obtained using the same method. In this case, the initial distribution is bimodal, suggesting the presence of multiple clusters. For k = 2, the local improvement is 1.48 and both distance distributions are unimodal, indicating that two clusters were identified. For k = 3, the local improvement is 1.10 and the distributions are remain unimodal, suggesting the presence of just two clusters Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Page 11 of 12 Fig. 9 Local improvement for k using weighted average distance for gene family COG1230. The relatively high local improvement at k = 2 and low local improvement for larger values of k suggests that there are two clusters in this space clusters under one criterion does not necessarily imply clusters using the other criterion. Further work is required to assess which linkage criteria are most meaningful and useful in practice. Funding g Funding for this research was provided by the R. Michael Shanahan Endowment, Harvey Mudd College, and the National Science Foundation under grant IIS-1905885 to RLH. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. Publication costs are funded by support from Harvey Mudd College. cluMPR software tool How- ever, the local improvement drops from 1.48 at k = 2 to 1.10 at k = 3 and the distributions remain unimodal at k = 3. Moreover, as shown in Fig. 9, the local improve- ment remains relatively close to 1 for larger values of k, further supporting the hypothesis that there are not more than two clusters in this case. Authors’ contributions RLH conceived the research. RLH and RM developed the algorithms. RM implemented the algorithm and performed the experiments. RLH and RM wrote the paper. Both authors read and approved the final manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. y y 4. Doyon J-P, Scornavacca C, Gorbunov KY, Szöllosi JG, Ranwez V, Berry V. An efficient algorithm for gene/species trees parsimonious reconciliation with losses, duplications and transfers. Comp Genom. 2011;6398:93–108. 4. Doyon J-P, Scornavacca C, Gorbunov KY, Szöllosi JG, Ranwez V, Berry V. An efficient algorithm for gene/species trees parsimonious reconciliation with losses, duplications and transfers. Comp Genom. 2011;6398:93–108. 5. Tofigh A, Hallett MT, Lagergren J. Simultaneous identification of duplications and lateral gene transfers. IEEE/ACM Trans Comp Bio Bioinfo. 2011;8(2):517–35. 5. Tofigh A, Hallett MT, Lagergren J. Simultaneous identification of duplications and lateral gene transfers. IEEE/ACM Trans Comp Bio Bioinfo. 2011;8(2):517–35. 6. Conow C, Fielder D, Ovadia Y, Libeskind-Hadas R. Jane: A new tool for cophylogeny reconstruction problem. Algoritm Mol Biol. 2010;5:16. 6. Conow C, Fielder D, Ovadia Y, Libeskind-Hadas R. Jane: A new tool for cophylogeny reconstruction problem. Algoritm Mol Biol. 2010;5:16. 7. Merkle D, Middendorf M, Wieseke N. A parameter-adaptive dynamic programming approach for inferring cophylogenies. BMC Bioinformatics. 2010;11:. https://doi.org/10.1186/1471-2105-11-s1-s60. 8. Bansal MS, Alm EJ, Kellis M. Reconciliation revisited: Handling multiple optima when reconciling with duplication, transfer, and loss. J Comput Biol. 2013;20(10):738–54. https://doi.org/10.1089/cmb.2013.0073. 9. Haack J, Ramirez A, Zupke E, Wu Y, Libeskind-Hadas R. Computing the diameter of the space of maximum parsimony reconciliations in the duplication-transfer-loss model. IEEE Trans Comput Biol Bioinforma. 2018. https://doi.org/10.1109/tcbb.2018.2849732. 10. Santichaivekin S, Mawhorter R, Libeskind-Hadas R. An Efficient Exact Algorithm for Computing All Pairwise Distances between Reconciliations in the Duplication-Transfer-Loss Model. BMC Bioinformatics. 2019. Accepted to appear in the Proceedings of RECOMB-CG 2019, Montpellier, France. 11. Nguyen T-H, Ranwez V, Berry V, Scornavacca C. Support measures to estimate the reliability of evolutionary events predicted by reconciliation methods. PLoS ONE. 2013;8(10):73667. 12. Grueter M, Duran K, Ramalingam R, Libeskind-Hadas R. Reconciliation reconsidered: In search of a most representative reconciliation in the duplication-transfer-loss model. In: Proceedings of the 17th Asia Pacific Bioinformatics Conference; 2019. https://doi.org/10.1109/tcbb.2019. 2942015. 13. Ozdemir A, Sheely M, Bork D, Cheng R, Hulett R, Sung J, Wang J, Libeskind-Hadas R. In: Figueiredo D, Martín-Vide C, Pratas D, Vega-Rodríguez MA, editors. Clustering the Space of Maximum Parsimony Reconciliations in the Duplication-Transfer-Loss Model. Received: 3 September 2019 Accepted: 14 November 2019 Received: 3 September 2019 Accepted: 14 November 2019 21. Huber KT, Moulton V, Sagot M.-F., Sinaimeri B. Geometric medians in reconciliation spaces of phylogenetic trees. Inf Process Lett. 2018;136: 96–101. 22. Huber KT, Moulton V, Sagot M-F, Sinaimeri B. Exploring and Visualizing Spaces of Tree Reconciliations. Syst Biol. 2018. https://doi.org/10.1093/ sysbio/syy075. References 1. Bansal MS, Alm EJ, Kellis M. Efficient algorithms for the reconciliation problem with gene duplication, horizontal transfer and loss. Bioinformatics. 2012;28(12):283–91. 1. Bansal MS, Alm EJ, Kellis M. Efficient algorithms for the reconciliation problem with gene duplication, horizontal transfer and loss. Bioinformatics. 2012;28(12):283–91. 23. Urbini L, Sinaimeri B, Matias C, Sagot M. Exploring the robustness of the parsimonious reconciliation method in host-symbiont cophylogeny. IEEE/ACM Trans Comput Biol Bioinforma. 2018;1:. https://doi.org/10.1109/ tcbb.2018.2838667. 2. Tofigh A. Using trees to capture reticulate evolution: Lateral gene transfers and cancer progression. Doctoral thesis, KTH Royal Institute of Technology. 2009. http://www.diva-portal.org/smash/record.jsf?pid= diva2%3A220830&dswid=-7963. 2. Tofigh A. Using trees to capture reticulate evolution: Lateral gene transfers and cancer progression. Doctoral thesis, KTH Royal Institute of Technology. 2009. http://www.diva-portal.org/smash/record.jsf?pid= diva2%3A220830&dswid=-7963. 24. Libeskind-Hadas R, Wu Y.-C., Bansal MS, Kellis M. Pareto-optimal phylogenetic tree reconciliation. Bioinformatics. 2014;30(12):87–95. 24. Libeskind-Hadas R, Wu Y.-C., Bansal MS, Kellis M. Pareto-optimal phylogenetic tree reconciliation. Bioinformatics. 2014;30(12):87–95. 3. Sjöstrand J, Tofigh A, Daubin V, Arvestad L, Sennblad B, Lagergren J. A bayesian method for analyzing lateral gene transfer. Syst Bio. 2014;63(3): 409–20. https://doi.org/10.1093/sysbio/syu007. 3. Sjöstrand J, Tofigh A, Daubin V, Arvestad L, Sennblad B, Lagergren J. A bayesian method for analyzing lateral gene transfer. Syst Bio. 2014;63(3): 409–20. https://doi.org/10.1093/sysbio/syu007. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. 18. Rousseeuw P. Silhouettes: A graphical aid to the interpretation and validation of cluster analysis. J Comput Appl Math. 1987;20(1):53–65. 19. Tibshirani R, Walther G, Hastie T. Estimating the number of clusters in a data set via the gap statistic. J R Stat Soc Ser B (Stat Methodol). 2001;63(2): 411–23. https://doi.org/10.1111/1467-9868.00293. https://rss.onlinelibrary.wiley.com/doi/pdf/10.1111/1467-9868.00293. Competing interests 20. Chen Z-Z, Deng F, Wang L. Simultaneous identification of duplications, losses, and lateral gene transfers. IEEE/ACM Trans Comput Biol Bioinforma. 2012;9(5):1515–28. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Availability of data and materials The software in this paper and the data used in the experiments are available at www.cs.hmc.edu/~hadas/clumpr. Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 Page 12 of 12 Mawhorter and Libeskind-Hadas BMC Bioinformatics (2019) 20:612 (2019) 20:612 Mawhorter and Libeskind-Hadas BMC Bioinformatics Publisher’s Note Cham: Springer; 2017, pp. 127–39. 14. Scornavacca C, Paprotny W, Berry V, Ranwez V. Representing a set of reconciliations in a compact way. J Bioinforma Comput Biol. 2013;11(02): 1250025. 15. David LA, Alm EJ. Rapid evolutionary innovation during an archaean genetic expansion. Nature. 2011;469:93–96. 16. Ma W, Smirnov D, Libeskind-Hadas R. DTL reconciliation repair. 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https://openalex.org/W4376107304	https://www.researchsquare.com/article/rs-2871677/latest.pdf	English	null	Comprehensive Study of Algal Blooms Variation in Jiaozhou Bay Based on Google Earth Engine and Deep Learning	Research Square (Research Square)	2,023	cc-by	11,984	ABSTRACT Jiaozhou Bay is one of China’s most important marine ecosystems, experiencing increasingly prominent harmful algal blooms due to water quality deterioration and eutrophication. This study utilized the ﬂoating algae index (FAI), which was calculated from MODIS and Sentinel-2 satellite image datasets, to analyze the spatial and temporal variation of harmful algae blooms in Jiaozhou Bay between 2000 and 2022. The study discovered that the frequency of planktonic algal outbreaks was low and constant until 2017, but has increased annually since then. Algae blooms are most common in the summer, and the outbreak area is primarily concentrated in the bay’s coast, middle and mouth, with obvious seasonal and spatial distribution characteristics. Several factors inﬂuencing algal outbreaks were identiﬁed in the study, including sea surface temperature, wind speed, air pressure, dissolved oxygen, nitrogen and phosphorus ratios, chemical oxygen demand (COD), and petroleum pollutants. Furthermore, according to the study, algal bloom outbreaks in Jiaozhou Bay are expected to remain high in 2023. Overall, the ﬁndings of this study provide crucial information for the management of water quality, as well as the prediction and prevention of future algal outbreaks in Jiaozhou Bay. Guan Bin1, Ning Shaowei2, Ding Xu2, Kang Dawei2, Song Jiale2,3,, and Yuan Hongwei4 1School of Computer Science and Information Engineering, Hefei University of Technology, Hefei 230009, China;guanbin@mail.hfut.edu.cn 2College of Civil Engineering, Hefei University of Technology, Hefei 230009, g g g y gy China;ning@hfut.edu.cn(N.S.);dingxu@mail.hfut.edu.cn(D.X);kangdawei@mail.hfut.edu.cn(K.D.) 3MOE Key Laboratory of Soft Soils and Geoenvironmental Engineering, Zhejiang University, Hangzhou 310058 China 4Anhui & Huaihe River Institute of Hydraulic Research, Key Laboratory of Water Conservancy and Water Resources of Anhui Province, Hefei 230088, China;yuanhw 1984@163.com Correspondence: songjiale@mail.hfut.edu.cn; Tel.: +86-15269251518 Keywords: Posted Date: May 10th, 2023 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on August 25th, 2023. See the published version at https://doi.org/10.1038/s41598-023-41138-w. Comprehensive Study of Algal Blooms Variation in Jiaozhou Bay Based on Google Earth Engine and Deep Learning Guan Bin1, Ning Shaowei2, Ding Xu2, Kang Dawei2, Song Jiale2,3,, and Y Introduction Algal blooms on a global scale have emerged as a rising environmental concern, which is primarily attributable to environmental stressors, such as eutrophication and oxygen depletion, that promote the overgrowth of phytoplankton or algae1,2. These ecological problems not only disrupt the balance of aquatic ecosystems, but also pose a signiﬁcant threat to human health and the economy. The consequences of algal bloom outbreaks can be profound, resulting in ﬁsh kills, mortality of aquatic vegetation, and degradation of water quality. When algal blooms decompose, they can cause oxygen depletion and the release of hazardous substances, thereby exacerbating their negative effects on the aquatic ecosystem. Research indicates that optical observation techniques are effective instruments for both the investigation and monitoring of Harmful Algal Blooms (HABs)3,4. Utilizing satellite imagery to monitor planktonic algae has proven useful in enhancing our understanding of the mechanisms underlying algal outbreaks5. Furthermore, it has been demonstrated that ocean color indices derived from spectral band differences provide reliable information on algal outbreaks in both open and coastal waters3 .It has been demonstrated that the Floating Algae Index (FAI) provides superior estimates of algal bloom coverage area6,7. In recent years, Jiaozhou Bay, located in the central Yellow Sea of China and on the southern coast of the Jiaodong Peninsula, has witnessed frequent outbreaks of green algae, which have had long-lasting effects on the coastal carbon cycle and ecosystem8–11. In addition, the bay has become signiﬁcantly impacted by industrial and domestic refuse discharges as a result of the expansion of economic activities and population. Therefore, nutrient concentrations in the bay have considerably increased, particularly from the Licun and Dagu rivers12. The high risk of harmful algal blooms (HABs) in Jiaozhou Bay, similar to the Gulf of Mexico, is mainly attributed to the bay’s rich nutrients and frequent water exchange with the Yellow Sea13.It has been found that the chlorophyll a concentration in the Jiaozhou Bay region is higher in summer, further corroborating the occurrence of algal blooms in the region14. The signiﬁcant changes in the shoreline of Jiaozhou Bay and the reduction of the bay area and tidal prism have led to a decrease in hydrodynamics, hindering the diffusion of nutrients within the bay but accelerating their accumulation, thus contributing to the emergence of planktonic algal blooms6. accumulation, thus contributing to the emergence of planktonic algal blooms6. accumulation, thus contributing to the emergence of planktonic algal blooms6. Introduction Earlier research on the community structure and species of planktonic algae in Jiaozhou Bay included collecting samples and performing microscopic observations to classify and identify them15,16.Several studies are currently being conducted to monitor algal bloom areas in lakes using optical remote sensing satellites and the FAI index, such as Taihu Lake, Dianchi Lake, and Chaohu Lake17–20. However, there are few studies on the surveillance of planktonic algae in semi-enclosed bays, such as Jiaozhou Bay. In this study, the spatial and temporal dynamics of algal blooms in the Jiaozhou Bay region from 2000 to 2022 were inverted using MODIS and Sentinel-2 optical remote sensing satellites. The study focuses on the following major issues: (1) The algal bloom extraction methods were constructed using the Google Earth Engine cloud platform (GEE) based on MODIS and Sentinel-2 datasets for the periods of 2000-2022 and 2015-2022, respectively. From 2000 to 2022, we mapped the algal bloom zone in Jiaozhou Bay and investigated its spatial and temporal sequence of outbreaks. (2) The causes of the algal bloom in Jiaozhou Bay were investigated using meteorological and nutrient factors, as well as regression analysis. (3) Deep neural network (DNN) and Seasonal Autoregressive Integrated Moving Average (SARIMA) deep learning model prediction methods were used to predict the change of pelagic algae in Jiaozhou Bay. Meteorological Data The meteorological data used in this investigation were collected from the Xiaomai Island Observatory and consisted of hourly observations of air temperature, sea surface temperature (SST), wind speed, wind direction, and air pressure from July 2010 to July 2022. These data were collected by the National Ocean Data Observation Centre (http://mds.nmdis.org.cn/). Monthly averages were calculated to investigate the meteorological factors that initiate harmful algal blooms (HABs) in Jiaozhou Bay. Sentinel-2 Data Sentinel-2 is a high-resolution multispectral imaging satellite equipped with a Multi-spectral Instrument (MSI) comprised of two identical satellites, Sentinel-2A and Sentinel-2B, which operate simultaneously25. It has a spatial resolution of 10 meters and a temporal resolution of 10 days for image acquisition for each satellite, with the two satellites observing every ﬁve days and alternating. The data were obtained from the Earth Engine Data Catalog platform (https://developers.google. com/earth-engine/datasets/catalog/sentinel-2). In this study, 460 scenes of 1C-level data from August 2015 to December 2021 and 246 scenes of 2A-level data from October 2018 to December 2021 were extracted after cloud ﬁltering with Google Earth Engine for the study of algal blooms in the Jiaozhou Bay region. MODIS Data MOD09GA.061 is a remote sensing data product gathered by the Moderate Resolution Imaging Spectroradiometer (MODIS) sensor aboard satellite. Once per day, a single image of the Earth’s surface is captured with a spatial resolution of 500 meters. This study selected MOD09GA.061 Terra Surface Reﬂectance Daily Global 500m satellite data from January 2000 until November 2022 in order to examine the evolution of algal blooms in the Jiaozhou Bay region. A total of 8216 images were ob- tained through data ﬁltering and used as one of the study’s data sources. The data were obtained from the Earth Engine Data Cata- log platform (https://developers.google.com/earth-engine/datasets/catalog/MODIS_061_MOD09GA MOD09GA.061 is a remote sensing data product gathered by the Moderate Resolution Imaging Spectroradiometer (MODIS) sensor aboard satellite. Once per day, a single image of the Earth’s surface is captured with a spatial resolution of 500 meters. This study selected MOD09GA.061 Terra Surface Reﬂectance Daily Global 500m satellite data from January 2000 until November 2022 in order to examine the evolution of algal blooms in the Jiaozhou Bay region. A total of 8216 images were ob- tained through data ﬁltering and used as one of the study’s data sources. The data were obtained from the Earth Engine Data Cata- log platform (https://developers.google.com/earth-engine/datasets/catalog/MODIS_061_MOD09GA Water Quality Monitoring data This study utilized measurements of dissolved inorganic nitrogen (DIN), dissolved inorganic phosphorus (DIP), chemical oxygen demand (COD), dissolved oxygen (DO), and petroleum pollutants for water quality monitoring. From 2014 to 2022, these measurements were obtained in the near-shore waters of Jiaozhou Bay. In addition, from 2014 to 2018, measurements of total nitrogen (TN), total phosphorus (TP), and silicate were collected from four inlet rivers in the Jiaozhou Bay area, including the Licun River, Dagu River, Moshui River and Haibo River. The information was gathered at the National Field Scientiﬁc Observation and Research Station for Marine Ecosystems in Jiaozhou Bay, Shandong Province(http: //jzb.cern.ac.cn/). Study Area y Jiaozhou Bay(120◦10′-120◦37′E,36◦06′-36◦25′W), located in the central Yellow Sea of China, is a semi-enclosed bay that covers the southern coast of the Jiaodong Peninsula in the city of Qingdao in Shandong Province, presenting a near trumpet shape, with a maximum length of about 40 km in the north-south direction and a maximum width of about 28 km in the east-west direction, with an average depth of 7 m and a maximum depth of 64 m. A total of 438 km2 makes it China’s third-largest bay (Figure 1). Jiaozhou Bay has a warm-temperate monsoon climate, meaning that its summers are affected by the southeast monsoon and its winters by the northwest monsoon. Several small-scale circulation currents, such as a counter-current and a rotating current, are caused by the Yellow Sea Current’s inﬂuence on the bay. Figure 1. Study area information including distribution of observatories and rivers discharged into Jiaozhou Bay and landuse around Jiaozhou Bay. Figure 1. Study area information including distribution of observatories and rivers discharged into Jiaozhou Bay and landuse around Jiaozhou Bay. Jiaozhou Bay (JZB) is a typical eutrophic ecosystem, where the chlorophyll a concentration is highest in the bay’s north- eastern and north-western regions and progressively decreases southward. August marks the annual maximum and maximal Jiaozhou Bay (JZB) is a typical eutrophic ecosystem, where the chlorophyll a concentration is highest in the bay’s north- eastern and north-western regions and progressively decreases southward. August marks the annual maximum and maximal 2/19 ﬂuctuations of chlorophyll a21. The total organic carbon (TOC), total nitrogen (TN), and carbon-to-nitrogen (C/N) ratios in the solid phase of Jiaozhou Bay sediments increase progressively, primarily as a result of the extensive pollution from human inputs via river discharges22 .Concentrations of phytoplankton are greatest in the northwestern and northern regions of the bay, near to the river headwaters, and decrease with increasing depth from the inner to the outer bay. Due to nutrient accumulation under south-eastern wind conditions, phytoplankton epidemics may occur near the northwestern coast23. Over the past three decades, JZB has experienced a considerable increase in pollutants, resulting in a decline in water quality24. ﬂuctuations of chlorophyll a21. Study Area The total organic carbon (TOC), total nitrogen (TN), and carbon-to-nitrogen (C/N) ratios in the solid phase of Jiaozhou Bay sediments increase progressively, primarily as a result of the extensive pollution from human inputs via river discharges22 .Concentrations of phytoplankton are greatest in the northwestern and northern regions of the bay, near to the river headwaters, and decrease with increasing depth from the inner to the outer bay. Due to nutrient accumulation under south-eastern wind conditions, phytoplankton epidemics may occur near the northwestern coast23. Over the past three decades, JZB has experienced a considerable increase in pollutants, resulting in a decline in water quality24. Processing of Sentinel-2 Satellite Data The GEE platform provides Sentinel-2 images in Level 1C and Level 2A formats. The ﬁrst is orthorectiﬁed and geometrically corrected but lacks atmospheric correction, whereas the second is ortho-corrected for bottom-of-atmosphere reﬂectance (BOA). Consequently, Level 2A images provide more realistic reﬂectance data, with more accurate color levels, greater brightness and contrast, and are generally more color sensitive. In the previous study, we found that atmospheric correction is predominantly responsible for the difference in quality. When extracting the area of algal blooms using the FAI threshold segmentation method, the area of Level 2A data was consistently greater than that of Level 1C data for bloom areas that exceeded a certain threshold. In this study, two images were extracted using 1C-Level and 2A-Level dataset from Sentinel-2 (Figure 2) on 10 July 2021, an algal bloom outbreak was observed, and analysis of the extracted area and color depth revealed that the FAI value for dateset 2A was greater than for dataset 1C. Consequently, when both were extracted above a particular threshold, the outbreak area of dataset 2A was greater than that of dataset 1C. On October 23, 2021, no algal bloom outbreak was detected. Nevertheless, it was discovered that the dataset 2A was more sensitive due to its own atmospheric correction procedure and Sentinel-2’s high resolution. This increased the number of false-positive planktonic algae identiﬁcations31,32. In contrast, fewer false positives were extracted from the dataset 1C. Although the threshold can be adjusted to reduce false positives, this may result in algal bloom area loss, which may not be desirable19. This study proposes the Single Threshold Multi-stage Weakening (STMW) method (as shown in Figure 4) to reduce the occurrence of false positives while mitigating the loss of algal bloom area. After determining the optimal threshold for the Sentinel-2 dataset, it was found that the optimal threshold for dataset 1C was larger than that for dataset 2A, and either 1C or 2A had the capability to extract the precise area. However, when both 1C and 2A utilized their optimal thresholds, their respective results were affected by false positives. As the analysis below demonstrates, applying the threshold for dataset 2A to 1C signiﬁcantly reduced the number of false positives and the resulting algal bloom area. Following an analysis, the optimal threshold for detecting the presence of a harmful algal bloom (HAB) using Sentinel 2 satellite level 2A data was determined to be 0.045. Processing of MODIS Satellite Data g In this study, we processed the MODIS satellite data using the Google Earth Engine cloud platform (GEE) (https://code. earthengine.google.com/)30. After reprojecting the MOD09GA dataset from ’SR-ORG:6974’ to ’EPSG:4326’, we clipped the images to our study area and kept the resolution constant at 500m. In order to investigate algal bloom outbreaks in the region, we additionally removed clouds from each image. During processing, we ﬁltered the data and selected only those with more than zero observations. Then, these particular data were declouded. For this research, we selected a band resolution of 500m, which may introduce some error when analyzing small areas such as Jiaozhou Bay. To prevent erroneous positives caused by the involvement of the ground in the calculation process, a special treatment was applied to the study area. Speciﬁcally, we selected only data within 1 kilometer of the coast. Ultimately, the algal bloom zone was extracted. Floating Algae Index(FAI) The Floating Algae Index (FAI) utilizes the red, near-infrared (NIR), and shortwave infrared (SWIR) bands to compute a threshold segmentation method that enables the extraction of areas of algal blooms in seawater7.FAI’s reduced susceptibility to environmental interference and enhanced advantages over other methods such as the Normalized Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI) have led to its increased use in the global study of algal blooms26–29. FAI = Rrc,NIR −R′ rc,NIR′ R′ rc,NIR = Rrc, Red +(Rrc,SWIR −Rrc, Red )×(λNIR −λRed )/(λSWIR −λRed ) (1) FAI = Rrc,NIR −R′ rc,NIR′ R′ rc,NIR = Rrc, Red +(Rrc,SWIR −Rrc, Red )×(λNIR −λRed )/(λSWIR −λRed ) (1) where Rrc,NIR,Rrc,Red and Rrc,SWIR are the reﬂectance in the NIR, Red and SWIR band after Rayleigh correction, respectively. λNIR, λRed and λSWIR are the central bands of the sensor’s corresponding bandwidth. where Rrc,NIR,Rrc,Red and Rrc,SWIR are the reﬂectance in the NIR, Red and SWIR band after Rayleigh correction, respectively. λNIR, λRed and λSWIR are the central bands of the sensor’s corresponding bandwidth. 3/19 Selection of the Optimal Threshold To accurately extract the area of algal colonies using FAI threshold segmentation, it is necessary to identify the optimal threshold that yields the most accurate results. However, it is not possible to manually determine the optimal threshold through point-by-point observation and experimentation on a single image. Similarly, manually determining the optimal threshold for all MODIS and Sentinel-2 images and then performing statistical analysis to identify the values would be a daunting task. The maximum gradient method introduced by Ma et al.19 is an effective alternative to manually determining the optimal threshold. However, it should be noted that this method requires a substantial quantity of human effort. In addition, it is essential to recognize that a large number of images without algal blooms will need to be analyzed and their optimal thresholds determined, which can be time-consuming and may affect the statistical regularity of the optimal thresholds for algal bloom outbreaks. This research differs from others in that it concentrates on continuously observing the extraction effect of various thresholds on an image of an algal outbreak (captured on 10 July 2021) in order to determine the optimal threshold as the starting point for analysis. Despite the possibility that the starting threshold does not entirely reﬂect the optimal extraction effect, it can still reﬂect the trend of algal bloom area change. The study then traversed all images that met the initial threshold in order to isolate the algal blooms region. By choosing the larger FAI area as the date of the algal bloom, a threshold for determining the image set was established. The study determined manually, with a minimum accuracy of 0.005, the optimal threshold value for each image in the set and analyzed the results. The most frequent threshold value was selected as the optimal threshold value, saving time and preventing the capture of images free of algal blooms. In the Jiaozhou Bay region, the optimal FAI extraction threshold was determined to be 0.05 for MODIS and 0.045 for Sentinel-2 using this method. Processing of Sentinel-2 Satellite Data We focused our analysis on the six months of January, February, March, October, November, and December for the years 2018-2022 because algal bloom outbreaks are known to occur primarily during the summer. Our goal was to extract data from datasets 1C and 2A with a threshold greater than 0.045 and then compare false positive rates during months when algal bloom outbreaks are uncommon. 4/19 Figure 2. Comparison of FAI index extracted from Sentinel-2 2A and 1C at the same threshold. Figure 2. Comparison of FAI index extracted from Sentinel-2 2A and 1C at the same threshold. The maximum area extracted from the level 2A data was only 31 km2, and the mean value was 5.6 km2. These results substantially affected our analysis of the area of the algal outbreak. Therefore, we chose to concentrate on the 1C data, which had a mean area value that was 93% less than that of dataset 2A. This signiﬁcantly reduced the impact of false positives and made it easier to differentiate between the real algal bloom outbreak and any false positives. To enhance our analysis even further, we decided to focus on a small rectangular area within Jiaozhou Bay. This allowed us to circumvent any confusion caused by false-positive results along the coast. In this tiny area, our analysis of level 1C data revealed a mean value that was 37.8% lower than level 2A data. This helped us to discount any false positives and obtain a more precise observation of the algal bloom outbreak’s extent. In order to reduce false positives, The optimal level 2A threshold 2019 2020 2021 2022 0.00 0.01 0.02 0.03 0.04 Algal Bloom Area(km2) (a) Difference Sentinel-2 Level 2A Sentinel-2 Level 1C 2019 2020 2021 2022 0 5 10 15 20 25 30 Algal Bloom Area(km2) (b) Difference Sentinel-2 Level 2A Sentinel-2 Level 1C Figure 3. Comparison of the loss of algal bloom area and false-positive reduction. (a) shows the histogram of false positive reduction, (b) shows the histogram of the loss of algal bloom area. (b) (a) Figure 3. Comparison of the loss of algal bloom area and false-positive reduction. (a) shows the histogram of false positive reduction, (b) shows the histogram of the loss of algal bloom area. was utilized to extract level 1C data, and statistical analysis was performed (As shown in Figure 3). Processing of Sentinel-2 Satellite Data While the number of false positives decreased substantially, the area of algal bloom also decreased noticeably. Due to the presence of false positives, it was difﬁcult to utilize level 2A data to evaluate the algal bloom. In contrast, the level 1C data eliminated the majority of false positives, making it simpler to observe the trend of the outbreak of algal bloom. The STMW method was developed to overcome the limitations of using either level 1C or level 2A data by combining the two. The 1C data can be used to identify 5/19 Figure 4. Flow chart for extracting the area of algal blooms using the GEE. Figure 4. Flow chart for extracting the area of algal blooms using the GEE. 6/19 the days of algal bloom outbreak, but the actual aera of algal bloom outbreak is calculated from the corresponding 2A data. This method extracts the area of the bloom from a copy of the level 2A data at its optimal threshold and utilizes the level 1C data to substantially reduce the number of false positives in the level 2A data. The STMW method utilizes the low sensitivity of the 1C data to eliminate the effect of false positives and precisely analyze the number of algal bloom outbreak days. Finally, it eliminates some outliers and reduce errors, thereby enhancing the detection accuracy of algal bloom areas. the days of algal bloom outbreak, but the actual aera of algal bloom outbreak is calculated from the corresponding 2A data. This method extracts the area of the bloom from a copy of the level 2A data at its optimal threshold and utilizes the level 1C data to substantially reduce the number of false positives in the level 2A data. The STMW method utilizes the low sensitivity of the 1C data to eliminate the effect of false positives and precisely analyze the number of algal bloom outbreak days. Finally, it eliminates some outliers and reduce errors, thereby enhancing the detection accuracy of algal bloom areas. Prediction the Area of Algae Bloom g Deep Neural Networks(DNN) Predicting the distribution of algal bloom remains difﬁcult due to their heterogeneity and unpredictability in response to complex environmental conditions33. This study employs deep learning techniques, particularly a deep neural network (DNN), to analyze the change of the distribution patterns of algal bloom. The analysis focuses exclusively on the effect of meteorological factors on the algal bloom’s area. It was widely acknowledged that DNNs were effective at predicting time series data34. Since 2010, the area of the algal bloom has been determined using MODIS, and the monthly x1 x2 x3 ... a(1) 1 a(1) 2 a(1) 3 a(1) 4 a(1) 10 ... a(2) 1 a(2) 2 a(2) 3 a(2) 8 ... a(3) 1 a(3) 2 a(3) 6 ... y1 ... Input Layer (Meteorological Data) Hidden Layers Output Layer (Algal Bloom Area) a(0) 1 a(0) 2 a(0) 3 a(0) 4 a(0) n a(1) m a(1) 3 a(1) 2 a(1) 1 w1,1 w1,1 w1,2 w1,2 w1,3 w1,3 w1,4 w1,4 w1,n w1,n ... ... = σ w1,0a(0) 0 +w1,1a(0) 1 +...+w1,na(0) n +b(0) 1 = σ n ∑ i=1 w1,ia(0) i +b(0) 1 !       a(1) 1 a(1) 2... a(1) m       = σ        w1,0 w1,1 ... w1,n w2,0 w2,1 ... w2,n ... ... ... ... wm,0 wm,1 ... wm,n            a(0) 1 a(0) 2... a(0) n       +       b(0) 1 b(0) 2... b(0) m         a(1) = σ W(0)a(0) +b(0) Figure 5. Structure diagram of deep neural network. his data has been calculated. The relationship between this area data and the three meteorological factors ressure, and SST was separately analyzed (Figure 11), and the results show that the change in algal blo ery obvious annual variation pattern and has a strong correlation with these three. This paper predicts the x1 x2 x3 ... a(1) 1 a(1) 2 a(1) 3 a(1) 4 a(1) 10 ... a(2) 1 a(2) 2 a(2) 3 a(2) 8 ... a(3) 1 a(3) 2 a(3) 6 ... y1 ... Prediction the Area of Algae Bloom However, the previous deep neural network (DNN) model had limitations in accurately predicting algal outbreaks under the inﬂuence of multiple factors in reality due to the issue of variability among indicators. Furthermore, because the input layer only used meteorological factors, the model lost data interpretability. So, we also use seasonal decomposition and the Seasonal Autoregressive Integrated Moving Average (SARIMA) model to control the objective pattern of algal development in order to improve prediction accuracy. The algal bloom area is analyzed and predicted in time series from an alternative perspective by exclusively combining the 22-year extraction results of the MODIS dataset35. The MODIS extraction results revealed an upward trend in algal bloom outbreaks, with signiﬁcantly stronger outbreaks occurring in the second and third quarters than the ﬁrst and fourth quarters. Despite the changes over time, the seasonal ﬂuctuations of the algal bloom aera did not differ signiﬁcantly during the previous and current observation periods. This study removes the inﬂuence of seasonality from the time series in order to investigate the seasonality of algal outbreaks and other masked characteristics. To accomplish this, we use an additive seasonal decomposition model to divide algal bloom outbreak time series into four components: irregular changes, seasonally adjusted series, seasonal adjustment factors, and trend cyclic components. These elements enable us to quantify the effects of different factors on algal bloom outbreaks. SARIMA (0, 0, 1) (0, 1, 1) was used in this study to analyze and predict based on the above patterns and after testing. SARIMA Model Changes in the time series of algal bloom area show that these outbreaks have strong seasonality, clear cyclical characteristics, and growth trends. However, the previous deep neural network (DNN) model had limitations in accurately predicting algal outbreaks under the inﬂuence of multiple factors in reality due to the issue of variability among indicators. Furthermore, because the input layer only used meteorological factors, the model lost data interpretability. So, we also use seasonal decomposition and the Seasonal Autoregressive Integrated Moving Average (SARIMA) model to control the objective pattern of algal development in order to improve prediction accuracy. The algal bloom area is analyzed and predicted in time series from an alternative perspective by exclusively combining the 22-year extraction results of the MODIS dataset35. Prediction the Area of Algae Bloom The MODIS extraction results revealed an upward trend in algal bloom outbreaks, with signiﬁcantly stronger outbreaks occurring in the second and third quarters than the ﬁrst and fourth quarters. Despite the changes over time, the seasonal ﬂuctuations of the algal bloom aera did not differ signiﬁcantly during the previous and current observation periods. This study removes the inﬂuence of seasonality from the time series in order to investigate the seasonality of algal outbreaks and other masked characteristics. To accomplish this, we use an additive seasonal decomposition model to divide algal bloom outbreak time series into four components: irregular changes, seasonally adjusted series, seasonal adjustment factors, and trend cyclic components. These elements enable us to quantify the effects of different factors on algal bloom outbreaks. SARIMA (0, 0, 1) (0, 1, 1) was used in this study to analyze and predict based on the above patterns and after testing. (2) 1−L12 yt = α0 +(1+θL) 1+ΘL12 εt where L is the lag operator, εt is a white noise series with variance σ2. Θ is the seasonal moving average term coefﬁcient, θ is the non-seasonal moving average term coefﬁcient and α0 is the intercept term. Results Comparison of Algal Bloom Observation Areas Using Different Satellites Figure 6. Example diagram of FAI extraction. (a) is the Sentinel-2 true color image on July 10, 2021, (b) and (c) are the FAI calculated from MODIS and Sentinel-2 at the same day. Comparison of Algal Bloom Observation Areas Using Different Satellites Prediction the Area of Algae Bloom Input Layer (Meteorological Data) Hidden Layers Output Layer (Algal Bloom Area) Input Layer (Meteorological Data) Hidden Layers a(0) 1 a(0) 2 a(0) 3 a(0) 4 a(0) n a(1) m a(1) 3 a(1) 2 a(1) 1 w1,1 w1,1 w1,2 w1,2 w1,3 w1,3 w1,4 w1,4 w1,n w1,n ... ... = σ w1,0a(0) 0 +w1,1a(0) 1 +...+w1,na(0) n +b(0) 1 = σ n ∑ i=1 w1,ia(0) i +b(0) 1 !       a(1) 1 a(1) 2... a(1) m       = σ        w1,0 w1,1 ... w1,n w2,0 w2,1 ... w2,n ... ... ... ... wm,0 wm,1 ... wm,n            a(0) 1 a(0) 2... a(0) n       +       b(0) 1 b(0) 2... b(0) m         a(1) = σ W(0)a(0) +b(0) Figure 5. Structure diagram of deep neural network. average of this data has been calculated. The relationship between this area data and the three meteorological factors of wind speed, air pressure, and SST was separately analyzed (Figure 11), and the results show that the change in algal bloom area exhibits a very obvious annual variation pattern and has a strong correlation with these three. This paper predicts the monthly average of this data has been calculated. The relationship between this area data and the three meteorological factors of wind speed, air pressure, and SST was separately analyzed (Figure 11), and the results show that the change in algal bloom area exhibits a very obvious annual variation pattern and has a strong correlation with these three. This paper predicts the monthly 7/19 mean algal bloom area in the study area using a DNN neural network model (Figure 5), with wind speed, air pressure, and SST serving as independent variables and algal bloom area serving as the dependent variable. mean algal bloom area in the study area using a DNN neural network model (Figure 5), with wind speed, air pressure, and SST serving as independent variables and algal bloom area serving as the dependent variable. g p g g p SARIMA Model Changes in the time series of algal bloom area show that these outbreaks have strong seasonality, clear cyclical characteristics, and growth trends. Comparison of Algal Bloom Observation Areas Using Different Satellites Figure 6. Example diagram of FAI extraction. (a) is the Sentinel-2 true color image on July 10, 2021, (b) and (c) are the FAI calculated from MODIS and Sentinel-2 at the same day. Multiple satellites were utilized to track the algal bloom in Jiaozhou Bay. Three images were selected for comparison on July 10, 2021: a Sentinel-2 true color image, a Sentinel-2 FAI index image, and a MODIS FAI index image. These images were contrasted to evaluate their ability to extract information about the algal bloom region (Figure 6).Using the extracted area from 8/19 the MODIS dataset, a 22-year time series of variations in the algal bloom area was generated(Figure 7,8). A sliding window method was used in order to better analyze the trend of changes in the algal bloom area in Jiaozhou Bay (Figure 7(b)).Prior to 2017, the area of algal bloom in Jiaozhou Bay remained relatively stable, according to the ﬁndings. However, there has been a general upward trend in the overall algal bloom area in Jiaozhou Bay over the next ﬁve years. Further examination of the annual changes in algal bloom reveals that algal bloom outbreaks are primarily concentrated in the summer, particularly from May to September. This ﬁnding is consistent with previous research on chlorophyll a concentrations in Jiaozhou Bay, which discovered that the peak concentration of chlorophyll a occurs during the summer months14. 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Algal Bloom Area(km2) (a) MODIS Trendline Annual Max 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 0 1 2 3 4 Algal Bloom Area(km2) (b) MODIS Trendline Figure 7. (a) Time series of algal blooms area in Jiaozhou Bay from 2000 to 2022. (b) Time series of algal blooms area processed by sliding window in Jiaozhou Bay from 2000 to 2022. Figure 7. (a) Time series of algal blooms area in Jiaozhou Bay from 2000 to 2022. (b) Time ser processed by sliding window in Jiaozhou Bay from 2000 to 2022. Figure 7. (a) Time series of algal blooms area in Jiaozhou Bay from 2000 to 2022. (b) Time series of algal blooms area processed by sliding window in Jiaozhou Bay from 2000 to 2022. Figure 8. Comparison of Algal Bloom Observation Areas Using Different Satellites Figure 10 depicts the frequency of spatial distribution characteristics of the algal bloom in Jiaozhou Bay. According to the ﬁndings, the coastal area of Jiaozhou Bay experienced a more severe outbreak of algal bloom, with the area of algal bloom decreasing from the coast to the bay’s inner sea. The outbreak was primarily concentrated in the bay’s coastal, central, and bay estuary regions. The outbreak of algal bloom in Jiaozhou Bay’s inner sea was not severe from 2000 to 2016. However, since 2017, the outbreak of algal bloom in Jiaozhou Bay’s inland area has become more severe and will not abate until 2021. value of algal bloom area of the MODIS data was higher than that of Sentinel-2, which can be attributed to the difference in resolution and sensors used by the two satellites. Figure 10 depicts the frequency of spatial distribution characteristics of the algal bloom in Jiaozhou Bay. According to the ﬁndings, the coastal area of Jiaozhou Bay experienced a more severe outbreak of algal bloom, with the area of algal bloom decreasing from the coast to the bay’s inner sea. The outbreak was primarily concentrated in the bay’s coastal, central, and bay estuary regions. The outbreak of algal bloom in Jiaozhou Bay’s inner sea was not severe from 2000 to 2016. However, since 2017, the outbreak of algal bloom in Jiaozhou Bay’s inland area has become more severe and will not abate until 2021. value of algal bloom area of the MODIS data was higher than that of Sentinel-2, which can be attributed to the difference in resolution and sensors used by the two satellites. Figure 10 depicts the frequency of spatial distribution characteristics of the algal bloom in Jiaozhou Bay. According to the ﬁndings, the coastal area of Jiaozhou Bay experienced a more severe outbreak of algal bloom, with the area of algal bloom decreasing from the coast to the bay’s inner sea. The outbreak was primarily concentrated in the bay’s coastal, central, and bay estuary regions. The outbreak of algal bloom in Jiaozhou Bay’s inner sea was not severe from 2000 to 2016. However, since 2017, the outbreak of algal bloom in Jiaozhou Bay’s inland area has become more severe and will not abate until 2021. ore severe and will not abate until 2021. Comparison of Algal Bloom Observation Areas Using Different Satellites orrelation Analysis eteorological Factors 0.00097 0.00098 0.00099 0.00100 Reciprocal of Air Pressure(1/hpa) 0.0 0.5 1.0 1.5 2.0 Algal Bloom Area(km2) (a) r = 0.55 N = 137 y=28839.611x-27.955 1:1 Lines 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 0.0 0.5 1.0 1.5 2.0 Area(km2) (b) 1000 1010 1020 1030 Air Pressure(hpa) 0 2 4 6 8 10 Month(2021) 0.00 0.25 0.50 0.75 1.00 1.25 Area(km2) (c) Area 1005 1010 1015 1020 1025 Air Pressure(hpa) Air Pressure 5 10 15 20 25 SST(¤C) 0.0 0.5 1.0 1.5 2.0 Algal Bloom Area(km2) (d) r = 0.74 N = 137 y=0.0447x-0.1335 1:1 Lines 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 0.0 0.5 1.0 1.5 2.0 Area(km2) (e) 5 10 15 20 25 SST(¤C) 0 2 4 6 8 10 Month(2021) 0.00 0.25 0.50 0.75 1.00 1.25 Area(km2) (f) Area 5 10 15 20 25 SST(¤C) SST 0.1 0.2 0.3 0.4 0.5 Reciprocal of Wind Speed(s/m) 0.0 0.5 1.0 1.5 2.0 Algal bloom area(km2) (g) r = 0.43 N = 137 y=1.887x-0.2165 1:1 Lines 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 0.0 0.5 1.0 1.5 2.0 Area(km2) (h) 2 3 4 5 6 7 Wind Speed(m/s) 0 2 4 6 8 10 Month(2021) 0.00 0.25 0.50 0.75 1.00 1.25 Area(km2) (i) Area 2.5 3.0 3.5 4.0 4.5 5.0 Wind Speed(m/s) Wind Speed gure 11. Analysis of the relationship between environmental factors and algal bloom outbreaks. (a)(d)(g) show scatter plo tween the algal bloom area and air pressure(countdown), sea surface temperature(SST), and wind speed (countdown), spectively, and (b)(e)(h) show monthly average series of the algal bloom area and the air pressure, SST and the wind spe r November 2010-2022, and d (c)(f)(i) show monthly average for 2021. Utilizing MODIS data, monthly averages of algal bloom area were calculated. The meteorological data was then analyzed termine the contributors to algal bloom outbreaks in Jiaozhou Bay. Comparison of Algal Bloom Observation Areas Using Different Satellites Monthly average algal bloom area from 2000 to 2022 (a), (b) shows the bubble matrix of algal bloom area, (c) shows the distribution of algal blooms occurring in different months. Figure 8. Monthly average algal bloom area from 2000 to 2022 (a), (b) shows the bubble matrix of algal bloom area, (c) shows the distribution of algal blooms occurring in different months. The extracted algal bloom area data from Sentinel-2 and MODIS were subsequently compared, followed by an examination of the differences and effects of extraction between the two satellites (Figure 9). Due to the discontinuity of the Sentinel-2 time series during the extraction procedure, the corresponding MODIS values were chosen for comparison based on the accessibility of Sentinel-2. Both MODIS and Sentinel-2 successfully captured the peak periods of high algal outbreaks, demonstrating the effectiveness of these two satellites in monitoring algal blooms. However, some false positives in MODIS data were observed during non-outbreak periods. Nonetheless, there was strong trend consistency between the two datasets. Furthermore, the 9/19 2016 2017 2018 2019 2020 2021 2022 2023 0 2 4 6 8 10 (a) Sentinel Modis 2016 2017 2018 2019 2020 2021 2022 2023 0 2 4 6 8 10 Algal Bloom Area(km2) (b) Sentinel Modis 2021-01 2021-03 2021-05 2021-07 2021-09 2021-11 2022-01 0 2 4 6 8 10 (c) Sentinel Modis Figure 9. Comparison of FAI index calculated by MODIS and Sentinel-2. (a) is the result from the original data without any processing, (b) is the result that eliminated area less than 0.7km2. (c) is the result for 2021. Figure 9. Comparison of FAI index calculated by MODIS and Sentinel-2. (a) is the result from the original data without any processing, (b) is the result that eliminated area less than 0.7km2. (c) is the result for 2021. 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 Figure 10. Spatial frequency distribution of the algal bloom area in Jiaozhou Bay from 2000 to 2022 extracted by MODIS . Figure 10. Spatial frequency distribution of the algal bloom area in Jiaozhou Bay from 2000 to 2022 extracted by MODIS . 10/19 value of algal bloom area of the MODIS data was higher than that of Sentinel-2, which can be attributed to the difference in resolution and sensors used by the two satellites. Comparison of Algal Bloom Observation Areas Using Different Satellites Examining the available data to ascertain the relationsh Correlation Analysis Meteorological Factors 0 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 0.0 0.5 1.0 1.5 2.0 Area(km2) (b) 1000 1010 1020 1030 Air Pressure(hpa) 0 2 4 6 8 10 Month(2021) 0.00 0.25 0.50 0.75 1.00 1.25 Area(km2) (c) Area 1005 1010 1015 1020 1025 Air Pressure(hpa) Air Pressure 0.00097 0.00098 0.00099 0.00100 0.0 0.5 1.0 1.5 2.0 Algal Bloom Area(km2) (a) r = 0.55 N = 137 y=28839.611x-27.955 1:1 Lines 0.000 0.000 0.000 0.001 Reciprocal of Air Pressure(1/hpa) Reciprocal of Air Pressure(1/hpa) 5 10 15 20 25 SST(¤C) 0.0 0.5 1.0 1.5 2.0 Algal Bloom Area(km2) (d) r = 0.74 N = 137 y=0.0447x-0.1335 1:1 Lines 5 10 15 20 25 SST(¤C) 0.0 0 2 4 6 8 10 Month(2021) 0.00 0.25 0.50 Area Area 5 10 SS SST 0.1 0.2 0.3 0.4 0.5 Reciprocal of Wind Speed(s/m) 0.0 0.5 1.0 1.5 2.0 Algal bloom area(km2) (g) r = 0.43 N = 137 y=1.887x-0.2165 1:1 Lines 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 0.0 0.5 1.0 1.5 2.0 Area(km2) (h) 2 3 4 5 6 7 Wind Speed(m/s) 0 2 4 6 8 10 Month(2021) 0.00 0.25 0.50 0.75 1.00 1.25 Area(km2) (i) Area 2.5 3.0 3.5 4.0 4.5 5.0 Wind Speed(m/s) Wind Speed 0.1 0.2 0.3 0.4 0.5 Reciprocal of Wind Speed(s/ 0.0 0.5 1.0 1.5 2.0 Algal bloom area(km2) (g) r = 0.43 N = 137 y=1.887x-0.2165 1:1 Lines Figure 11. Analysis of the relationship between environmental factors and algal bloom outbreaks. (a)(d)(g) show scatter plots between the algal bloom area and air pressure(countdown), sea surface temperature(SST), and wind speed (countdown), respectively, and (b)(e)(h) show monthly average series of the algal bloom area and the air pressure, SST and the wind speed for November 2010-2022, and d (c)(f)(i) show monthly average for 2021. Utilizing MODIS data, monthly averages of algal bloom area were calculated. The meteorological data was then analyzed to determine the contributors to algal bloom outbreaks in Jiaozhou Bay. Examining the available data to ascertain the relationship 11/19 between these variables and the area of the algal bloom outbreak. The results show a weak negative correlation between wind speed and algal bloom area, which is consistent with previous ﬁndings (Figure 11)36. Comparison of Algal Bloom Observation Areas Using Different Satellites Simultaneously, our analysis reveals a signiﬁcant inverse relationship between air pressure and algal bloom outbreaks, as well as a signiﬁcant positive relationship between SST and algal bloom outbreaks. The coefﬁcients of correlation between environmental factors and algal bloom outbreaks were calculated (Table 1). These ﬁndings suggest that algal bloom outbreaks are more likely when wind speed is low, air pressure is low, and temperature is high. With these environmental factors in mind, appropriate measures to control algal bloom outbreaks can be taken. After analyzing the spatial distribution characteristics of the algal bloom area in Jiaozhou Bay, our ﬁndings indicate that the algal bloom pandemic is more severe in the bay’s coastal regions. The area of algal bloom decreases progressively from the coast to the bay’s inner waters, with the majority of outbreak areas concentrated in the coast, middle, and estuary. From 2000 to 2016, the outbreak of algal bloom in Jiaozhou Bay’s inner waters was not severe. However, beginning in 2017, the outbreak of algal bloom in the bay’s inner waters grew steadily worse until 2021, when it was ultimately mitigated. Table 1. Correlation coefﬁcients between the algal bloom area and three meteorological factors: air pressure, wind speed and SST. Air Pressure SST Wind Speed Pearson’s r2 0.30 0.55 0.18 ρ1 -0.58 0.8 -0.32 1 Here ρ represents the Spearman’s rank correlation coefﬁcient cients between the algal bloom area and three meteorological factors: air pressure, wind speed and . Correlation coefﬁcients between the algal bloom area and three meteorological factors: air pressur Seawater Quality Factors y In this study, seawater quality data collected in Jiaozhou Bay between 2014 and 2022 were used to investigate the relationship between water quality and algal bloom outbreaks in conjunction with algal bloom area extracted from MODIS and Sentinel-2 data. The MODIS satellite images in the period that covered the date of water quality sampling (5 to 7 days before and after sampling)were chosen for extracting the algal bloom areas, and then extracted areas were averaged in this time period for comparison analysis. As Figure 12 shows, there was an obvious correlation between the algal blooms area and petroleum pollutants. This could be attributed to the fact that petroleum hydrocarbons nourish algal organisms. In contrast, there was an inverse relationship between algal bloom area and dissolved oxygen (DO), which may be a result of oxygen depletion in the water column caused by algal growth. The previously described approach was not suitable for Sentinel-2 satellite data, due to its long sampling interval. Consequently, the monthly and annual averages of the algal bloom area derived from Sentinel-2 satellite data were utilized to analyze the correlation with the corresponding water quality data. The results (Figure 13) indicate a certain correlation between algal bloom area, DIN/DIP, and COD concentrations. The results of their regression analysis are presented in Table 2. Table 2. Regression results Signiﬁcant indicators Coefﬁcient Std. err. t P>\|t\| DIN/DIP 0.02 0.006 2.88 0.028 SST 0.06 0.005 12.47 0 Petroleum 97.9 30.7 3.18 0.005 DO -0.71 0.21 -3.33 0.004 Predicted Results Predicted Results Figure 14(a) demonstrates that the results from the DNN neural network model are in close agreement with the actual values, indicating the model’s robustness and consistent trend throughout. Due to the unavailability of meteorological data during those periods, some monthly area data could not be used in the forecast. However, since the prediction is based solely on meteorological factors, it is impossible to accurately foresee abrupt increases in algal bloom area due to other factors. By integrating the water quality composition of seawater, the model’s results can be made more accurate. After optimizing the model’s hyperparameters, its performance was assessed and summarized (Table 3). The ﬁnal test set has an R2 value of 0.65, indicating that the accuracy of the predictions is acceptable. Predicted Results Figure 14(a) demonstrates that the results from the DNN neural network model are in close agreement with the actual values, indicating the model’s robustness and consistent trend throughout. Due to the unavailability of meteorological data during those periods, some monthly area data could not be used in the forecast. However, since the prediction is based solely on meteorological factors, it is impossible to accurately foresee abrupt increases in algal bloom area due to other factors. By integrating the water quality composition of seawater, the model’s results can be made more accurate. After optimizing the model’s hyperparameters, its performance was assessed and summarized (Table 3). The ﬁnal test set has an R2 value of 0.65, indicating that the accuracy of the predictions is acceptable. 12/19 2015 2016 2017 2018 2019 2020 2021 2022 0 1 2 3 4 5 Algal Bloom Area(km2) (a) Area from Modis 5 6 7 8 9 DO(mg/L) Dissolved oxygen (DO) 2015 2016 2017 2018 2019 2020 2021 2022 0 1 2 3 4 5 Algal Bloom Area(km2) (b) 0.010 0.015 0.020 0.025 0.030 Petroleum(mg/L) Petroleum-based pollutants Figure 12. Time series of petroleum pollutants, DO and algal bloom area extracted from MODIS. Area from Modis Dissolved oxygen (DO) Petroleum-based pollutants rea from Modis Dissolved oxygen (DO) Petroleum-based pollutants Dissolved oxygen (DO) Area from Modis Figure 12. Predicted Results Time series of petroleum pollutants, DO and algal bloom area extracted from MODIS 2016 2017 2018 2019 2020 2021 2022 0.1 0.2 0.3 0.4 0.5 0.6 Algal Bloom Area(km2) (a) Annual average area form Sentinel 5 10 15 20 25 30 DIN/DIP DIN/DIP 2016 2017 2018 2019 2020 2021 2022 0.0 0.5 1.0 1.5 Algal Bloom Area(km2) (b) Area form Sentinel 0.8 1.0 1.2 1.4 1.6 COD(mg/L) COD Figure 13. Yearly time series of DIN/DIP, COD and algal bloom area extracted from Sentinel-2 data. 5 6 Annual average area form Sentinel Figure 13. Yearly time series of DIN/DIP, COD and algal bloom area extracted from Sentinel-2 data. Table 3. DNN model evaluation. MSE RMSE MAE MAPE R2 Training Sets 0.097 0.311 0.219 66.763 0.541 Cross-validation Sets 0.105 0.314 0.231 151.714 0.467 Test Sets 0.055 0.234 0.183 65.706 0.651 Table 3. DNN model evaluation. Figure 14. Prediction of algal bloom area by DNN (a) and SARIMA (b). Figure 14. Prediction of algal bloom area by DNN (a) and SARIMA (b). According to the predicted results of the SARIMA model (Figure 14(b)), the outbreak of algal bloom is extremely seasonal. On the one hand, it is due to the cyclical changes in temperature accompanying the seasons, and on the other hand, it is considered that in summer, the peak of this ﬁshing season leading to the disruption of the nitrogen-phosphorus ratio balance in the water column. Since algal blooms have a strong correlation with temperature, nitrogen and phosphorus ratios, this leads to seasonal outbreaks of algal blooms. We observe an upward trend in algal bloom outbreaks, with the second and third quarters exhibiting substantially stronger outbreaks than the ﬁrst and fourth quarters. We utilized an additive model of seasonal decomposition to quantify the impact of seasonal factors on algal bloom outbreaks. Table 4. Seasonal factors for the variation of algal bloom area Table 4. Seasonal factors for the variation of algal bloom area Month 1 2 3 4 5 6 7 8 9 10 11 12 SF∗ -0.22 -0.29 -0.30 -0.26 0.03 0.12 0.30 0.39 0.40 0.03 -0.09 -0.11 The Seasonal Factor Table 4. Seasonal factors for the variation of algal bloom area The seasonal factors are positive for the months of May to October and negative for the months of January to April and November to December, according to the results shown in Table 4. Predicted Results This suggests that algal bloom outbreaks are more severe in the second and third quarters than in the ﬁrst and fourth. The most intense outbreak occurred in September, with an area larger than the annual average of 0.398 km2, while the smallest occurred in March, with an area smaller than the annual average of 0.298 km2. We removed outliers from two decades of historical algal bloom outbreak data before iteratively estimating the parameters to obtain an iterative expression for the time series (2). All signiﬁcance levels are less than 5% based on the estimation results (Table 5), indicating a good ﬁt. yt = 0.015+yt−12 +εt −0.217εt−1 +0.689εt−12 −0.150εt−13 (3) (3) yt = 0.015+yt−12 +εt −0.217εt−1 +0.689εt−12 −0.150εt−13 where yt and yt−12 are the actual observed area of algal blooms in periods t and t-12, respectively. εi is the white noise series with variance σ2, which represents the difference between the predicted value and the observed value in period i. In this equation i takes t,t-1,t-12,t-13. where yt and yt−12 are the actual observed area of algal blooms in periods t and t-12, respectively. εi is the white noise series with variance σ2, which represents the difference between the predicted value and the observed value in period i. In this equation i takes t,t-1,t-12,t-13. Discussion Nevertheless, despite our best efforts to modify the threshold value, non-algal material may still be involved, resulting in a high value of algal bloom area. Although red and green light band of MODIS have some degree of accuracy, NIR and blue light have a high degree of uncertainty and may not be suitable for monitoring algal blooms at a large scale37. In contrast, the 10-meter resolution Sentinel satellite is unstable during the extraction procedure. Using the FAI threshold extraction method, the algal bloom region can be extracted from certain images with relatively high precision. However, because of its long resampling interval, it is unsuitable for analyzing the algal bloom’s area changes. In addition, its higher resolution makes it susceptible to the inﬂuence of coastal infrastructure on ocean color, resulting in erroneous estimates of the extracted algal bloom area38. 0 2 4 6 8 10 12 Bloom Area derived from Sentinel-2 (km2) 0 2 4 6 8 10 12 Bloom Area derived from MODIS (km2) N = 35 RMSE = 1.1km2 R2 = 0.67 y=1.39x-0.37 1:1 Lines Figure 15. Comparison of algal bloom area derived from MODIS and Sentinel-2 data. Figure 15. Comparison of algal bloom area derived from MODIS and Sentinel-2 data. The algal bloom data extracted from the MODIS dataset were analyzed in this study using the average of observed non-zero data covering the time of water quality observation (within the range of 5 to 7 days before and after the observation time). This was due to the time dispersion in water quality data measurements, which were conducted three to four times a year. Because of their temporal inconsistency, using the monthly average of algal bloom area to correlate with the monthly average of water quality data may result in errors. While it would be ideal to use the one-day resampling nature of the MODIS satellite to match the precise date of water quality data collection, the de-cloud processing for the MODIS satellite dataset makes it impossible to make the period of satellite observation coincide precisely with the period of water quality monitoring. As a result, the average value of the observed water bloom area before and after 5–7 days of the water quality monitoring date is used as the data corresponding to the water quality monitoring data, so that the observation times of the two sets of data correspond as closely as feasible. Discussion For comparative analysis, we obtained MODIS datasets corresponding to the Sentinel-2 data. To reduce the impact of false positives during the extraction process, we excluded data with minor values based on predetermined thresholds and only For comparative analysis, we obtained MODIS datasets corresponding to the Sentinel-2 data. To reduce the impact of false positives during the extraction process, we excluded data with minor values based on predetermined thresholds and only 14/19 Table 5. Evaluation of estimation results Table 5. Evaluation of estimation results Parameter Estimation Result Standard Error t Signiﬁcance α0 * 0.015 0.004 3.28 0.001 θ * -0.22 0.06 -3.46 0.001 Θ * 0.69 0.05 12.66 0 * α0,θ,Θ are estimates of the parameters to be estimated in Equation 2 retained data collected during the period of algal bloom. The data was then ﬁltered to yield 35 corresponding data points. We calculated an R2 value of 0.67 after analyzing the correlation between the two datasets using scatter plots (Figure 15). Due to the 500m resolution of the MODIS satellite’s observational pixel, the vast majority of algal bloom could be included, even at the boundary. Nevertheless, despite our best efforts to modify the threshold value, non-algal material may still be involved, resulting in a high value of algal bloom area. Although red and green light band of MODIS have some degree of accuracy, NIR and blue light have a high degree of uncertainty and may not be suitable for monitoring algal blooms at a large scale37. In contrast, the 10-meter resolution Sentinel satellite is unstable during the extraction procedure. Using the FAI threshold extraction method, the algal bloom region can be extracted from certain images with relatively high precision. However, because of its long resampling interval, it is unsuitable for analyzing the algal bloom’s area changes. In addition, its higher resolution makes it susceptible to the inﬂuence of coastal infrastructure on ocean color, resulting in erroneous estimates of the extracted algal bloom area38. retained data collected during the period of algal bloom. The data was then ﬁltered to yield 35 corresponding data points. We calculated an R2 value of 0.67 after analyzing the correlation between the two datasets using scatter plots (Figure 15). Due to the 500m resolution of the MODIS satellite’s observational pixel, the vast majority of algal bloom could be included, even at the boundary. Discussion The 5-day resampling time for the Sentinel-2 satellite emphasizes the discontinuity of the data following de-clouding processing. As a result, the analysis can only be performed using the monthly averaged water bloom area value based on Sentinel-2 observations compared to monthly water quality observation data, resulting in some error creation. In 15/19 addition, it is optimal to collect water quality data before and after the algal bloom separately for comparative analysis in order to investigate the cause of algal blooms with precision. The inconsistency between satellite sampling and water quality monitoring leads to a lack of correlation between the two sets of data, which can also result in a degree of analytical error. Algal blooms are caused by a combination of their own physiological mechanisms and environmental factors such as water temperature, wind speed, atmospheric pressure, and the availability of nutrients. Nitrogen and phosphorus have been identiﬁed as signiﬁcant contributors to algal bloom outbreaks39. Research indicates that silicate and phosphorus are the primary limiting factors for planktonic algae expansion in the Jiaozhou Bay region40. In terms of nutrient sources, it has been found that the majority of Jiaozhou Bay’s total DIN and DIP discharges come from land-based sources, accounting for 93% and 98%, respectively41,42.Land-based pollutants enter Jiaozhou Bay via the inlet rivers, the majority of which are presently ﬂow-interrupted. Four tributary rivers, namely the Licun River, the Dagu River, the Ink River, and the Haibe River, were chosen for analysis in 2014 and 2015, when they had water ﬂow. The ratios of various nutrients were determined by analyzing the data on the total nitrogen, total phosphorus, and silicate content of the four rivers at their entrances to Jiaozhou Bay. The N/P ratio was 25.8±17.1, the N/Si ratio was 3.35±1.35, and the Si/P ratio was 8.9±6.4. It is evident that the N/Si values tend to remain stable in Jiaozhou Bay, while the ﬂuctuation of N/P ratio is the highest. Furthermore, P has a more signiﬁcant impact on the growth of planktonic algae than Si in Jiaozhou Bay. Some studies have also indicated that the Jiaozhou Bay area has been transformed into a "phosphorus-limited" environment43,44, which is consistent with the ﬁndings of the present study. Researchers investigating 17 lakes around the world have shown that the lower the N/P ratio, the better the algal growth45, but some studies have also shown that algal outbreaks occur when the N/P ratio is high, i.e. Discussion the lower the N/P ratio is a result of the outbreak rather than the cause of the outbreak46. It should be noted that The N and P needs of algae vary depending on the environment and the species. The analysis of this study concluded that there is a positive correlation between the N/P ratio and the area of algal blooms. The results also demonstrated that the N/Si value tends to be stable, indicating that the algae in Jiaozhou Bay are highly sensitive to changes in phosphorus levels. The highest value of observed DIN/DIP reached 387, and the N/P ratio at several observation sites was greater than 100 in summer, exceeding the Redﬁeld ratio47. However, the ratio decreased in winter. For example, in 2019, during a severe algal bloom, the average N/P ratio in August was 62, while the average in October was 14.9, approaching the Redﬁeld ratio. These ﬁndings suggest that sudden increases in phosphorus content due to external factors could trigger large outbreaks of algal blooms, leading to a decrease in the nitrogen-phosphorus ratio. In this study, a positive correlation was observed between the area of algal bloom and COD content. This can be attributed to the release of organic matter by the algae during their growth cycle, particularly during the normal growth and metabolism stages when extracellular organic matter (EOM) is released into the water column, leading to a signiﬁcant increase in COD content. Other research48 conﬁrms this idea, indicating that the abrupt spike in COD concentration is the result of an algal bloom epidemic. However, due to the inﬂuence of the long resampling interval for Sentinel-2 observation and nutrient concentration measurement , monthly average data was used for the analysis, and thus the lag in COD content was not very clear. Nevertheless, the study still demonstrates a correlation between COD and the area of algal bloom. Conclusion It is essential to examine the long-term time series of algal blooms in order to comprehend the ecological changes in Jiaozhou Bay and to mitigate the risks posed by algal blooms. Using MODIS and Sentinel-2 satellite datasets, this study observed the spatial and temporal variations of algal blooms from 2000 to 2022, analyzed the factors driving the outbreak of algal blooms in the Jiaozhou Bay region, and predicted the changes of algal blooms in the region using deep learning and SARIMA models. The study found that the incidence of algal blooms in Jiaozhou Bay was comparatively stable from 2000 to 2016, but has increased since 2017, especially during the summer, indicating a more pronounced seasonality. The algal bloom outbreak areas were primarily concentrated along the bay’s coast, in the midsection of Jiaozhou Bay, and at the estuary of the bay. Meteorological factors such as sea surface temperature, wind speed, and air pressure, as well as water quality factors such as dissolved oxygen, nitrogen to phosphorus ratio, chemical oxygen demand, and petroleum pollutants, have been identiﬁed as important drivers of algal bloom outbreaks. 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Xu, X. et al. Study on the relationship and cause of blue-green algae metabolism and chemical oxygen demand in dianchi(in chinese). Environ. Sci. 36, 1685–1691 (2015). 48. Xu, X. et al. Study on the relationship and cause of blue-green algae metabolism and chemical oxygen demand in dianchi(in chinese). Environ. Sci. 36, 1685–1691 (2015). Acknowledgements The authors thank the Google Earth Engine cloud platform for providing Sentinel-2 and MODIS satellite data and the cloud computing platform. We also thank the National Ocean Data Observation Centre for providing meteorological data, and the National Field Scientiﬁc Observation and Research Station for Marine Ecosystems in Jiaozhou Bay, Shandong Province for providing water quality monitoring data. This research was funded by National Training Program of Innovation and Entrepreneurship for Undergraduates (grant number 202210359109,202110359102) and Natural Science Foundation of Anhui Province (grant number 2208085US15). Data Availability The water quality monitoring datasets of Jiaozhou Bay used during the current study are not publicly available due to the provision of data provider (Jiaozhou Bay Marine Ecosystem Research Station, Chinese Ecosystem Research Network) but are available from the corresponding author on reasonable request. Author contributions statement Conceptualization, G.B and N.S.; Methodology, N.S.; Software, D.X. and K.D.; Data Curation, Y.htbp.; Writing-original draft preparation, G.B., D.X. and K.D.; Writing-review and editing, N.S.;Supervision, S.J.. All authors have read and agreed to the published version of this manuscript. 19/19
https://openalex.org/W2996368815	https://scholarworks.utrgv.edu/cgi/viewcontent.cgi?article=1151&context=som_pub	English	null	Diet-induced leukocyte telomere shortening in a baboon model for early stage atherosclerosis	Scientific reports	2,019	cc-by	10,134	Diet-induced leukocyte telomere shortening in a baboon model for Diet-induced leukocyte telomere shortening in a baboon model for early stage atherosclerosis early stage atherosclerosis Follow this and additional works at: https://scholarworks.utrgv.edu/som_pub Part of the Diseases Commons Part of the Diseases Commons University of Texas Rio Grande Valley University of Texas Rio Grande Valley ScholarWorks @ UTRGV ScholarWorks @ UTRGV School of Medicine Publications and Presentations School of Medicine 12-12-2019 Diet-induced leukocyte telomere shortening in a baboon model for Diet-induced leukocyte telomere shortening in a baboon model for early stage atherosclerosis early stage atherosclerosis Genesio M. Karere Michael C. Mahaney The University of Texas Rio Grande Valley Deborah E. Newman Angelica M. Riojas Clint Christensen See next page for additional authors University of Texas Rio Grande Valley University of Texas Rio Grande Valley ScholarWorks @ UTRGV ScholarWorks @ UTRGV University of Texas Rio Grande Valley University of Texas Rio Grande Valley ScholarWorks @ UTRGV ScholarWorks @ UTRGV School of Medicine Publications and Presentations School of Medicine Recommended Citation Recommended Citation Karere, G.M., Mahaney, M.C., Newman, D.E. et al. Diet-induced leukocyte telomere shortening in a baboon model for early stage atherosclerosis. Sci Rep 9, 19001 (2019). https://doi.org/10.1038/ s41598-019-55348- This Article is brought to you for free and open access by the School of Medicine at ScholarWorks @ UTRGV. It has been accepted for inclusion in School of Medicine Publications and Presentations by an authorized administrator of ScholarWorks @ UTRGV. For more information, please contact justin.white@utrgv.edu, william.flores01@utrgv.edu. Genesio M. Karere, Michael C. Mahaney, Deborah E. Newman, Angelica M. Riojas, Clint Christensen, Shifra Birnbaum, John L. VandeBerg, and Laura Cox Authors Authors This article is available at ScholarWorks @ UTRGV: https://scholarworks.utrgv.edu/som_pub/152 www.nature.com/scientificreports OPEN y g Genesio M. Karere 1,4, Michael C. Mahaney2, Deborah E. Newman3,4, Angelica M. Riojas1 Clint Christensen4, Shifra Birnbaum4, John L. VandeBerg2 & Laura Cox1,3,4 Reported associations between leukocyte telomere length (LTL) attrition, diet and cardiovascular disease (CVD) are inconsistent. This study explores effects of prolonged exposure to a high cholesterol high fat (HCHF) diet on LTL in a baboon model of atherosclerosis. We measured LTL by qPCR in pedigreed baboons fed a chow (n = 105) or HCHF (n = 106) diet for 2 years, tested for effects of diet on LTL, and association between CVD risk factors and atherosclerotic lesions with LTL. Though not different at baseline, after 2 years median LTL is shorter in HCHF fed baboons (P < 0.0001). Diet predicts sex- and age-adjusted LTL and LTL attrition (P = 0.0009 and 0.0156, respectively). Serum concentrations of CVD biomarkers are associated with LTL at the 2-year endpoint and LTL accounts approximately 6% of the variance in aortic lesions (P = 0.04). Although heritable at baseline (h2 = 0.27, P = 0.027) and after 2 years (h2 = 0.46, P = 0.0038), baseline LTL does not predict lesion extent after 2 years. Atherogenic diet influences LTL, and LTL is a potential biomarker for early atherosclerosis. Prolonged exposure to an atherogenic diet decreases LTL and increases LTL attrition, and shortened LTL is associated with early-stage atherosclerosis in pedigreed baboons. Telomeres are non-coding DNA sequence repeats at the ends of eukaryotic chromosomes (e.g., TTAGGG in ver- tebrates); they are involved in maintaining genetic stability and integrity by providing protection from damage and fusion. In dividing somatic cells, telomeric regions, like other chromosomal regions, are replicated by DNA polymerase. However, DNA polymerase cannot fully replicate the 3′ end of linear DNA molecules, resulting in progressive shortening of telomeres with repeated cell division. In vertebrates, the telomerase enzyme complex adds DNA to the ends of chromosomes, but is typically only active in certain types of cells, including stem cells, germline cells, granulosa cells, early embryos1. In differentiated cells, telomere attrition typically leads to senes- cence or programmed cell death when mean telomere length reaches a critical value2,3. Over the past 2 decades, recognition of telomere dynamics and attrition as fundamental features of cellular senescence has motivated exten- sive research into their causal roles and potential utility as biomarkers and therapeutic targets for specific cancers4. Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 Materials and Methods d bj d Study subjects and treatment. We used blood samples drawn from live baboons for DNA isolation. All health care, maintenance, and research procedures involving the baboons (Papio hamadryas) in this study were managed by the veterinary resources staff of the Southwest National Primate Research Center (SNPRC), Texas Biomedical Research Institute (Texas Biomed), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All research procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Texas Biomed. All experiments were performed in accord- ance with relevant guidelines and regulations. g g Baboons from which data were collected for this study were members of a large, six-generation pedigreed breeding colony developed and maintained at SNPRC. The study described in this paper utilizes data from 2 groups of baboons distinguished by diet. Baboons in the control group were fed a baseline diet (chow), low in cholesterol (0.021 mg/kcal) and fat (10% kcal), throughout the study. Baboons in the experimental diet group were fed the chow diet prior to beginning a 2-year dietary challenge during which they were fed a diet high in cholesterol (approximately 1.865 mg/kcal) and fat (40% kcal; HCHF). The diets used in this study have been described in more detail elsewhere19. All baboons in this study were fed ad libitum. In summary, the control group was fed chow (LCLF) diet for 2 years while the experimental group was fed HCHF diet for the same duration.h The mean age of animals in diet challenged group (n = 106; 46 females, 60 males) was 10.8 years, with ages ranging from approximately 6 to 17 years. For the control group, we selected age-sex matched adult baboons (n = 106; 47 females, 58 males) from the colony; mean age = 10.9 years and an age range from approximately 6 to 17 years. Blood collection. Blood samples (10 ml) were collected through femoral artery venipuncture into EDTA-containing tubes and processed using standard procedures; buffy coats (leukocytes) were stored at −80 °C. For the experimental diet group, samples were collected and processed at 3 time-points: 1–2 weeks prior to the beginning of the 2-year HCHF diet challenge (while on chow), 7 weeks, and at the end of 2-year period during which they were fed the HCHF diet. For the control group, samples were collected at 2 time-points inter-spaced by 2 years. DNA isolation. www.nature.com/scientificreports/ environmental factors that increase systemic inflammation, oxidative stress and cellular aging. Research results consistent with each of these hypotheses may be found readily in the epidemiological literature.i yp y y p g While some studies in human cohorts and families provide evidence that a biologically significant proportion of the inter-individual variance in mean telomere length is attributable to genetic effects8–10, there also is evidence that environmental factors, including lifestyle variables which are known to affect atherosclerosis risk, can affect leukocyte telomere length (LTL) as well11. Many of these effects on both LTL and atherosclerosis are mediated or hypothesized to be mediated, by, systemic oxidative stress and inflammation12. In that case, shortened telomeres, are potentially useful as biomarkers for the cumulative burden imposed by oxidative stress and inflammation on the vasculature – a burden which may result in accelerated atherosclerosis. Key among lifestyle factors that are known to influence systemic inflammation and oxidative stress is diet composition13. While the results of all stud- ies are not in complete concordance, a few cross-sectional human studies suggest that some putative atherogenic dietary components, particularly saturated fats, as well as cholesterol and other dietary risk factors for cardiomet- abolic disease, also may contribute to LTL attrition15–17. y Previously, we have shown that prolonged (2-year) exposure to a diet high in cholesterol and fat increases circulating concentrations and/or activity of intrinsic atherosclerosis risk factors, and reliably induces early-stage atherosclerosis in pedigreed baboons from a single, large, six-generation pedigree, which has been extensively characterized at multiple levels of biological organization14,18. Here, we take advantage of biomaterials obtained from those same baboons during that study to test the hypothesis that exposure to that atherogenic diet decreases LTL and increases LTL attrition independent of the effects of age and sex. We also test for associations of LTL and LTL attrition with circulating atherosclerosis risk factors previously found to predict extent of atherosclerotic lesions in these animals, as well with the extent of lesions themselves. OPEN p y p g pi A large number of epidemiological reports also implicate shorter telomeres as risk factors for many age-related pathologies, including but not limited to components of metabolic disease – i.e., type-2 diabetes5, obesity6, and cardiovascular disease (CVD)7. More specifically, shorter telomere length has been associated with coronary artery disease, as well as atherosclerosis, its consequent complications, disease endpoints, e.g., stroke, and subse- quent mortality8–14.h The work reported in this paper focuses on the relationship between telomere length and atherosclerosis. A number of mechanisms have been advance to explain the observed associations with atherosclerosis. An oversim- plification would organize them into two very general categories. In one, critically shortened telomeres, regard- less of when or how established, accelerate disease progression and development of more widely disseminated, later-stage atherosclerosis, as well as consequent CVD through effects on numbers and function of, for example, endothelial progenitor cells needed to effect vascular repair and plaque stabilization7. The other explains the cor- relation between telomere shortening and atherosclerosis as just that: correlated responses to shared exposures to 1Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA. 2South Texas Diabetes and Obesity Institute and Department of Human Genetics, University of Texas Rio Grande Valley School of Medicine, Brownsville, Texas, USA. 3Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, Texas, USA. 4Department of Genetics, Texas Biomedical Research Institute, San Antonio, Texas, USA. email: gkarere@wakehealth.edu Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ p p g All reactions were performed in triplicate, and the cycling profile was Stage 1: 95 °C for 15 min; Stage 2: 2 cycles at 94 °C for 15 s, 49 °C for 15 s; and Stage 3: 32 cycles at 94 °C for 15 s, 62 °C for 10 s, 74 °C for 15 s, 84 °C for 10 s and 88 °C for 15 s. Signals for Telomere repeat Ct values were acquired at 74 °C, and 88 °C for LIPG Ct values. Measurement of telomere length. After thermal cycling, we used MyiQ software (Bio-Rad iQ5 2.0 Standard Edition Optical System Software) to generate 2 adjusted standard curves per plate from which telomere length and LIPG copy number (Fig. 1a,b). The MyiQ software generated Ct values matching the DNA content of telomere repeats and LIPG for each of the five standard DNA concentrations. For experimental samples, we used BioRad CFX Manager software together with standard curves to estimate the amount of the standard DNA that matched the experimental sample for the copy number of the telomere (T) and LIPG copy number (S) per sample. We report the mean telomere length of experimental sample/Telomere length of reference sample (T/S) ratio, which is proportional to the mean telomere length per sample. CVD related biomarkers. In this study we used data on 13 circulating risk factors which we earlier had reported were significantly correlated with extent of atherosclerotic lesions following the 2-year HCHF diet chal- lenge. Assay methods are described in detail elsewhere21. Data obtained from samples collected at baseline and baseline plus 7 weeks into the challenge were analyzed. Atherosclerotic lesion assessments. The approach used to assess lesion development in 3 major arter- ies, the aortic arch, thoracic section of the descending aorta, and the common iliac artery has been described in detail elsewhere21,22. Briefly, baboons were humanely euthanized after 2 years on the HCHF diet and the 3 arterial sections were harvested in the course of standard necropsy procedures. Following dissection, preparation, and application of a lipophilic stain, lesions (fatty streaks and/or raised fibrous plaques) were visually identified and photographed, and the photographs were imported into an image analysis system and percent area covered by lesions was quantified as previously described23. Note: Raised lesions most resembled AHA lesion Types Va or Vc24. Analytical methods. Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 Materials and Methods d bj d We isolated DNA from 100ul of leukocytes using phenol-chloroform method. Briefly, we mixed cells with lysis buffer and proteinase K and incubated overnight at 55 °C. We transferred lysate to a Phase Lock Gel (PLG) tube (Eppendorf) and centrifuged at 1,500 × g for 2 min at room temperature. Organic extraction was performed twice using phenol, and then with an equal volume of chloroform:isoamyl alcohol (24:1). After the mixture was centrifuged at 1,500 × g for 6 min, the aqueous layer was transferred to a 2 ml tube. To precipi- tate DNA, we mixed the aqueous layer with 1 volume of 3 M NaOAc and 2.5 volumes of cold 100% ethanol, and inverted the tube 6–8 times. The precipitated DNA was transferred to a new tube containing 70% ethanol, centri- fuged for 10 min at 10,000 rpm, and then ethanol was aspirated. The DNA pellet was air-dried, re-suspended in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5) and stored at −80 °C. QPCR multiplexing. Relative telomere length was determined using Bio-Rad MyiQ Single Color Real-Time PCR Detection System. DNA samples from diet challenged and control groups for time-points 0 and 2 years were multiplexed side by side in the same 96-well plate to measure the amount of telomere repeats and of a single copy gene (baboon Endothelial lipase; LIPG). Each PCR reaction (20ul) contained 25 ng of DNA, 1X Platinum SYBR qPCR SuperMix-UDG with ROX (MM) and 200 nM of each primer pair for telomere and LIPG. Telomere primer sequences were telg (ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAG. TGT) and talc (TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA). LIPG primer sequences were forward (CGGCGGCGGGCGGCGCGGGCTGGGCGGCACTGACTCCAATCGCTTCA) and reverse (GCCCGGCCCGCCGCGCCCGTCCCGCCGATTACAACGGTTCTTGCGGC). The telomere primers were designed such that the telomere amplicon melted at a lower temperature than the LIPG amplicon, and generate a Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Adjusted standard curves for measurements of (left) baboon leukocyte telomere length, (right) single copy gene (baboon endothelial lipase, LIPG). Figure 1. Adjusted standard curves for measurements of (left) baboon leukocyte telomere length, (right) single copy gene (baboon endothelial lipase, LIPG). single fixed-length PCR product (79 bp) as described20. In addition, each plate contained 81-fold serial dilutions comprising five concentrations of a reference (standard) DNA that ranged from 2.22 to 180 ng together with 1X MM and 200 nM of either telomere or LIPG primer pair for generation of telomere and LIPG standard curves. All reactions were performed in triplicate, and the cycling profile was Stage 1: 95 °C for 15 min; Stage 2: 2 cycles at 94 °C for 15 s, 49 °C for 15 s; and Stage 3: 32 cycles at 94 °C for 15 s, 62 °C for 10 s, 74 °C for 15 s, 84 °C for d f l f T l l d d f l single fixed-length PCR product (79 bp) as described20. In addition, each plate contained 81-fold serial dilutions comprising five concentrations of a reference (standard) DNA that ranged from 2.22 to 180 ng together with 1X MM and 200 nM of either telomere or LIPG primer pair for generation of telomere and LIPG standard curves. All ti f d i t i li t d th li fil St 1 95°C f 15 i St 2 2 gi g comprising five concentrations of a reference (standard) DNA that ranged from 2.22 to 180 ng together with 1X MM and 200 nM of either telomere or LIPG primer pair for generation of telomere and LIPG standard curves. All reactions were performed in triplicate, and the cycling profile was Stage 1: 95 °C for 15 min; Stage 2: 2 cycles at 94 °C for 15 s, 49 °C for 15 s; and Stage 3: 32 cycles at 94 °C for 15 s, 62 °C for 10 s, 74 °C for 15 s, 84 °C for 10 s and 88 °C for 15 s. Signals for Telomere repeat Ct values were acquired at 74 °C, and 88 °C for LIPG Ct values. Results i Descriptive statistics and initial impressions. Baseline. Initial examination and analyses of raw (untransformed) data (Table 1) shows the 2 cohorts to be well matched on LTL prior to the 2-year diet chal- lenge when both were consuming the LCLF diet. Median LTL in the 2 cohorts is not different (sexes combined: P = 0.8204; females: P = 0.5548; males: P = 0.4256). Between cohorts. For both cohorts, the within-cohort com- parisons of the sexes indicate that the median LTL in female baboons is greater than that in males (control: P = 0.0094; challenge: P = 0.0076). Baseline plus 2 years. When compared to those at baseline, observations made at time point 2 suggest an effect of the HCHF diet. The difference between median LTL at the 2 time points was not significant in the con- trol cohort, which had consumed the LCLF diet for 2 years (combined sexes: P = 0.4057; females: P = 0.1042; males: P = 0.7220); but it was in the cohort that had consumed the atherogenic HCHF diet (combined sexes: P = 0.0000007; females: P = 0.0076; males: P = 0.0008). Median LTL in the control cohort was longer than that in the experimental cohort (sexes combined: P = 0.0005). p At this time point, the results of within-cohort comparisons of the sexes are similar to those at baseline. However, while median LTL in females is absolutely greater than that in males in both cohorts, that difference is statistically significant only in the experimental cohort (control: P = 0.2132; experimental: P = 0.0069). Sex and age effects on inter-individual variation in LTL: Separate cohorts. We maximized a model with sex and age terms on the i-normalized LTL data for each of the cohorts separately at each time point to estimate and test the significance of mean effects that might underly the observations above. Baseline. Sex, age, and an age-by-sex interaction all exerted significant mean effects on LTL in the control cohort (P = 0.004; P = 0.00002; and 0.026, respectively). In the experimental cohort, the mean effects of sex and age were significant (P = 0.028; P = 0.025) but no age-by-sex interaction was detected. In both cohorts the signs of the mean effects were as expected: sex (female) and age were, respectively, positively and negatively correlated with LTL. Baseline plus 2 years. www.nature.com/scientificreports/ www.nature.com/scientificreports/ T/S Ratio Control Cohort Baseline (chow diet) Baseline + 2 Years (chow diet) Females Males Total Females Males Total Mean 1.07 0.97 1.01 0.97 0.91 0.94 Median 0.96 0.87 0.91 0.91 0.89 0.90 SD 0.27 0.23 0.25 0.21 0.12 0.17 Minimum 0.78 0.73 0.73 0.77 0.74 0.74 Maximum 1.59 1.61 1.61 1.75 1.56 1.75 Range 0.81 0.88 0.88 0.98 0.82 0.91 Experimental Cohort Mean 0.97 0.91 0.94 0.89 0.86 0.87 Median 0.97 0.90 0.92 0.89 0.84 0.86 SD 0.13 0.09 0.12 0.08 0.08 0.08 Minimum 0.78 0.77 0.77 0.74 0.74 0.74 Maximum 1.47 1.31 1.47 1.15 1.16 1.16 Range 0.69 0.54 0.70 0.41 0.42 0.42 Table 1. Telomere-to-Single Copy Gene (T/S) Ratios in Pedigreed Baboons: Descriptive Statistics. T/S Ratio Control Cohort Baseline (chow diet) Baseline + 2 Years (chow diet) Females Males Total Females Males Total Mean 1.07 0.97 1.01 0.97 0.91 0.94 Median 0.96 0.87 0.91 0.91 0.89 0.90 SD 0.27 0.23 0.25 0.21 0.12 0.17 Minimum 0.78 0.73 0.73 0.77 0.74 0.74 Maximum 1.59 1.61 1.61 1.75 1.56 1.75 Range 0.81 0.88 0.88 0.98 0.82 0.91 Experimental Cohort Mean 0.97 0.91 0.94 0.89 0.86 0.87 Median 0.97 0.90 0.92 0.89 0.84 0.86 SD 0.13 0.09 0.12 0.08 0.08 0.08 Minimum 0.78 0.77 0.77 0.74 0.74 0.74 Maximum 1.47 1.31 1.47 1.15 1.16 1.16 Range 0.69 0.54 0.70 0.41 0.42 0.42 Table 1. Telomere-to-Single Copy Gene (T/S) Ratios in Pedigreed Baboons: Descriptive Statistics. Table 1. Telomere-to-Single Copy Gene (T/S) Ratios in Pedigreed Baboons: Descriptive Statistics. www.nature.com/scientificreports/ Summaries of raw data are presented by cohort, time-point, and sex as means, medi- ans, standard deviations, and ranges. The data are in a sense derived “opportunistically” for this particular study. That is, the study is not one in which cases and controls could be selected based on baseline LTL and matched for sex and age. Further, normality of distributions could not be guaranteed. As some departures from univariate normality (e.g., skew) affect the mean more than the median, we instead use the latter as the measure of central tendency in our initial comparisons of raw data. We employ a Wilcoxon-Mann-Whitney U test of medians that implements an exact permutation approach which is robust to the presence of outliers25. Further, because the data come from related animals (from the large, six-generation pedigree alluded to ear- lier), assumptions of independence of observations on which many statistical tests rely also cannot be guaranteed (note: mean kinship coefficient between all pairs of animals within each of the two cohorts is approximately 0.14, and for the combined cohort, 0.17 – i.e., between half and full siblings). To address this potential bias, in all remaining analyses we utilize a maximum likelihood-based variance decomposition approach (SOLAR26) which accounts for kinship in data from pedigrees of arbitrary size and complexity. To address possible departures from multivariate normality, data analyzed using this approach are i-normalized quantile scores (i.e., inverse Gaussian normalization), the distribution of which are symmetric about the mean and median. We use this approach to decompose the phenotypic covariance among related animals into genetic and environmental components and then model the phenotype of an individual as a general linear function of the trait, its mean, covariates and their regression coefficients, plus additive genetic values and non-genetic deviations. Here we test for effects of covari- ates on the phenotype by comparing the maximum likelihood of a model in which the mean effect is estimated to that of a model where that covariate’s effect is constrained to equal zero (the null model). Results i The estimated heritability also was significant (h2 = 0.46 ± 0.24). Diet effects on change in LTL. As should be expected from the results of our comparisons of median LTL in the 2 cohorts at time point 2 (above), change in LTL (ΔLTL), or telomere attrition itself, is greater in animals who consumed the HCHF diet for 2 years than in the controls who ate the LCLF diet during the same interval (P = 0.006). These analyses of the raw data disclose no significant between-sex difference in ΔLTL within either of the cohorts (control: P = 0.3681; experimental: P = 0.1554). p We used the approach described above in 2 series of analyses of ΔLTL: one with data from the separate cohorts and the other with data from the cohorts combined. The first series of ΔLTL was calculated as the dif- ference between the i-normalized in raw LTL from the individual cohorts and the second from the cohorts com- bined. 1) Separate cohorts. Our analysis of the control cohort data finds a significant mean positive effect of age in the control cohort (P = 0.003); that is, ΔLTL was greater in older control baboons. This effect accounts for approximately 20% of the variance in ΔLTL in that cohort. Analysis of data from the experimental cohort finds no significant evidence for a significant mean effect of sex or age on ΔLTL. 2) Combined cohorts. To test for an effect of diet on change in LTL within this same framework, we calculated ΔLTL as the difference between the i-normalized sex- and age-adjusted residuals for LTL at the 2 time points. As inferred from comparisons of median ΔLTL in the preceding analyses (above), the mean effect of diet on ΔLTL is positive; i.e., ΔLTL is greater in animals fed the HCHF diet for approximately 2-years (P = 0.0156). We observed no evidence of a significant additive genetic effect (heritability) on ΔLTL in either of the cohorts. Biomarkers of cardiovascular disease risk and LTL. Results from analyses of well-recognized CVD risk factors, 13 circulating biomarkers of lipoprotein metabolism, oxidative stress, and inflammation assayed in samples collected at 3 time-points, i.e., time-point 0, 0 + 7 weeks, and 0 + 2 years are presented in Table 3. Results i MLE ± s.e.: maximum likelihood estimates of parameters and their standard errors. P: Probability a parameter estimate equals zero (by likelihood ratio test). Sex: A dichotomous (0, 1) variable. Estimate of mean effect of being female. Age: Continuous variable (decimal years). Diet: Dichotomous variable (0, 1). Estimate of mean effect of HCHF diet. h2: Heritability or proportion of residual phenotypic variance in the phenotype due to the additive effects of genes. Baseline Baseline + 2 years Parameter MLE ± S.e. P MLE ± s.e. P βsex 0.516 ± 0.141 0.0003 0.218 ± 0.169 0.2975 βage −0.089 ± 0.040 0.0274 −0.048 ± 0.031 0.1976 βdiet N/A N/A −0.486 ± 0.143 0.0009 h2 0.270 ± 0.128 0.0266 0.457 ± 0.242 0.0038 Table 2. Mean effects of sex, age, and diet and additive effects of genes on LTL in pedigreed baboons at two timepoints: Cohorts combined. Baseline: diet is LCLF for both; Baseline + 2 years: Control group fed LCLF diet and experimental group fed HCHF atherogenic challenge diet for 2 years. MLE ± s.e.: maximum likelihood estimates of parameters and their standard errors. P: Probability a parameter estimate equals zero (by likelihood ratio test). Sex: A dichotomous (0, 1) variable. Estimate of mean effect of being female. Age: Continuous variable (decimal years). Diet: Dichotomous variable (0, 1). Estimate of mean effect of HCHF diet. h2: Heritability or proportion of residual phenotypic variance in the phenotype due to the additive effects of genes. Table 2. Mean effects of sex, age, and diet and additive effects of genes on LTL in pedigreed baboons at two timepoints: Cohorts combined. Baseline: diet is LCLF for both; Baseline + 2 years: Control group fed LCLF diet and experimental group fed HCHF atherogenic challenge diet for 2 years. MLE ± s.e.: maximum likelihood estimates of parameters and their standard errors. P: Probability a parameter estimate equals zero (by likelihood ratio test). Sex: A dichotomous (0, 1) variable. Estimate of mean effect of being female. Age: Continuous variable (decimal years). Diet: Dichotomous variable (0, 1). Estimate of mean effect of HCHF diet. h2: Heritability or proportion of residual phenotypic variance in the phenotype due to the additive effects of genes. for 2 years. The estimated mean effect of diet, independent of age and sex, was significant (P = 0.000857); and, consistent with our earlier observations, LTL was smaller in baboons who had been fed HCHF diet for 2 years. Results i None of the serum biomarkers assayed in the time-point 0 samples are correlated with LTL at 2 years; however, 4 of them, quantified in samples obtained at 7 weeks, are correlated with LTL at 2 years. Sex and age adjusted concentrations of very low plus low density lipoprotein cholesterol (V + LDLC) and apolipoprotein E (apo E) are negatively correlated with and account for approximately 5.8% to 4.6% of the variance in LTL following the 2-year challenge (P = 0.015 and P = 0.032). The mean effects of the activity of paraoxonase (PON1], an inhibitor of high density lipoprotein oxi- dation, and adjusted total antioxidant status (TAS], a measure of peroxyl-scavenging capacity27, on mean LTL are positive (P = 0.035 and P = 0.025, respectively]. On average, they are correlated with longer LTL. PON1 and TAS biomarkers each account for approximately 5% of the variance in LTL. There is no evidence for any suggestively significant (0.5 ≤ P ≤ 0.10] effects of any other of the 13 CVD-related biomarkers analyzed in this study. Association of LTL and LTL attrition with extent of atherosclerotic lesions. We find significant evidence that LTL is associated with the extent of atherosclerotic lesions quantified as the percent area in a section of one of the three arteries harvested during necropsy from baboons following the 2-year atherogenic diet chal- lenge: the descending aorta. LTL accounted for approximately 6% (P = 0.010) of the variance in lesion extent in that vessel. The relationship is negative (r = −0.247), with smaller ratios being associated with greater proportions of arterial area covered by an atherosclerotic lesion (Table 4). We detect a similarly small but significant associ- ation between the magnitude of LTL attrition (ΔT/S ratio) and the extent of atherosclerotic lesions in the com- mon iliac artery. ΔLTL accounts for approximately 4% (P = 0.036) of the variance in lesion extent in that vessel (Table 5). However, LTL at baseline is not predictive of extent of atherosclerotic lesions in any of the three arteries (for aortic arch, P = 0.89; common iliac artery, P = 0.20; and descending aorta, P = 0.28) in the current study. Results i For the control group, at time point 2, only the mean effects of an age-by-sex interaction are statistically significant (P = 0.031), the net effect of which may underlie that apparent decrease in difference between LTL in females and males (Table 1). While the signs are in the expected orientations – i.e., sex is posi- tively correlated (P = 0.113) with LTL and age is negatively correlated (P = 0.232] – the magnitudes of each are not large. Analyses of time point 2 data for experimental animals reveals a small, suggestively significant sex effect (P = 0.056) and no age effect (P = 0.266). Diet, sex and age effects on inter-individual variation in LTL: Combined cohorts. Baseline. We maximized a quantitative genetic model for LTL on data from both cohorts at baseline when all animals had been fed the LCLF diet. In addition to the additive genetic and effects of unmeasured environmental and non-additive genetic factors, this model contained sex and age as covariates. Likelihood ratio tests showed that each exerted a significant mean effect on LTL (Psex = 0.000304 and Page = 0.0273) and the effects of each exhibited the expected directionality reported in the literature and seen in our raw data: i.e., mean LTL is greater in female baboons than in males and smaller in older individuals (Table 2). Also, the estimated proportion of the residual phenotypic variance in LTL attributable to the effects of genes, the heritability, was significant, but modest (h2 = 0.27 ± 0.19, P = 0.027). Baseline plus 2 years. We applied the same approach to sex- and age-adjusted residuals of data collected from both cohorts after the control and experimental cohorts had been fed the LCLF and HCHF diets, respectively, Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 www.nature.com/scientificreports/ Baseline Baseline + 2 years Parameter MLE ± S.e. P MLE ± s.e. P βsex 0.516 ± 0.141 0.0003 0.218 ± 0.169 0.2975 βage −0.089 ± 0.040 0.0274 −0.048 ± 0.031 0.1976 βdiet N/A N/A −0.486 ± 0.143 0.0009 h2 0.270 ± 0.128 0.0266 0.457 ± 0.242 0.0038 Table 2. Mean effects of sex, age, and diet and additive effects of genes on LTL in pedigreed baboons at two timepoints: Cohorts combined. Baseline: diet is LCLF for both; Baseline + 2 years: Control group fed LCLF diet and experimental group fed HCHF atherogenic challenge diet for 2 years. Discussion R2: proportion of the variance in lesion extent attributable to ΔLTL; correlation between ΔLTL and lesion extent; P(r = 0): probability that the correlation between ΔLTL on extent of lesion (r) equals zero, which is equivalent to the probability that the mean effect of ΔLTL (β) on lesion extent equals zero in the model. Trait R2 r P(r = 0) Aortic arch 0.0034 −0.059 0.595 Common iliac artery 0.0408 −0.202 0.036 Descending aorta 0.0190 −0.137 0.170 Sum of lesion extent at three sites 0.0114 −0.106 0.279 Mean lesion extent at three sites 0.0114 −0.109 0.266 Table 5. Correlations between ΔLTL and extent of atherosclerotic lesions at three sites. R2: proportion of the variance in lesion extent attributable to ΔLTL; correlation between ΔLTL and lesion extent; P(r = 0): probability that the correlation between ΔLTL on extent of lesion (r) equals zero, which is equivalent to the probability that the mean effect of ΔLTL (β) on lesion extent equals zero in the model. Table 5. Correlations between ΔLTL and extent of atherosclerotic lesions at three sites. R2: proportion of the variance in lesion extent attributable to ΔLTL; correlation between ΔLTL and lesion extent; P(r = 0): probability that the correlation between ΔLTL on extent of lesion (r) equals zero, which is equivalent to the probability that the mean effect of ΔLTL (β) on lesion extent equals zero in the model. factors and modifiable extrinsic environmental factors already known to increase risk for the disease. High fat diet is one of the major traditional environmental risk factors for atherosclerosis and one relatively novel intrinsic risk factor for atherosclerosis is telomere length15. factors and modifiable extrinsic environmental factors already known to increase risk for the disease. High fat diet is one of the major traditional environmental risk factors for atherosclerosis and one relatively novel intrinsic risk factor for atherosclerosis is telomere length15. g Based on our study of 2 cohorts of pedigreed baboons, which for 2 years consumed either a control diet, low in cholesterol and fat, or an experimental HCHF diet, known to be atherogenic, we derive 4 inferences. The 2 most salient of these are that 1) prolonged consumption of the HCHF diet leads to significantly lower median LTL, independent of the effects of aging and 2) shorter LTL and the decrease in LTL (ΔLTL) following the diet challenge are associated with the extent of early atherogenic lesions. Discussion For table column headers, R2: proportion of the variance in LTL attributable to the biomarker; correlation between the biomarker and LTL; P(r = 0): probability that the correlation between the biomarker and LTL equals zero, which is equal the probability that mean effect of the biomarker (β) on LTL equals zero in the model. Trait R2 r P(r = 0) Aortic arch 0.0157 −0.125 0.201 Common iliac artery 0.0027 −0.052 0.596 Descending aorta 0.0609 −0.247 0.010 Sum of lesion extent at three sites 0.0219 −0.148 0.130 Mean lesion extent at three sites 0.0220 −0.148 0.130 Table 4. Correlations between LTL and extent of atherosclerotic lesions at three sites. R2: proportion of the variance in lesion extent attributable to LTL; correlation between LTL and lesion extent; P(r = 0): probability that the correlation between LTL on extent of lesion (r) equals zero, which is equivalent to the probability that the mean effect LTL (β) on lesion extent equals zero in the model. Trait R2 r P(r = 0) Aortic arch 0.0157 −0.125 0.201 Common iliac artery 0.0027 −0.052 0.596 Descending aorta 0.0609 −0.247 0.010 Sum of lesion extent at three sites 0.0219 −0.148 0.130 Mean lesion extent at three sites 0.0220 −0.148 0.130 Table 4. Correlations between LTL and extent of atherosclerotic lesions at three sites. R2: proportion of the variance in lesion extent attributable to LTL; correlation between LTL and lesion extent; P(r = 0): probability that the correlation between LTL on extent of lesion (r) equals zero, which is equivalent to the probability that the mean effect LTL (β) on lesion extent equals zero in the model. Table 4. Correlations between LTL and extent of atherosclerotic lesions at three sites. R2: proportion of the variance in lesion extent attributable to LTL; correlation between LTL and lesion extent; P(r = 0): probability that the correlation between LTL on extent of lesion (r) equals zero, which is equivalent to the probability that the mean effect LTL (β) on lesion extent equals zero in the model. Trait R2 r P(r = 0) Aortic arch 0.0034 −0.059 0.595 Common iliac artery 0.0408 −0.202 0.036 Descending aorta 0.0190 −0.137 0.170 Sum of lesion extent at three sites 0.0114 −0.106 0.279 Mean lesion extent at three sites 0.0114 −0.109 0.266 Table 5. Correlations between ΔLTL and extent of atherosclerotic lesions at three sites. Discussion In the last few decades, major risk factors for atherosclerosis have been identified and many novel intrinsic risk factors continue to be nominated. For any of these, finding evidence to support an association with vascular pathology and to implicate a mechanism by which their effects are manifested will be a key step to assessing the likelihood that further study ultimately could result in the development of diagnostic, preventive, therapeutic, or prognostic approaches. Other key steps will be elucidating interactions between some of these novel intrinsic risk Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 www.nature.com/scientificreports/ Trait Baseline Baseline + 7 weeks Baseline + 2 years R2 r P(r = 0) R2 r P(r = 0) R2 r P(r = 0) HDLC 0.0164 0.090 0.373 0.008 0.089 0.378 0.010 0.102 0.313 V + LDLC 0.0260 −0.160 0.112 0.058 −0.241 0.015 0.005 −0.069 0.498 TG 0.0020 −0.040 0.693 0.016 0.126 0.212 0.002 −0.048 0.635 apo A1 0.0037 0.192 0.055 0.002 0.045 0.657 0.000 0.003 0.976 apo B 0.0049 0.070 0.488 0.004 0.020 0.843 0.001 0.030 0.767 apo E 0.00008 0.009 0.929 0.046 −0.214 0.032 0.002 −0.046 0.649 CRP 0.0002 0.013 0.897 0.015 0.122 0.226 0.004 −0.062 0.540 oxLDL 0.0044 −0.066 0.514 0.012 0.110 0.272 0.0003 0.018 0.859 IL8 0.0060 −0.077 0.446 0.002 0.045 0.667 0.010 −0.101 0.317 LpPLA2 0.0034 0.058 0.567 0.012 0.110 0.276 0.004 −0.061 0.547 PON1 0.0163 0.127 0.207 0.045 0.212 0.035 0.001 0.038 0.707 TAS 0.0106 0.103 0.378 0.050 0.224 0.025 0.007 0.085 0.400 VWF 0.0005 0.023 0.820 0.014 0.118 0.242 0.001 0.027 0.790 Table 3. Correlations: LTL and biomarkers of lipid metabolism, inflammation and oxidative stress by time on HCHF diet. Abbreviations: For traits, TSC: total serum cholesterol, HDLC: high density lipoprotein cholesterol, V + LDLC: very low + low density lipoprotein cholesterol (V + LDLC), TG: triglycerides, apo A1: apolipoprotein A1, apo B: apolipoprotein B, apo E: apolipoprotein E, CRP: C-reactive protein, IL8: interleukin 8, LpPLA2: lipoprotein associated phospholipase 2 activity, PON1: paraoxonase 1 activity, TAS: total antioxidant status, and VWF: von Willebrand factor. For table column headers, R2: proportion of the variance in LTL attributable to the biomarker; correlation between the biomarker and LTL; P(r = 0): probability that the correlation between the biomarker and LTL equals zero, which is equal the probability that mean effect of the biomarker (β) on LTL equals zero in the model. Discussion Trait Baseline Baseline + 7 weeks Baseline + 2 years R2 r P(r = 0) R2 r P(r = 0) R2 r P(r = 0) HDLC 0.0164 0.090 0.373 0.008 0.089 0.378 0.010 0.102 0.313 V + LDLC 0.0260 −0.160 0.112 0.058 −0.241 0.015 0.005 −0.069 0.498 TG 0.0020 −0.040 0.693 0.016 0.126 0.212 0.002 −0.048 0.635 apo A1 0.0037 0.192 0.055 0.002 0.045 0.657 0.000 0.003 0.976 apo B 0.0049 0.070 0.488 0.004 0.020 0.843 0.001 0.030 0.767 apo E 0.00008 0.009 0.929 0.046 −0.214 0.032 0.002 −0.046 0.649 CRP 0.0002 0.013 0.897 0.015 0.122 0.226 0.004 −0.062 0.540 oxLDL 0.0044 −0.066 0.514 0.012 0.110 0.272 0.0003 0.018 0.859 IL8 0.0060 −0.077 0.446 0.002 0.045 0.667 0.010 −0.101 0.317 LpPLA2 0.0034 0.058 0.567 0.012 0.110 0.276 0.004 −0.061 0.547 PON1 0.0163 0.127 0.207 0.045 0.212 0.035 0.001 0.038 0.707 TAS 0.0106 0.103 0.378 0.050 0.224 0.025 0.007 0.085 0.400 VWF 0.0005 0.023 0.820 0.014 0.118 0.242 0.001 0.027 0.790 Table 3. Correlations: LTL and biomarkers of lipid metabolism, inflammation and oxidative stress by time on HCHF diet. Abbreviations: For traits, TSC: total serum cholesterol, HDLC: high density lipoprotein cholesterol, V + LDLC: very low + low density lipoprotein cholesterol (V + LDLC), TG: triglycerides, apo A1: apolipoprotein A1, apo B: apolipoprotein B, apo E: apolipoprotein E, CRP: C-reactive protein, IL8: interleukin 8, LpPLA2: lipoprotein associated phospholipase 2 activity, PON1: paraoxonase 1 activity, TAS: total antioxidant status, and VWF: von Willebrand factor. For table column headers, R2: proportion of the variance in LTL attributable to the biomarker; correlation between the biomarker and LTL; P(r = 0): probability that the correlation between the biomarker and LTL equals zero, which is equal the probability that mean effect of the biomarker (β) on LTL equals zero in the model. Table 3. Correlations: LTL and biomarkers of lipid metabolism, inflammation and oxidative stress by time on HCHF diet. Abbreviations: For traits, TSC: total serum cholesterol, HDLC: high density lipoprotein cholesterol, V + LDLC: very low + low density lipoprotein cholesterol (V + LDLC), TG: triglycerides, apo A1: apolipoprotein A1, apo B: apolipoprotein B, apo E: apolipoprotein E, CRP: C-reactive protein, IL8: interleukin 8, LpPLA2: lipoprotein associated phospholipase 2 activity, PON1: paraoxonase 1 activity, TAS: total antioxidant status, and VWF: von Willebrand factor. Discussion Additionally, we infer that 3) variation in inflammation and oxidative stress contribute to variation in LTL shortening and 4) the additive effects of genes account for a significant proportion of observed variation in LTL before and after prolonged consumption of the HCHF diet. Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 www.nature.com/scientificreports/ The central finding of this study is that prolonged exposure to the atherogenic HCHF diet results in significant LTL attrition. Rodent models have previously demonstrated a similar finding, however this is the first study to show the relationship between HCHF diets and LTL attrition in primates11. While consistent with expectations based on epidemiological studies of human populations and cohorts in which consumption of diets that vary with respect to the relative proportions of dietary fat (particularly saturated fats and polyunsaturated fats) is correlated with variation in LTL11,13,16,17,28, we interpret the results of our study to be indicative of a causal relationship. Support for this inference derives from 2 principal observations: 1) decreased LTL is independent of age and sex effects in the challenge animals and 2) there is no significant change in LTL over the same period in the control animals. The effect of the 2-year exposure to the HCHF diet on LTL is substantial. As much as 40% of the pheno- typic variance in LTL is due to the effects of the HCHF diet.hl ypf There is general agreement that inflammation and oxidative stress contribute to LTL attrition29 and the HCHF diet used in this study certainly is pro-inflammatory. Associations between LTL at 2 years and 4 biomarkers of inflammation and/or oxidative stress implicate these processes in the observed diet-induced LTL: i.e., serum concentrations of 2 biomarkers of lipid metabolism, V + LDLC and apo E, and the activities of 2 indicators of resistance to oxidative stress, PON1 and TAS, which, respectively, are negatively and positively correlated with LTL. The effects of these biomarkers are relatively small, with each accounting for 4% to 6% of the variance in LTL at 2 years. The fact that it is the 7-week values for the biomarkers, and not those from time-point 0 or 2 years, for which we detect effects on LTL at 2 years would be consistent with a causal relationship. Discussion While the work reported here cannot conclusively demonstrate such a relationship, we note that elevated biomarkers of lipid metabolism, inflammation, and oxidative stress measured at this same time-point (i.e., 7 weeks into the HCHF diet challenge) also were stronger predictors of the extent of atherosclerotic lesions at 2 years in same pedigreed baboons than those assayed at other time-points19. y p While both LTL and ΔLTL are significantly correlated with the extent of atherosclerotic lesions in 2 major arteries, the effect sizes implicated are small, accounting for between 4% and 6% of the variance in the descending aorta and common iliac artery, respectively. Nonetheless, the signs of the correlations are consistent with expec- tations: shorter LTL and greater LTL attrition (over the 2-year period) are associated with increased lesion extent. Based on the results of studies in humans, other researchers have suggested that short LTL at any time may be a more important risk factor for atherosclerosis than LTL attrition. One of these is that inter-individual variation in LTL, which has a significant heritable component, is established by rapid attrition in early life, after which age-related attrition rates do not differ significantly regardless of differences in risk30. The other is that short LTL at any time increases susceptibility to atherogenic factors and that LTL attrition is associated with increased sever- ity of atherosclerosis. Our study is not designed as an explicit test or either hypothesis. y y g p yp Our study design also does not allow us to determine when during the 2-year diet challenge this association is established – i.e., when shorter LTL marks an increase in risk for atherosclerosis. However, an earlier study in which baboons from this same breeding colony consumed the same HCHF diet for only 7 weeks found increased prevalence of senescence in vascular endothelial cells31, suggesting that diet-induced LTL attrition, a biomarker of, if not a contributor to, cellular senescence could also begin very soon after starting the atherogenic diet. g yt g g Again, the focus of our research is early-stage atherosclerosis. We are not aware of a previous report of a rela- tionship between LTL and early-stage atherosclerosis in either humans or nonhuman primates. Discussion Those human stud- ies to which we alluded earlier in this paper find that short LTL is associated with clinically appreciable indicators of CVD, examples of which include, but are not limited to, angiographically detected severe triple-vessel coronary artery disease15; coronary artery calcium32; and complicated carotid artery plaques21,33; as well as with compos- ite measures of cardiovascular health34,35. But those observations reflect well-developed, clinically appreciable, later-stage atherosclerosis in large-scale cross-sectional cohort studies of adults. Citing the results of 2 such stud- ies36,37, Riezschel et al.7 posit that the short LTL-atherosclerosis association does not extend to early-stage disease.f y g We believe the different picture painted by our results lies in large part in the experimental study design, which enhances our ability to quantify and control and many factors of interest with greater precision than is possible in most epidemiological studies in human populations/cohorts. These include, for example, having confidence in the compositions of both the baseline and experimental diets, which are fully defined and completely uniform throughout the course of the diet challenge; minimization of exposures to extraneous “lifestyle” and other envi- ronment factors; accuracy of the additive genetic “background;” and the validity of the data on the presence, size, and nature of atherosclerotic lesions, as they were obtained by direct observation (see earlier publication21). Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 Summary and Conclusion We have shown that a diet previously demonstrated to be atherogenic in captive baboons from a pedigreed breed- ing colony affects LTL and that the effects of diet are in addition to those of aging. We also have shown that diet-induced shorter LTL is negatively associated with extent of vascular lesion development in early-stage ather- osclerosis. Both observations have been made in the same individuals, in the course of the same study.i To our knowledge this is the first prospective, longitudinal, experimental study of its kind in a primate species. Although highly informative, such study designs are impractical, if not impossible, in humans as they require accurate knowledge of and control of composition and consumption of a diet known to reliably induce the disease state of interest, the ability to control for background genetic variation in order to maximize the signal-to-noise ratio, and the ability to accurately assess the pre-clinical disease state. But as we have shown here, in the case of early-stage atherosclerosis, all of these issues can be addressed so that such studies can be conducted success- fully with a relevant animal model for early-stage atherosclerosis: the pedigreed baboon. Given its phylogenetic proximity and consequent genetic, physiological, and anatomical similarity to our own species, this nonhuman primate model can be used profitably to investigate the role(s) of telomeres and/or telomere attrition in vascular changes accompanying early stages of, or even presaging, atherogenesis in humans. Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8 www.nature.com/scientificreports/ Data availability y For the current study, datasets generated during and/or analyzed are available upon request from the corresponding author on reasonable request. Received: 9 April 2019; Accepted: 26 November 2019; Published: xx xx xxxx Received: 9 April 2019; Accepted: 26 November 2019; Published: xx xx xxxx References ll l ff f h d k f h l f l d f h d h . A. et al. Selected methodologic aspects of the International Ather 2. Guzman, M. A. et al. Selected methodologic aspects of the International Atherosclerosis Project. 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Short Telomere Load, Telomere Length, and Subclinical Atherosclerosis: The PESA Study. Journal of the American College of Cardiology 67, 2467–2476, https://doi.org/10.1016/j.jacc.2016.03.530 (2016). p g j p ( ) 7. Fernandez-Alvira, J. M. et al. Short Telomere Load, Telomere Length, and Subclinical Atherosclerosis: The PESA Study. Journal o the American College of Cardiology 67, 2467–2476, https://doi.org/10.1016/j.jacc.2016.03.530 (2016). Acknowledgementsh g This study received support from the following: National Institutes of Health (Grant number: P01 HL028972) received by JLV; NIH Office of the Director (Grant number: P51 OD011133) received by JLV; NIH Office of the Director (Grant number: P51 RR013986) received by JLV; (Grant number: C06 RR14578; C06 RR15456; C06 RR013556; C06 RR017515). The funder supported the infrastructure that was used during the investigation of the work reported in the manuscript. h y pp g ( ) received by JLV; NIH Office of the Director (Grant number: P51 OD011133) received by JLV; NIH Office of the Director (Grant number: P51 RR013986) received by JLV; (Grant number: C06 RR14578; C06 RR15456; C06 RR013556; C06 RR017515). The funder supported the infrastructure that was used during the investigation of received by JLV; NIH Office of the Director (Grant number: P51 OD011133) received by JLV; NIH Office of the Director (Grant number: P51 RR013986) received by JLV; (Grant number: C06 RR14578; C06 RR15456; C06 RR013556; C06 RR017515) The funder supported the infrastructure that was used during the investigation of Competing interestsh p g The authors declare no competing interests. Author contributions Conceptualization or design of the work, G.M.K., M.C.M., J.L.V., L.A.C. Acquisition and analysis of data, G.M.K., M.C.M., D.E.N., C.C., S.B. Interpretation of data, G.M.K., M.C.M., J.L.V., L.A.C.; Drafting of the manuscript or substantial revision, G.M.K., M.C.M., L.A.C., A.M.R. Additional information Correspondence and requests for materials should be addressed to G.M.K. Correspondence and requests for materials should be addressed to G.M.K. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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. 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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) 2019 Scientific Reports \| (2019) 9:19001 \| https://doi.org/10.1038/s41598-019-55348-8
https://openalex.org/W2034149560	http://www.vliz.be/imisdocs/publications/298227.pdf	English	null	Registry of non-native species in the Two Seas region countries (Great Britain, France, Belgium and the Netherlands)	NeoBiota	2,014	cc-by	6,670	Registry of non-native species in the Two Seas region countries (Great Britain, France, Belgium and the Netherlands) Alexandra Zieritz1, Belinda Gallardo1, David C. Aldridge1 1 Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) Corresponding author: Belinda Gallardo (galla82@hotmail.com) Citation: Zieritz A, Gallardo B, Aldridge DC (2014) Registry of non-native species in the Two Seas region countries (Great Britain, France, Belgium and the Netherlands). NeoBiota 23: 65–80. doi: 10.3897/neobiota.23.5665 Abstracth This dataset represents a registry of species that are not native but recorded to live in the wild of at least one of the four countries that comprise the Two Seas Area, i.e. Great Britain, France, Belgium and the Netherlands. For each of the 6,661 species, subspecies and hybrids listed, we provide detailed information on its status in each country, taxonomic affiliation and environment inhabited. The data were collected by review of 36 web- and print-based sources over an eight-month period. Further systematic scanning of three of the most relevant scientific journals, i.e. Neobiota, Aquatic Invasions and BioInvasions Records, recovered 19 additional relevant publications from which information was included in the registry. As a result, the registry will serve as a basis for developing effective, cross-boundary strategies to manage and control non-native species, which can have severe ecological and economic impacts. The registry can fur ther be used as a general reference for both scientists and practitioners, as well as a tool to assess reliability and comprehensiveness of other well-known databases such as the DAISIE portal. Copyright Alexandra Zieritz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Regi NeoBiota 23: 65–80 (2014) doi: 10.3897/neobiota.23.5665 http://neobiota.pensoft.net/ Regi NeoBiota 23: 65–80 (2014) doi: 10.3897/neobiota.23.5665 http://neobiota.pensoft.net/ Regi NeoBiota 23: 65–80 (2014) doi: 10.3897/neobiota.23.5665 http://neobiota.pensoft.net/ species in the Tw DATA PAPER Keywords Belgium, English Channel, exotic species, France, Great Britain, invasive species, Netherlands Copyright Alexandra Zieritz 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. Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) 66 Definition of terms Native species Refers to a species that has been observed in the form of a naturally occurring and self-sustaining population in historical times. Non-native species (NS) Refers to a species that has been introduced through human action outside its natural present or historical range. This term includes species whose main pathway of introduction is human-related although they have entered a country through natural spread from one or more neighbouring countries. However the term excludes species expanding their range without direct human action, as in the case of migration or species expanding because of climate change or habitat modification, even if these changes are caused by humans. Invasive non-native species (INS) Refers to a non-native species that adversely affects the regions and habitats it invades environmentally, economically and/or ecologically. This term therefore excludes non-native species that do not pose any significant threat to biodiversity conservation. Definition of terms Introduction The region comprising the British Channel and southern part of the North Sea, as well as coastal areas of Great Britain, France, Belgium and The Netherlands (also re ferred to as The Two Seas region) has a long history of trade and travel, and includes important commercial ports such as Southampton, Felixstowe, Le Havre, Antwerp and Rotterdam ("World Shipping Council. Top 50 World Container Ports" ; Enshaei and Mesbahi 2009). These intensive activities across national borders have led to the introduction of numerous exotic animal, plant and other species to this area, both from other European regions and further afield (Holdich and Pöckl 2007; Gherardi et al. 2009; Keller et al. 2009). Invasive species do not know political borders, which is why cooperation and col laboration between countries is key in the fight against devastating and costly non- native, invasive species. Efficient cross-border communication and knowledge transfer would guarantee that knowledge on the vectors, impacts and control options for non- native species gained in one country informs decisions on management and control for non-native species in other countries. It can further help to raise the alarm on species that are likely to spread from one country to another, prompting preventive action plans. International cooperation in environmental politics can facilitate development and implementation of sustainable cross-border management practices for non-native species (Essl et al. 2011).i Examples exist where international cooperation has significantly improved the prevention of non-native species’ spread. These include the Inter-American Invasive Species Network (IABIN-13N, http://i3n.iabin.net/) that supports the detection and management of invasive alien species in the Americas, and the Trilateral Committee for Wildlife and Ecosystem Conservation and Management (http://www.trilat.org), which addresses environmental challenges common to Canada, United States and Mexico (Simpson et al. 2006; Simpson et al. 2009). Registry of non-native species in the Two Seas region countries 67 With regard to the Two Seas region, the European-funded ‘Interreg Two Seas Pro gramme’ promotes cross-border cooperation between Great Britain, France, Belgium and The Netherlands (see http://www.interreg4a-2mers.eu for more information). Within this initiative, the RINSE (Reducing the Impacts of Non-native Species in Europe) project aims to develop cross-border tools to improve the prioritisation and targeting of non-native species. As a minimum requirement, such a regional approach to invasive species’ management requires an up-to-date and comprehensive registry of non-native species containing information on the current status of each non-native species in each of the four Two Seas region countries. B. Data set description The dataset consists of 1 file, containing two worksheets. Worksheet “Registry” con tains a 10 × 6,662 matrix of text values, Worksheet “Summary of data” contains three summarising tables in the form of three 2 × 33, 2 × 5 and 8 × 5 matrices of text and numeric values. The file is labelled as Table_RegistryNonNativeSpecies.xls A. Data set identity A. Data set identity Registry of non-native species in the Two Seas region countries (Great Britain, France, Belgium and the Netherlands) 2. Originators The project was conducted within and on behalf of the European Union funded RINSE (Reducing the Impacts of Non-native Species in Europe) Project. Methodol ogy was developed by the three authors Alexandra Zieritz, Belinda Gallardo and David C. Aldridge. Data collection was done by Alexandra Zieritz and Belinda Gallardo. 1. Principal investigators Alexandra Zieritz. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) Belinda Gallardo. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) David C. Aldridge. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) David C. Aldridge. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) 1. Identity We collected information on the status of non-native species in each of the four coun tries Great Britain, France, Belgium and the Netherlands. The taxonomic affiliation and environment inhabited by each species is also provided. Introduction To facilitate application in reli able horizon-scanning and similar exercises, and allow for meaningful comparisons of inventories between countries and taxa, it is crucial that this database is as comprehen sive as possible but not skewed towards particular countries or taxa. p p Unfortunately, currently available databases are unsuitable for such a purpose. For example, the number of species listed in freely accessible online databases dif fers considerably between databases as well as between the four countries of con cern. Thus, the primary data portal for non-native species in Europe, DAISIE (De livering Alien Invasive Species Inventories for Europe; http://www.europe-aliens. org/), features 2,471 non-native species for Belgium, 2,075 of which are terrestrial plants, but only 881 species for the slightly larger Netherlands. In contrast, the Dutch Biodiversity registry (http://www.nederlandsesoorten.nl) lists 925 non-na tive species, and only 101 invasive non-natives are highlighted by the Belgian in formation system Harmonia (http://ias.biodiversity.be). The Great Britain Invasive Non-Native Species Secretariat (NNSS) database (http://www.nonnativespecies. org) comprises over 3,000 species. No comparable initiatives exist in France. Such enormous discrepancies between inventories of neighbouring countries that would be expected to host comparable numbers and sets of non-native species are unlikely to be real but probably root in different experts providing the data for different countries and databases.h The present dataset aims at providing a registry of non-native species in the Two Seas region that is comprehensive and not biased towards particular countries or taxa. We achieve this by integrating information from a total of 55 national and interna tional print- and online-sources on the presence of non-native terrestrial, marine and freshwater species in the four Two Seas region countries. In addition, for each taxon, the registry provides information on its taxonomic classification, current distribution and environment inhabited. Potential utilities of this registry include developing na tional checklists of non-native species, and analysing spatial patterns of distribution of species. Furthermore, the database offers a general point of reference for both scientists and practitioners working on non-natives in the Two Seas region and adjacent coun tries. Finally, the registry could act as a tool to assess reliability and comprehensiveness of other databases from which data was retrieved. This could be done by, for example, comparing number and identities of non-native species listed by a given source data base to those of the present registry. Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) 68 Metadata Data set descriptors 3. Period of study Data was collected from 01/06/2012 to 27/02/2014. Collected data correspond to contemporary species records. Registry of non-native species in the Two Seas region countries 69 4. Objectives The primary objective of the present work was to compile a registry of non-native species present in the four countries comprising the Two Seas region, i.e. the English Channel and the Southern part of the North Sea. The registry will serve as a basis for developing effective, cross-boundary strategies to manage and control non-native species, which can have severe ecological and economic impacts. It can further be used as a general reference for both scientists and practitioners, as well as a tool to assess reliability and comprehensiveness of other well-known databases such as the DAISIE portal. 5. Source of funding INTERREG IVA 2-Seas Programme. Funded by the European Regional Development Fund (ERDF). Project: RINSE, Reducing the Impacts of Non-Native species in Europe. Work Package 1 subproject: Targeting and Prioritisation for Non-Native species into the RINSE area. Summary of the RINSE project RINSE (Reducing the Impacts of Non-native Species in Europe) is a European Project which investigates best strategies of managing non-native species (NS) across the Two Seas Programme area. The project specifically aims to i) develop cross-border tools to improve prioritisation and targeting of NS, so that scarce resources can be directed towards the species and sites of greatest concern, ii) enhance the capacity to address NS within a range of target stakeholders, and iii) develop new approaches and best prac tices for the management of NS, by delivering field trials and demonstration projects. RINSE works across borders to share best practice and adopt strategic approaches to tackle the threats posed by non-native species (NS). b. Geography Location b. Geography Location Countries comprising the Two Seas Programme area (i.e. the English Channel and the Southern part of the North Sea): Great Britain, France, Belgium and The Netherlands. B. Specific subproject description 1. Site description. a. Site type The region includes terrestrial, marine and freshwater habitats. d. Geology, landform d. Geology, landform The region includes various geological types, ranging from Pre-Cambrian, to Car boniferous, Cretaceous and Tertiary rocks. b. Data collection period, frequency, etc. Basic data collection period was 01/06/2012 to 17/01/2013. Additional systematic scanning of the three journals Neobiota, Aquatic Invasions and BioInvasions Re cords was performed in January and February 2014. c. Habitath The region includes terrestrial, marine and freshwater habitats. Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) 70 e. Watersheds, hydrology , y gy The main river systems in the area include the Thames, Loire, Seine, Meuse and Rhine. f. Climate 2. Experimental or sampling design a. Design characteristics Basic data were collected by systematic review of 36 web- and print-based sources over an eight-month period (see Table 1). Additional systematic scanning of three scientific journals, i.e. Neobiota, Aquatic Invasions and BioInvasions Records, re covered 19 additional relevant publications from which information was included in the registry. 3. Research methods Harmonia database" Other abbreviations: na, not applicable because no data on presence of species within the respective phylum/division in the four countries were available from this source; N, source not used with regard to respective phylum/division; Y, all taxa of respective phylum that this source lists to be present in one or more of the four countries were included; Y ex T, all taxa except terrestrial ones of respective phylum that this source lists to be present in one or more of the four countries were included. Phylum DAISIE ISSG CABI FAO NOBANIS NNSS Waarnemingen Waarneming Naturalis BFIS Gollasch et al. 3. Research methods A number of online and print data sources were used to obtain information on non- native species present in the four Two Seas region countries. In total, the basic data were gathered from 36 sources, including the 12 listed in detail in Table 1 used for all animal phyla and plant divisions, and additional references for particular groups of organisms (see references). Selection of the databases included in this work was done with the help of consulting experts within the European RINSE (Reducing the Im pacts of Non-native Species in Europe) project. j Particular care was thereby taken to avoid and counteract any bias towards par ticular countries. For example, the lack of a national database on non-native species in France was targeted by inclusion of an additional 11 grey-literature sources from France, which we obtained through our local RINSE partners ("Le Conservatoire Botanique National de Bailleul. Liste des plantes exotiques considérées comme enva hissantes en Picardie"; Agence de l’eau Artois Picardie and Conservatoire Botanique National de Bailleul 2005; Agence de l’eau Rhin Meuse 2005; Costa 2005; Delbart et al. 2007; Conseil General du Finistere 2008; Lacroix et al. 2008; Paradis et al. 2008; Zambettakis and Magnanon 2008; Reseau regional des Gestionnaires des Milieux Aquatiques Paca 2009; Hudin and Vahrameev 2010). Registry of non-native species in the Two Seas region countries 71 Table 1. The main 12 web- and print-based sources per taxa used for compiling the registry of non-native species in the Two Seas region countries Great Britain, France, Belgium and the Netherlands. Acronyms and abbreviations of online databases: "DAISIE - Delivering Alien Invasive Species Inven tories for Europe", "ISSG - Invasive Species Specialist Group. Global Invasive Species Database", "CABI - Centre for Agricultural Bioscience International. Invasive Species Compendium. Wallingford, UK: CAB International", "FAO - Food and Agriculture Organisation (United Nations). Fisheries and Aquaculture top ics. Introduction of species. Database on Introductions of Aquatic Species. In: FAO Fisheries and Aquaculture Department. Rome", "NOBANIS - North European and Baltic Network on Invasive Alien Species. Gateway to Information on Invasive Alien species in North and Central Europe", "NNSS - GB Non-native Species Secretariat. GB Non-native Species Information Portal", "BFIS - Belgian Forum on Invasive Species. Har monia database", "Waarnemingen. Belgian daughter website of the Global Biodiversity Recording Project", "Waarneming. Dutch daughter website of the Global Biodiversity Recording Project", "Naturalis. Nederlands Soortenregister, version 2.0", "BFIS - Belgian Forum on Invasive Species. 3. Research methods (2009) Wolff (2005) Entoprocta na na na na na Y Y Y Y na na na Nemertea Y na na na na na Y Y na na na na Mollusca Y Y Y Y na Y Y Y Y na Y Y Annelida Y Y Y Y na Y Y Y Y na Y Y Nematoda Y na Y na na Y Y Y Y na Y Y Arthropoda Y Y Y Y na Y ex T N N Y Y Y Y Chordata Y Y Y Y Y Y Y Y Y Y Y Y Additional 17 sources were used for following groups: Additional 17 sources were used for following groups: • Angiospermae: Agence de l’eau Artois Picardie and Conservatoire Botanique National de Bailleul (2005), Agence de l’eau Rhin Meuse (2005), Costa (2005), Delbart et al. (2007), Conseil General du Finistere (2008), Lacroix et al. (2008), Paradis et al. (2008), Zambettakis and Magnanon (2008), Reseau regional des Gestionnaires des Milieux Aquatiques Paca (2009), Hudin and Vahrameev (2010), "DAISIE - Delivering Alien Invasive Species Inventories for Europe. 100 of The Worst", "EPPO - European and Mediterranean Plant Protection Organisation. EPPO list of invasive alien plants", "Le Conservatoire Botanique National de Bail leul. Liste des plantes exotiques considérées comme envahissantes en Picardie", "Q-bank. Invasive Plants database. Comprehensive databases on quarantine plant pests and diseases" • Arthropoda: Rabitsch (2008), Roques et al. (2010), "DAISIE - Delivering Alien Invasive Species Inventories for Europe. 100 of The Worst" • Arthropoda: Rabitsch (2008), Roques et al. (2010), "DAISIE - Delivering Alien Invasive Species Inventories for Europe. 100 of The Worst" p ph • Heterokontophyta, Chlorophyta, Rhodophyta, Lycopodiophyta, Pteridophyta and Pinophyta: Plantlife (2010) p ph • Heterokontophyta, Chlorophyta, Rhodophyta, Lycopodiophyta, Pteridophyta and Pinophyta: Plantlife (2010) h • Heterokontophyta, Chlorophyta, Rhodophyta, Lycopodiophyta, Pteridophyta and Pinophyta: Plantlife (2010) Furthermore, all volumes of the three journals Neobiota (vol. 9–20), Aquatic Inva sions (vol. 1–8) and BioInvasions Records (vol. 1–2 and vol 3 in press articles) available by February 2014 were thoroughly and systematically scanned for relevant studies that potentially provided further information on species’ presence in the RINSE countries. This was done by reading the titles and, in case that indicated potential relevance to our database, reading the abstract and complete manuscript. In total, the following 19 studies were thereby included in the registry: Copp et al. (2006), Kerckhof et al. (2007), Sjøtun et al. 3. Research methods (2009) Wolff (2005) Viruses, Bacteria, Protista Viruses na na Y na na Y na na N na na na Firmicutes na na na na na Y na na N na na na Proteobacteria na Y Y na na na na na N na na na Cercozoa Y na na na na Y na na N na Y Y Algae Dinoflagellata Y na na na na Y Y Y N na Y Y Haptophyta Y na na na na Y Y Y N na Y na Heterokontophyta Y Y Y Y na Y Y Y N na Y Y Chlorophyta Y Y Y Y na Y Y Y N na Y Y Rhodophyta Y Y Y Y na Y Y Y N na Y Y Plantae Marchantiophyta Y na na na na na N N N na na na Bryophyta Y na Y na na na N N N na na na Lycopodiophyta Y na na na na Y N N N na na na Pteridophyta Y na Y Y na Y N N N Y na na Pinophyta Y na Y na na N N N N na na na Angiospermae Y Y Y Y Y Y ex T N N N Y Y Y Fungi Chytridiomycota Y na na na na na N N N na na na Zygomycota na na na na na Y N N N na na na Ascomycota Y Y Y na na na N N N na na na Basidiomycota Y Y Y na na na N N N na na na Animalia Porifera Y na na na na Y Y Y Y na Y Y Cnidaria Y na Y Y na Y Y Y Y na Y Y Ctenophora Y na Y na na Y Y Y Y na Y na Platyhelminthes Y na na na na Y Y Y Y na Y Y Rotifera Y na na na na na Y Y Y na na na Bryozoa Y Y Y na na Y Y Y Y na Y Y Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) 72 Phylum DAISIE ISSG CABI FAO NOBANIS NNSS Waarnemingen Waarneming Naturalis BFIS Gollasch et al. 3. Research methods (2008), Kai and Soes (2009), Wijnhoven and Dekker (2010), Zięba et al. (2010), Vaate and Beisel (2011), Brylinski et al. (2012), Faasse and Gian grande (2012), Marescaux et al. (2012), Faasse (2013a), Faasse (2013b), Heiler et al. (2013), Kessel et al. (2013), Lavesque et al. (2013), Minchin et al. (2013), Pinder et al. (2013), Scalone and Rabet (2013), and Soors et al. (2013). Registry of non-native species in the Two Seas region countries 73 Finally, apart from collecting basic data on non-native species present in at least one of the four Two Seas region countries, additional presence in the other three countries of concern was checked using the following seven geographic distribution gateways: "GBIF Finally, apart from collecting basic data on non-native species present in at least one of the four Two Seas region countries, additional presence in the other three countries of concern was checked using the following seven geographic distribution gateways: "GBIF g g g g p g y - Global Biodiversity Information Facility. GBIF Data Portal", Hopkins (2012), "In tergovernmental Oceanographic Commission of UNESCO. The Ocean Biogeographic Information System OBIS", "Muséum national d'Histoire naturelle. INPN Inventaire national du Patrimoine Naturel", "NBN - National Biodiversity Network. National Biodiversity Network's Gateway", "NLBIF - Netherlands Biodiversity Information Fa cility. Data portal of the Dutch national node of the Global Biodiversity Information Facility (GBIF)", and Verloove (2006). This was done for all taxa except those terrestrial Angiospermae and Pinophyta that were listed as present by the DAISIE portal but not by any of the other databases consulted. In the registry, these species are indicated by the phrase “data based solely on DAISIE portal” in the final column (headed “Notes”). - Global Biodiversity Information Facility. GBIF Data Portal", Hopkins (2012), "In tergovernmental Oceanographic Commission of UNESCO. The Ocean Biogeographic Information System OBIS", "Muséum national d'Histoire naturelle. INPN Inventaire national du Patrimoine Naturel", "NBN - National Biodiversity Network. National Biodiversity Network's Gateway", "NLBIF - Netherlands Biodiversity Information Fa cility. Data portal of the Dutch national node of the Global Biodiversity Information Facility (GBIF)", and Verloove (2006). This was done for all taxa except those terrestrial Angiospermae and Pinophyta that were listed as present by the DAISIE portal but not by any of the other databases consulted. In the registry, these species are indicated by the phrase “data based solely on DAISIE portal” in the final column (headed “Notes”). Principal investigators: Alexandra Zieritz. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) Belinda Gallardo. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) g g g David C. Aldridge. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) Associated investigator: Eduard Jones. Aquatic Ecology Group, Department of Zoology, University of Cam bridge, Downing St. CB2 3EJ, Cambridge (UK) 3. Research methods i After compilation of the database was completed, we checked for errors through the process of blind repetition of data-compilation for 1% of the dataset (i.e. for 34 species or 136 data points (34 species × 4 countries)). This revealed an error rate of 0.007% (i.e. 1 of 136 data points was incorrect).h The present database will be sustained in the future by periodically conducting a systematic literature review on new invasions in the four countries. This could be done, for example, by a Web of Science or Google Scholar search using keyword combina tions such as “non-native OR exotic OR invasive AND Britain OR UK OR Neth erlands OR France OR Belgium”, and/or a systematic scanning of the most relevant journals such as Nebiota, Aquatic Invasions and BioInvasions Records. David C. Aldridge. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) A. Status 1. Latest update. 28/02/2014. 2. Metadata status Metadata are complete. Supervisor: David C. Aldridge. Aquatic Ecology Group, Department of Zoology, University of Cambridge, Downing St. CB2 3EJ, Cambridge (UK) 74 Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) B. Accessibility B. Accessibility 1. Storage location and medium. Original data files exist on the authors’ personal computers in MS Excel® format. 2. Contact Persons Alexandra Zieritz: alexandra.zieritz@cantab.net Belinda Gallardo: galla82@hotmail.com 3. Copyright restrictions None. 4. Proprietary restrictions None. b. Citation Data were provided by the RINSE (Reducing the Impacts of Non-native Species in Europe) project (http://www.rinse-europe.eu/). 1. Storage location and medium. Original data files exist on the authors’ personal computers in MS Excel® format. 2. Contact Persons Alexandra Zieritz: alexandra.zieritz@cantab.net Belinda Gallardo: galla82@hotmail.com Data set status and accessibility Data set status and accessibility A. Status 1. Latest update. 28/02/2014. 2. Metadata status Metadata are complete. Data set status and accessibility A. Status 1. Latest update. 28/02/2014. 2. Metadata status Metadata are complete. 1. Identityh The data set comprises one file (MS Excel® document) named Table_RegistryNonNa tiveSpecies.xls. The file contains two worksheet: 1.1. The “Registry” worksheet comprises the registry itself, listing all non-native species that were recorded as non-native in at least one of the four countries of the Two Seas region (Great Britain, France, Belgium and Netherlands). For each species, the phylum/division, class, genus and species name, environment, as well as its status in each of the four countries is given. 1.1. The “Registry” worksheet comprises the registry itself, listing all non-native species that were recorded as non-native in at least one of the four countries of the Two Seas region (Great Britain, France, Belgium and Netherlands). For each species, the phylum/division, class, genus and species name, environment, as well as its status in each of the four countries is given. 1.2. The “Summary of data” worksheet provides 3 tables, grouping the non-native species of the registry according to their 1.2.1. Phyla, 1.2.2. Presence in each Two Seas region country, and 1.2.3. Environment inhabited. Three simple graphs visualising these tables are also provided. 1.2. The “Summary of data” worksheet provides 3 tables, grouping the non-native species of the registry according to their 1.2.1. Phyla, 1.2.2. Presence in each Two Seas region country, and 1.2.3. Environment inhabited. Three simple graphs visualising these tables are also provided. Registry of non-native species in the Two Seas region countries 75 75 hi 4. Header information hi 4. Header information A single header row includes the species’ phylogenetic classification (i.e. four headers: phylum/division, class, genus and species name), status in the four countries inves tigated (i.e. present, native, extinct or not confirmed), environment (i.e. terrestrial, freshwater, marine, freshwater+terrestrial, marine+freshwater or terrestrial+marine), and Notes. 2. Sizeh The size of the file is 389 KB. The table lists 6,661 species, subspecies and hybrids. In total and including headers, the “Registry” worksheet therefore contains 66,620 cells. The “Summary of data” worksheet contains 92 cells. h 3. Format and storage modehi The file type is MS Excel®. No compression scheme was employed. Acknowledgements RINSE is funded by the European Union Interreg IVA 2 Mers Seas Zeeën Programme, with the support of the European Regional Development Fund. The programme pro motes cross-border cooperation between the coastal regions of four Member States: France (Nord-Pas de Calais), England (SW, SE), Belgium (Flanders) and The Neth erlands (South coastal area). This data paper reflects the authors’ views and the Pro gramme Authorities are not liable for any use that may be made of the information contained therein. B. Variable information 1. Variable identity 2. Variable definition 3. Units of measure-ment 4a. Storage type 4b. List and definition of variable codes Phylum / Division Taxonomic phylum or division of species N/A Character N/A Class Taxonomic class of species N/A Character incertae sedis - taxonomic placement currently unresolved Genus Genus name N/A Character N/A Species Species name N/A Character sp. - taxon not identified to species level Great Britain Status of species in Great Britain N/A Character extinct - non-native species was present in the wild in GB/France/Belgium/Netherlands at some time but is no longer present in the respective country native - species native to GB/France/Belgium/ Netherlands not confirmed - presence of non-native species not confirmed for GB/France/Belgium/ Netherlands present - non-native species has been recorded in the wild in GB/France/Belgium/Netherlands and is likely to exist there at this time present/extinct - non-native species listed as “present” by one source but as “extinct” by another source present/native - non-native species listed as “present” by one source but as “native” by another source France Status of species in France N/A Character Belgium Status of species in Belgium N/A Character Netherlands Status of species in the Netherlands N/A Character Environment Environment(s) inhabited by species N/A Character N/A Notes Additional notes to data source N/A Character data based solely on DAISIE portal - taxon listed as present by the DAISIE portal but not by any of the other databases consulted; no additional portal was consulted regarding geographical distribution (also see Methods section) 76 Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) 5. Data format a. Columns Start column, end column Start column = Phylum/Division, End column = Notes a. Columns Start column, end column Start column = Phylum/Division, End column = Notes a. Columns Start column, end column Start column = Phylum/Division, End column = Notes References Agence de l’eau Artois Picardie, Conservatoire Botanique National de Bailleul (2005) Les espèces végétales invasives des milieux aquatiques et humides du Bassin Artois Picardie, 37 pp. Agence de l’eau Rhin Meuse (2005) Plantes invasives des milieux aquatiques et des zones hu mides du Nord-est de la France. Une menace pour notre environnement, 20 pp. BFIS - Belgian Forum on Invasive Species. Harmonia database. http://ias.biodiversity.be [accessed 2013] Brylinski J-M, Antajan E, Raud T, Vincent D (2012) First record of the Asian copepod Pseudo diaptomus marinus Sato, 1913 (Copepoda: Calanoida: Pseudodiaptomidae) in the south ern bight of the North Sea along the coast of France. 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Supplementary material 1 Supplementary material 1 Registry of non-native species in the Two Seas region countries (Great Britain, France, Belgium and the Netherlands) References Zięba G, Copp GH, Davies GD, Stebbing P, Wesley KJ, Britton JR (2010) Recent releases and dispersal of non-native fishes in England and Wales, with emphasis on sunbleak Leucaspius delineatus (Heckel, 1843). Aquatic Invasions 5: 155–161. doi: 10.3391/ai.2010.5.2.04 Alexandra Zieritz et al. / NeoBiota 23: 65–80 (2014) 80 Registry of non-native species in the Two Seas region countries (Great Britain, France, Belgium and the Netherlands) g Authors: Alexandra Zieritz, Belinda Gallardo, David C. Aldridge Authors: Alexandra Zieritz, Belinda Gallardo, David C. Aldridge Data type: occurrence Authors: Alexandra Zieritz, Belinda Gallardo, David C. Aldridge Data type: occurrencehi Authors: Alexandra Zieritz, Belinda Gallardo, David C. Aldridge D Data type: occurrenceh yp Explanation note: The MS Excel file contains two worksheets:h Explanation note: The MS Excel file contains two worksheets:h 1. The “Registry” worksheet comprises lists 661 non-native species that were recorded as non-native in at least one of the four countries Great Britain, France, Belgium and the Netherlands. For each species, the phylum/division, class, genus and spe cies name, environment and status in each country is given.h 2. The “Summary of data” worksheet provides 3 tables, grouping the non-native spe cies of the registry according to their phyla, presence in each country, and environ ment inhabited. Three simple graphs visualising these tables are also provided.h h Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
https://openalex.org/W3124083453	https://www.auctoresonline.org/uploads/articles/1625315811pdf.pdf	English	null	Laser Therapy in the Complex Prevention and Treatment of Covid-19 (Preliminary Results)	International journal of clinical case reports and reviews	2,021	cc-by	3,494	Abstract This article presents preliminary results of treatment of 51 patients with COVID 19 (Moscow Region, Russia). These patients were subjected to various schemes of immune stimulation for the prevention and treatment of this disease. Were compared- Percutaneus laser therapy (PLT), Intravenous Laser Blood Irradiation (ILBI), Drug stimulation and their combination. The results showed: 1. In the treatment of COVID-19, the use of various types of immunomodulation and anticoagulants proved to be most effective. 2. The combination of ILBI and TLT with immunomodulators proved to be the most effective in the prevention and treatment of COVID-19. 2. The combination of ILBI and TLT with immunomodulators proved to be the most effective in the preventio COVID-19. 2. The combination of ILBI and TLT with immunomodulators proved to be the most effective in the prevention and treatment of COVID-19. 3. Immediate use of immunomodulators at the very beginning of COVID-19 reduces the severity of the disease, and facilitates its course. 3. Immediate use of immunomodulators at the very beginning of COVID-19 reduces the severity of the disease, and facilitates its course. 3. Immediate use of immunomodulators at the very beginning of COVID-19 reduces the severity of the dise its course. Background and Aims: We started to use laser therapy in 1988. When used in different categories of cancer patients, it was found that various types of laser radiation stimulate the immune system. We started to use this peculiarity of laser therapy to boost the immune status of sickly patients with weakened immune systems, as well as for prevention and treatment of respiratory viral infections (e.g. influenza, parainfluenza, acute respiratory infections). We performed various types of Immunostimulation in 51 patients from Russia and evaluated its influence both on the morbidity and the course of COVID-19. Rationale: Laser radiation (890-910 nm) stimulates cell immunity, increasing the amount of active T-lymphocytes. The wavelength of 630-640 nm is the most effective for irradiation both the blood and the vascular walls. At this wavelength photons are absorbed by oxygen, microcirculation improves, decrease blood viscosity, and direct impact on the nerve and muscle elements of the vascular wall influences the activity of the vascular and nervous systems. Conclusion::The laser therapy practice we have been exercising for over 30 years has shown that it produces good immunostimulating effects. Abstract The use of various laser therapy methods combined with immunomodulatory drugs allow to reduce the number of patients infected with COVID-19, and reduce the severity of the disease. Key words: immunostimulation; percutaneus laser therapy (plt); intravenous laser blood irradiation (ilbi); immunomodulatory drugs; covid-19; respiratory virus infection phenomenon as a new form of parainfluenza. Discussions with pediatricians and general practitioners confirmed our assumptions. Laser Therapy in the Complex Prevention and Treatment of Covid-19 (Preliminary Results) V.A. Mikhaylov Eternity Medicine Institute, Dubai V.A. Mikhaylov Eternity Medicine Institute, Dubai Corresponding Author: V.A. Mikhaylov, M.D. Ph.D., Kooperative str, 2/14, fl.96, 119048, Moscow, Russia, Eternity Medicine Institute, KG Tower, PO Box 120618, Dubai Marina, UAE. Received date: December 15, 2020; Accepted date: January 04, 2021; Published date: January 08, 2021 15, 2020; Accepted date: January 04, 2021; Published date: January 08, 2021 Citation: V.A. Mikhaylov. (2021) Laser Therapy in the Complex Prevention and Treatment of Covid-19 (Preliminary Results). International Journal of Clinical Case Reports and Reviews. 6(2); DOI:10.31579/2690-4861/101 Copyright: © 2021 V.A. Mikhaylov, 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. Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 Methods of Immunostimulation: The patients were distributed by their gender as follows (Table 2). The patients were distributed by their gender as follows (Table 2). Stage I: ILBI (November-December, 2019). ILBI was performed according to the standard method (10 sessions) (A puncture of the ulnar vein with one-time sterile catheter was performed. The catheter consisted of a thin needle with a monofilament through which intravascular irradiation came out (produced by Polironic). After each session of irradiation, the catheter was rejected. We divided all the patients who had fallen ill according to the severity of the disease: Mild degree: fever up to 38-39˚ C, dry cough, weakness, sweating, difficulty breathing (from 1 to 3 days). Cough from 5 to 7 days. The patients had no need to be hospitalized. Medium degree: fever up to 38-390 C, weakness, sweating, difficulty breathing (for 4-5 days). Cough up to 7-10 days. The patients had no need to be hospitalized. Stage II: Percutaneous laser therapy (Percutaneus laser therapy, hereinafter “PLT”) (mid-late January, 2020). We performed percutaneous to be hospitalized. Age Below 40 40-50 50-60 60-80 Over 80 1 group - 1 3 2 2 2 group 1 - 4 4 2 3 group 3 7 5 5 4 4 group 2 2 2 2 - Table 1: Distribution of the patients with viral infections by age 1 group 2 group 3 group 4 group Male 4 6 11 5 Female 4 5 13 3 Table 2: Distribution of patients with viral infections by gender Severe degree: fever up to 39 ˚ C or higher, weakness, sweating. Pulmonary insufficiency and heart failure. Severe symptoms of intoxication. Dry barking cough. Patients needed to be hospitalized. Immediately, Amixin was taken according to the application scheme, and vitamin C. In order to prevent secondary infections, Biseptol was taken according to the application scheme. On the next day the patient’s state improved: the temperature dropped to 37,8˚ C. The weakness and the sweating lessened by day 5. The dry cough remained for 2 weeks, though. The 2nd course of PLT was performed, Amixin was re-taken according to the application scheme, as well as vitamin C. Gradually the cough lessened. The cough finally disappeared only 4 weeks after the onset of the disease. The treatment was performed at home in self-isolation. Contact persons were not identified. Two patients fell ill in Group 1. Results 2. Semi-conductor (diode) laser – Mulat, wavelengths 640 nm nm, 1-2 mW. 2. Semi-conductor (diode) laser – Mulat, wavelengths 640 nm nm, 1-2 mW. All patients were divided into the following groups: Group 1: 8 patients (ILBI + PLT + immunomodulatory drugs). 3. Frequency-modulated diode laser – Magic Beam, 640 nm, 1-2 mW. Group 2: 11 patients (PLT + immunomodulatory drugs). Equipment for PLT: Group 3: 24 patients (received only medical immunostimulation). 1. GaAs semi-conductor laser Uzor (wavelength 890 nm, pulsed mode, pulse power 5-10 W. 1. GaAs semi-conductor laser Uzor (wavelength 890 nm, pulsed mode, pulse power 5-10 W. Group 4: 8 patients (immunomodulatory drugs according to application the scheme + vitamin C). All had the signs of acute respiratory disease. 2. Semiconductor laser Mustang (wavelength 910 nm, pulsed mode, pulse power 10 W. 2. Semiconductor laser Mustang (wavelength 910 nm, pulsed mode, pulse power 10 W. The patients were distributed by their age as follows (Table 1). Introduction: In September 2019 we paid attention that there evolved an acute respiratory infection which had a clinical course different from regular flu clinic and the acute respiratory viral infections. The main manifestation of this infection were long-term and severe pneumonias, which did not respond well to the standard therapy. The computer tomography revealed interstitial edemas of the tissue which is an attribute of viral pneumonias. This was first noted with children (within September-October 2019), then with adults (within October-December, 2019). Initially we treated this Up to date, there are no clear criteria for COVID-19 treatment, while the treatment methods used before, appeared to be inefficient. We therefore chose to exercise those methods of laser therapy that could be efficient in the prevention and treatment of COVID-19. ILBI has the direct impact on all blood components, and the vascular wall. The most significant among these effects are improved microcirculation, improved rheological blood properties, as well as blood clotting reduction. For the studies of this method I received an award (Ming Chien Kao Awards 2015) [1, 2, 3]. Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 Page 1 of 4 Copy rights@ V.A. Mikhaylov et.al. International Journal of Clinical Case Reports and Reviews laser stimulation using the standard method during 5 days. This technique is used for Immunostimulation in cancer patients [5, 6]. For immunostimulation we used the PLT schemes, which we had used for the treatment of cancer patients, respiratory viral infections [4, 5]. The obtained results showed that the use of combined Immunostimulation (laser + medication) could reduce the number of infected patients, as well as reduce the disease severity. Stage III: After 2 weeks, immunomodulatory drugs (Tirolone, Levamisole) were introduced according to the application schemes. Patients of group 4: With the first signs of the disease these patients received Tilorone according to the application scheme (from 3 to 5 tablets subject to the disease severity). Vitamin C (ascorbic acid) 200 mg 3 times a day. In case the treatment appeared to be ineffective, the patients called an ambulance and were hospitalized when necessary. Equipment for ILBI: The main laser therapy systems are used for ILBI: The main laser therapy systems are used for ILBI: The main laser therapy systems are used for ILBI: 1. Helium-neon laser -Alok-1, wavelength 632.8 nm, 1-2 mW. ( 1. Helium-neon laser -Alok-1, wavelength 632.8 nm, 1-2 mW. ( Methods of Immunostimulation: Both patients got sick during February, 2020 after their contacts with those infected with COVID-19 (one after contacts with the relatives who had come back from the UK, the other fell ill after a trip to Spain). There was detected a mild degree of the disease, the recovery came after 3 or 4 days. Five patients fell ill in Group 2, among which 4 persons with a mild degree of the disease (2 patients in November 2019, 1 patient in December, 2019, 2 patients in the end of January-beginning of February, 2020). Group 3: 6 persons fell ill (4 persons (end of January-beginning of February, 2020, 2 persons in mid-March, 2020). Group 4: 8 patients (4 persons in the end of January, 2 persons in the beginning of February, 2020; 2 persons in the beginning-middle of March, 2020) were infected from relatives and acquaintances. For 2 persons the contacts were not identified. Severe degree: 1 person fell ill (November, 2019). A 17-year-old male patient fell ill abruptly. The temperature rose to 39,2˚ C. The patient developed a severe dry cough, chills, weakness, sweating. A 17-year-old male patient fell ill abruptly. The temperature rose to 39,2˚ C. The patient developed a severe dry cough, chills, weakness, sweating. Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 Page 2 of 4 Page 2 of 4 Copy rights@ V.A. Mikhaylov et.al. International Journal of Clinical Case Reports and Reviews diagnosis of COVID-19 was confirmed. The diagnosis is COVID-19, mild degree. The results obtained for morbidity are presented in Table 3. Table 3: Distribution of patients with viral infections by disease degree Groups (number of patients ) A number of performed tests Results of testing Number of sick patients Reliability of the performed tests, % outpatients in the hospital positive negative outpatients in the hospital Group 1 (8) 6 (2) 2 3 (1) 2 (1) 2 75,67% 95,45% Group 2 (11) 7 (1) 6 (1) 6 (1) 4 Group 3 (24) 14 (4) 6 6 (1) 12 (2) 9 Group 4 (8) 10 (2) 8 8 2 (2) 8 Table 4: Results of testing patients for COVID-19 Table 4: Results of testing patients for COVID-19 Number of sick patients The distribution of patients according to the degree of the disease mild medium severe Group 1 25% 25% - - Group 2 36,36% 27,27% 9,09% Group 3 37,5% 37,5% - - Group 4 100% 87,5% 12,5% Table 5: Distribution of COVID-19 patients by number and severity of the disease (%) Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 The results obtained for morbidity are presented in Table 3. diagnosis of COVID-19 was confirmed. The diagnosis is COVID-19, mild degree. 1* - (17-year-old male). The severity of the disease was assessed by the duration of dry cough and weakness which lasted for 4 weeks. The patient was not hospitalized. Starting February, 2020 the patients were tested for COVID-19. The test results are presented in Table 4. The patients are herewith distributed by the number and the degrees of COVID-19 severity as follows (Table 5): 1** - (54-year-old male). The patient got infected at work from a COVID- 19 colleague. The Patient was tested for COVID-19, however the test came back negative. After 3 days the temperature increased to 39,2˚ C, chills and weakness developed. There was no cough. On the 4th day in the morning the temperature dropped to 36,0˚ C. The patient developed a tachycardia and a slight shortness of breath. Taking into account concomitant diseases (hypertension, obesity of the 2nd degree, coronary heart disease, vascular atherosclerosis), the patient was hospitalized. The We did not take into account the number of hospitalized patients (i.e., 1 patient). This patient was hospitalized not due to the severe grade of the COVID-19 disease, but due to his concomitant diseases. Even more so, the diagnosis made in the hospital was a mild grade of COVID-19. ( yp , y g , y heart disease, vascular atherosclerosis), the patient was hospitalized. The At the time of writing (May 15, 2020), there were no other cases of the disease. The results obtained for morbidity are presented in Table 3. Severity of the disease Mild Medium grade Severe degree Group 1 2 - - Group 2 3 - 1* Group 3 9 (1*) - - Group 4 7 1 - Table 3: Distribution of patients with viral infections by disease degree Groups (number of patients ) A number of performed tests Results of testing Number of sick patients Reliability of the performed tests, % outpatients in the hospital positive negative outpatients in the hospital Group 1 (8) 6 (2) 2 3 (1) 2 (1) 2 75,67% 95,45% Group 2 (11) 7 (1) 6 (1) 6 (1) 4 Group 3 (24) 14 (4) 6 6 (1) 12 (2) 9 Group 4 (8) 10 (2) 8 8 2 (2) 8 Table 4: Results of testing patients for COVID-19 Number of sick patients The distribution of patients according to the degree of the disease mild medium severe Group 1 25% 25% - - Group 2 36,36% 27,27% 9,09% Group 3 37,5% 37,5% - - Group 4 100% 87,5% 12,5% Table 5: Distribution of COVID-19 patients by number and severity of the disease (%) Discussion For over 30 years we have been using laser energy of various wavelengths to prevent and treat various diseases [6, 7, 8, 9, 10]. N i f d di ti (890 910 ) t t d l i t Ministry of Health of Russia (there have been 6 updates of the recommendations for the treatment of COVID-19 since January, 2020). Since recently all patients in Moscow hospitals were obliged to take anticoagulants and immunomodulatory drugs. Conclusion: 1. In the treatment of COVID-19, the use of various types of immunomodulation and anticoagulants proved to be most effective. 9. Mikhaylov V.A. A newly discovered way of the function of cardio-vascular system and the latest theory of the development of Hypertension and other cardio-vascular diseases. Adstr. “2nd International Conference on Hypertension & Healthcare”, Amsterdam, Netherlands, 2017, September 11-13. 2. The combination of ILBI and TLT with immunomodulators proved to be the most effective in the prevention and treatment of COVID-19. 3. Immediate use of immunomodulators at the very beginning of COVID- 19 reduces the severity of the disease, and facilitates its course. 3. Immediate use of immunomodulators at the very beginning of COVID- 19 reduces the severity of the disease, and facilitates its course. 10. Mikhaylov V.A. A newly discovered way of the function of cardio-vascular system and the latest theory of the development of Hypertension and other cardio-vascular diseases. EC Cardiology (ECCY), Volume 5, Issue 4 (Page No: 179-187). Discussion Using ILBI to treat acute infections was a relative contraindication [11]. 3. Mikhaylov V.A. (2016) Ming Chien Kao Awards 2015 Laser Therapy, mar. 25(1); 9-10. The clinical picture of COVID-19 failed to fit the picture of regular respiratory infections. It was characterized by high aggressiveness, atypical course and lack of treatment effects. The effect of frosted glass denoting the diffuse interstitial edema of the lung tissue was noted on Computed Tomography (CT) scans. All these indicated a systemic lesion of the lung tissue. ILBI's main systemic effects were reduced blood clotting and improved microcirculation. In this case, these effects could be the main ones in the complex treatment of COVID-19. In May, 2020 anticoagulants were introduced in Moscow hospitals. This proved that our choice had been correct. 4. Mikhailov V. A., Zakharov S. D., Skobelkin O. K., Eliseenko V. I., Chaitsev V. G., Silkina G. I., Denisov I. N. (1990) Activation of the immune system in cancer patients with low-energy laser radiation in the preoperative period. Collection of scientific and practical conference reports. Low-energy laser radiation in medical practice, Khabarovsk. 5. Mikhailov V.A., Derbenjev V.A., Denisov I.N. (1999) The use of low level laser therapy in the treatment of some pulmonary diseases (10 –years’ experience). 14 th. International Congress Laser Medicine ICLM 99, Florence, 28-31 October, Suppl. Laser Journal. In conclusion I would like to note that the presented material describes a small amount of cases, therefore the results may statistically be unreliable, while a short observation period does not provide long-term treatment results. I would just like to share the experience which was gained in the treatment of COVID-19. I trust this will help other researchers, and can support further improvements aimed at the COVID-19 treatment effectiveness. 6. Mikhailov V. A Skobelkin O. K. Low-energy laser in preoperative period in oncologic patients. Proceeding of the International Conference. Tashkent. 40-44. 7. Mikhailov V.A. (1998) Principles of treatment and laser therapy. Proceedings 2 Congress World Association for Laser Therapy. sept. 2-5, Kansas-Sity, USA, p.76-77. 8. Mikhailov V.A. (2000) Results of clinical study of use low level laser therapy (LLLT) for the treatment of the malignant tumors of a gastro-intestinal system. LASERS IN MEDICINE, EMLA.437- 455. Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 Discussion Ministry of Health of Russia (there have been 6 updates of the recommendations for the treatment of COVID-19 since January, 2020). Since recently all patients in Moscow hospitals were obliged to take anticoagulants and immunomodulatory drugs. For over 30 years we have been using laser energy of various wavelengths to prevent and treat various diseases [6, 7, 8, 9, 10]. Near infrared radiation range (890-910 nm) can penetrate deeply into tissues, and have a local effect on the work of various tissues and organs. This allows you to influence the pathological focus and the work of various body systems. The use of this wavelength allows stimulating cellular immunity for up to 3 months as an average. The identical results in Groups 2 and 3 are explained by the fact that the patients’ response to the introduction of immunomodulatory drugs (Group 3) is stronger than in patients receiving PLT(Group 2). The body responds to the introduction of immunomodulatory drugs with a faster production of interferons and the activation of cellular immunity. The body responds to PLT in a slower manner. As a rule, the effect comes slower, and reaches its maximum in 2 weeks. The duration of the effect is longer and lasts up to 3 months. This allows limiting the number of complications and to reduce the severity of the disease. Using ILBI (630-640 nm) allows to directly influence the parameters of all blood cells, blood plasma, the process of clotting and the work of nerve and muscle elements of the vascular wall. The best results with the combined use of ILBI (Group 1) can be explained by a direct impact on blood cells, improvement of microcirculation and rheological properties of blood by reducing blood clotting. Our assumptions were confirmed by the recommendations of the The scheme (PLT + immunomodulatory drugs) has been used for the treatment of acute respiratory infections for the past 5 years. As a rule, in 70-80% of cases, a positive effect was observed. The disease was stopped Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 Page 3 of 4 Copy rights@ V.A. Mikhaylov et.al. International Journal of Clinical Case Reports and Reviews within 2 or 3 days. We have never used ILBI in these cases. Using ILBI to treat acute infections was a relative contraindication [11]. within 2 or 3 days. We have never used ILBI in these cases. References: 1. Mikhailov V. (2009) Development and clinical application of Intravenous Laser Blood irradiation (ILBI). Laser Therapy. 18(2); 69- 84. 11. Mikhailov V. Intravenous Laser Blood Irradiation. Greece, 2007, p.103 2. Mikhaylov V.A. (2015) Use of intravenous laser blood irradiation (ILBI) at 630-640 nm to prevent vascular diseases and to increase life expectancy. Laser Therapy, vol. 24(1); 15-26. Auctores Publishing – Volume 6(2)-0101 www.auctoresonline.org ISSN: 2690-4861 Page 4 of 4 Page 4 of 4

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